Genetica e studi molecolari (aprile 2003 - aprile 2012)

Non-chemotactic influence of CXCL7 on human phagocytes. Modulation of antimicrobial activity against L. pneumophila

González-Cortés C, Diez-Tascón C, Guerra-Laso JM, González-Cocaño MC, Rivero-Lezcano OM.

Unidad de Investigación, Complejo Asistencial Universitario de León, Spain. omrivero@iecscyl.com

Immunobiology. 2012 Apr;217(4):394-401.

ABSTRACT: We have investigated the role of CXCL7 in the immune response of human phagocytes against the intracellular bacteria Mycobacterium tuberculosis and Legionella pneumophila. We have observed that polymorphonuclear neutrophil (PMN) chemotaxis induced by the supernatants of infected monocyte derived macrophages (MDM) may be attributed to CXCL8 rather than CXCL7, although both chemokines are present in large quantities. We have also found that CXCL7 is present not only in the supernatants of MDM, but also in the supernatants of PMN of some, but not all, individuals. Western blot analysis revealed that, in both MDM and PMN supernatants appeared two bands with molecular weights consistent with the platelet basic protein (PBP) and the neutrophil activating protein-2 (NAP-2) sizes. Regarding the influence on infected cells, recombinant NAP-2 enhanced the antimicrobial activity of IFNγ activated MDM against L. pneumophila, but not against M. tuberculosis. In addition, U937 cells transfected with a NAP-2 construct inhibited the intracellular multiplication of L. pneumophila, supporting its role in the modulation of the antimicrobial activity. Finally, U937 cells transfected with the NAP-2 construct showed an adherence that was dramatically enhanced when the substrate was fibronectin. We conclude that human phagocytes produce CXCL7 variants that may have a significant influence on the immune response against bacterial pathogens.

 

Importance of the icmN gene in the growth of Legionella pneumophila in amoebic cells at low temperature

Qin T, Iida K, Piao Z, Shiota S, Ren H, Shao Z, Yoshida S.

Department of Bacteriology, Faculty of Medical Sciences, Division of Oral Infectious Diseases and Immunology, Faculty of Dental Science, Kyushu University, Higashi-ku, Fukuoka 812-8582, Japan. qintian@icdc.cn

Can J Microbiol. 2012 Apr;58(4):490-501.

ABSTRACT: Legionella pneumophila grows in amoebae and has achieved the ability to grow at various temperatures, although the mechanisms controlling this ability remain poorly understood. The Icm/Dot type IVB secretion system is composed of more than 25 proteins and is known to be essential for intracellular growth. The role of the icmN gene in intracellular multiplication and the effects of culture temperatures on it are not precisely understood. We conducted our investigation using an icmN mutant made by gene replacement mutagenesis. Intracellular growth of the mutant was impaired both in mammalian macrophages and amoeba at 37 °C. In particular, intracellular growth in amoebae was completely impaired at 25 °C. It was found that genes from icmN to icmC formed an operon, i.e., icmN, -M, -L, -E, -G, -C,, and the promoter activity of the icmN operon was stronger at 25 than at 37 °C. It was suggested that icmM and its downstream genes had a secondary promoter that enables icmN mutant grow in amoebae at lower temperatures and macrophages at 37 °C. These results show that the icmN promoter has a low temperature inducible nature, and gene products of the icmN operon require high expression for bacterial proliferation at low temperatures within amoeba.

 

Preventing bacterial DNA release and absent in melanoma 2 inflammasome activation by a Legionella effector functioning in membrane trafficking

Ge J, Gong YN, Xu Y, Shao F.

National Institute of Biological Sciences, Beijing 102206, China. shaofeng@nibs.ac.cn

Proc Natl Acad Sci U S A. 2012 Apr 17;109(16):6193-8.

ABSTRACT: Legionella pneumophila, the causative agent of Legionnaires' pneumonia, resides in a distinct vacuole structure called Legionella-containing vacuole (LCV). The LCV resists fusion with the lysosome and permits efficient bacterial replication in host macrophages, which requires a Dot/Icm type IVB secretion system. Dot/Icm-translocated effector SdhA is critical for L. pneumophila intracellular growth and functions to prevent host cell death. Here, we show that the absence of SdhA resulted in elevated caspase-1 activation and IL-1β secretion as well as macrophage pyroptosis during Legionella infection. These inflammasome activation phenotypes were independent of the established flagellin-NAIP5-NLRC4 axis, but relied on the DNA-sensing AIM2 inflammasome. We further demonstrate that Legionella DNA was released into macrophage cytosol, and this effect was significantly exaggerated by the absence of SdhA. SdhA bears a functional Golgi-targeting GRIP domain that is required for preventing AIM2 inflammasome activation. Ectopically expressed SdhA formed a unique ring-shape membrane structure, further indicating a role in membrane trafficking and maintaining LCV membrane integrity. Our data together suggest a possible link, mediated by the function of SdhA, between LCV trafficking/maturation and suppression of host innate immune detection.

 

The major facilitator superfamily-type protein LbtC promotes the utilization of the legiobactin siderophore by Legionella pneumophila

Chatfield CH, Mulhern BJ, Viswanathan VK, Cianciotto NP.

Department of Microbiology and Immunology, Northwestern University Medical School, Chicago, IL 60611, USA. n-cianciotto@northwestern.edu

Microbiology. 2012 Mar;158(Pt 3):721-35.

ABSTRACT: The Gram-negative bacterium Legionella pneumophila elaborates the siderophore legiobactin. We previously showed that cytoplasmic LbtA helps mediate legiobactin synthesis, inner-membrane LbtB promotes export of legiobactin, and outer-membrane LbtU acts as the ferrisiderophore receptor. RT-PCR analyses now identified lbtC as an iron-repressed gene that is the final gene in an operon containing lbtA and lbtB. In silico analysis predicted that LbtC is an inner-membrane protein that belongs to the major facilitator superfamily (MFS). Although capable of normal growth in standard media, lbtC mutants were defective for growth on iron-depleted agar media. While producing normal levels of legiobactin, lbtC mutants were unable to utilize supplied legiobactin to stimulate growth on iron-depleted media and displayed an impaired ability to take up radiolabelled iron. All lbtC mutant phenotypes were complemented by reintroduction of an intact copy of lbtC. When a cloned copy of both lbtC and lbtU was introduced into a heterologous bacterium (Legionella longbeachae), the organism acquired the ability to utilize legiobactin to grow better on low-iron media. Together, these data indicate that LbtC is involved in the uptake of legiobactin, and based upon its predicted location is most likely the mediator of ferrilegiobactin transport across the inner membrane. The data are also a unique documentation of how an MFS protein can promote bacterial iron-siderophore import, standing in contrast to the vast majority of studies which have defined ABC-type permeases as the mediators of siderophore import across the Gram-negative inner membrane or the Gram-positive cytoplasmic membrane.

 

Legionella pneumophila LidA affects nucleotide binding and activity of the host GTPase Rab1

Neunuebel MR, Mohammadi S, Jarnik M, Machner MP.

Cell Biology and Metabolism Program, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, USA. machnerm@mail.nih.gov

J Bacteriol. 2012 Mar;194(6):1389-400.

ABSTRACT: Legionella pneumophila, the causative agent of a severe pneumonia known as Legionnaires' disease, intercepts material from host cell membrane transport pathways to create a specialized vacuolar compartment that supports bacterial replication. Delivery of bacterial effector proteins into the host cell requires the Dot/Icm type IV secretion system. Several effectors, including SidM, SidD, and LepB, were shown to target the early secretory pathway by manipulating the activity of the host GTPase Rab1. While the function of these effectors has been well characterized, the role of another Rab1-interacting protein from L. pneumophila, the effector protein LidA, is poorly understood. Here, we show that LidA binding to Rab1 stabilized the Rab1-guanosine nucleotide complex, protecting it from inactivation by GTPase-activating proteins (GAPs) and from nucleotide extraction. The protective effect of LidA on the Rab1-guanine nucleotide complex was concentration dependent, consistent with a 1:1 stoichiometry of the LidA-Rab1 complex. The central coiled-coil region of LidA was sufficient for Rab1 binding and to prevent GAP-mediated inactivation or nucleotide extraction from Rab1. In addition, the central region mediated binding to phosphatidylinositol 3-phosphate and other phosphoinositides. When bound to Rab1, LidA interfered with the covalent modification of Rab1 by phosphocholination or AMPylation, and it also blocked de-AMPylation of Rab1 by SidD and dephosphocholination by Lem3. Based on these findings, we propose a role for LidA in bridging the membrane of the Legionella-containing vacuole (LCV) with that of secretory transport vesicles surrounding the LCV.

 

Draft genome sequences of two Legionella dumoffii strains, TEX-KL and NY-23

Qin T, Cui Y, Cen Z, Liang T, Ren H, Yang X, Zhao X, Liu Z, Xu L, Li D, Song Y, Yang R, Shao Z, Song Y.

State Key Laboratory for Infectious Disease Prevention and Control and National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, People's Republic of China. songyajun88@yahoo.com.cn

J Bacteriol. 2012 Mar;194(5):1251-2.

ABSTRACT: Legionella (Fluoribacter) dumoffii is one of the agents causing Legionnaires' disease. Here, we used Illumina second-generation sequencing technology to decipher for the first time the whole-genome sequences of two strains of this species, TEX-KL and NY-23. The assembly results for both strains consist of one chromosome and two plasmids.

 

Whole-genome sequence of the human pathogen Legionella pneumophila serogroup 12 strain 570-CO-H

Amaro F, Gilbert JA, Owens S, Trimble W, Shuman HA.

Department of Microbiology, University of Chicago, Chicago, Illinois, USA. hshuman@bsd.uchicago.edu

J Bacteriol. 2012 Mar;194(6):1613-4.

ABSTRACT: We present the genomic sequence of the human pathogen Legionella pneumophila serogroup 12 strain 570-CO-H (ATCC 43290), a clinical isolate from the Colorado Department of Health, Denver, CO. This is the first example of a genome sequence of L. pneumophila from a serogroup other than serogroup 1. We highlight the similarities and differences relative to six genome sequences that have been reported for serogroup 1 strains.

 

Post-translational modifications of host proteins by Legionella pneumophila: a sophisticated survival strategy

Rolando M, Buchrieser C.

Institut Pasteur, Biologie des Bactéries Intracellulaires, 75724 Paris, France. cbuch@pasteur.fr

Future Microbiol. 2012 Mar;7(3):369-81.

ABSTRACT: Eukaryotic proteins are tightly regulated by post-translational modifications, leading to a very subtle degree of regulation in time and space. Pathogen-mediated post-translational modifications are key strategies to modulate host factors by targeting central signaling pathways in the host cell. Legionella pneumophila, an intracellular pathogen that coevolved with protozoan hosts, encodes a large arsenal of secreted effectors conferring the ability to evade host cellular defenses and to manipulate them to promote invasion and intracellular replication. Conservation of many signaling pathways of protozoa in human macrophages confers the ability of L. pneumophila to infect humans, causing a severe pneumonia called legionnaires' disease. Most of the secreted proteins are delivered by the Dot/Icm type IV secretion system and several of these have been shown to act on different cellular pathways critical for infection. Moreover, multiple effectors target a single host function to orchestrate bacterial survival. In this review, we focus on those effectors in the repertoire of L. pneumophila proteins that target key cellular pathways by specific post-translational modifications.

 

Structural insights into a unique Legionella pneumophila effector LidA recognizing both GDP and GTP bound Rab1 in their active state

Cheng W, Yin K, Lu D, Li B, Zhu D, Chen Y, Zhang H, Xu S, Chai J, Gu L.

State Key Laboratory of Microbial Technology, Shandong University, Jinan, China. lcgu@sdu.edu.cn

PLoS Pathog. 2012 Mar;8(3):e1002528.

ABSTRACT: The intracellular pathogen Legionella pneumophila hijacks the endoplasmic reticulum (ER)-derived vesicles to create an organelle designated Legionella-containing vacuole (LCV) required for bacterial replication. Maturation of the LCV involved acquisition of Rab1, which is mediated by the bacterial effector protein SidM/DrrA. SidM/DrrA is a bifunctional enzyme having the activity of both Rab1-specific GDP dissociation inhibitor (GDI) displacement factor (GDF) and guanine nucleotide exchange factor (GEF). LidA, another Rab1-interacting bacterial effector protein, was reported to promote SidM/DrrA-mediated recruitment of Rab1 to the LCV as well. Here we report the crystal structures of LidA complexes with GDP- and GTP-bound Rab1 respectively. Structural comparison revealed that GDP-Rab1 bound by LidA exhibits an active and nearly identical conformation with that of GTP-Rab1, suggesting that LidA can disrupt the switch function of Rab1 and render it persistently active. As with GTP, LidA maintains GDP-Rab1 in the active conformation through interaction with its two conserved switch regions. Consistent with the structural observations, biochemical assays showed that LidA binds to GDP- and GTP-Rab1 equally well with an affinity approximately 7.5 nM. We propose that the tight interaction with Rab1 allows LidA to facilitate SidM/DrrA-catalyzed release of Rab1 from GDIs. Taken together, our results support a unique mechanism by which a bacterial effector protein regulates Rab1 recycling.

 

Reversible phosphocholination of Rab proteins by Legionella pneumophila effector proteins

Goody PR, Heller K, Oesterlin LK, Müller MP, Itzen A, Goody RS.

Department of Physical Biochemistry, Max Planck Institute of Molecular Physiology, Dortmund, Germany. Department of Tropical Medicine and Infectious Diseases, University of Rostock, Rostock, Germany. aymelt.itzen@tum.de

EMBO J. 2012 Feb 3;31(7):1774-84.

ABSTRACT: The Legionella pneumophila protein AnkX that is injected into infected cells by a Type IV secretion system transfers a phosphocholine group from CDP-choline to a serine in the Rab1 and Rab35 GTPase Switch II regions. We show here that the consequences of phosphocholination on the interaction of Rab1/Rab35 with various partner proteins are quite distinct. Activation of phosphocholinated Rabs by GTP/GDP exchange factors (GEFs) and binding to the GDP dissociation inhibitor (GDI) are strongly inhibited, whereas deactivation by GTPase activating proteins (GAPs) and interactions with Rab-effector proteins (such as LidA and MICAL-3) are only slightly inhibited. We show that the Legionella protein lpg0696 has the ability to remove the phosphocholine group from Rab1. We present a model in which the action of AnkX occurs as an alternative to GTP/GDP exchange, stabilizing phosphocholinated Rabs in membranes in the GDP form because of loss of GDI binding ability, preventing interactions with cellular GTPase effectors, which require the GTP-bound form. Generation of the GTP form of phosphocholinated Rab proteins cannot occur due to loss of interaction with cellular GEFs.

 

The protein SdhA maintains the integrity of the Legionella-containing vacuole

Creasey EA, Isberg RR.

Department of Molecular Biology and , Tufts University School of Medicine, Boston, MA 02111, USA. ralph.isberg@tufts.edu

Proc Natl Acad Sci U S A. 2012 Feb 28;109(9):3481-6.

ABSTRACT: Legionella pneumophila directs the formation of a specialized vacuole within host cells, dependent on protein substrates of the Icm/Dot translocation system. Survival of the host cell is essential for intracellular replication of L. pneumophila. Strains lacking the translocated substrate SdhA are defective for intracellular replication and activate host cell death pathways in primary macrophages. To understand how SdhA promotes evasion of death pathways, we performed a mutant hunt to identify bacterial suppressors of the ΔsdhA growth defect. We identified the secreted phospholipase PlaA as key to activation of death pathways by the ΔsdhA strain. Based on homology between PlaA and SseJ, a Salmonella protein associated with vacuole degradation, we determined the roles of SdhA and PlaA in controlling vacuole integrity. In the absence of sdhA, the Legionella-containing vacuole was unstable, resulting in access to the host cytosol. Both vacuole disruption and host cell death were largely dependent on PlaA. Consistent with these observations, the ΔsdhA strain colocalized with galectin-3, a marker of vacuole rupture, in a PlaA-dependent process. Access of ΔsdhA strains to the macrophage cytosol triggered multiple responses in the host cell, including degradation of bacteria, induction of the type I IFN response, and activation of inflammasomes. Therefore, we have demonstrated that the Legionella-containing vacuole is actively stabilized by the SdhA protein during intracellular replication. This vacuolar niche affords the bacterium protection from cytosolic host factors that degrade bacteria and initiate immune responses.

 

The Legionella pneumophila effector VipA is an actin nucleator that alters host cell organelle trafficking

Franco IS, Shohdy N, Shuman HA.

Department of Microbiology and Immunology, Columbia University Medical Center, New York, New York, USA. irinafranco@hotmail.com

PLoS Pathog. 2012 Feb;8(2):e1002546.

ABSTRACT: Legionella pneumophila, the causative agent of Legionnaires' disease, invades and replicates within macrophages and protozoan cells inside a vacuole. The type IVB Icm/Dot secretion system is necessary for the translocation of effector proteins that modulate vesicle trafficking pathways in the host cell, thus avoiding phagosome-lysosome fusion. The Legionella VipA effector was previously identified by its ability to interfere with organelle trafficking in the Multivesicular Body (MVB) pathway when ectopically expressed in yeast. In this study, we show that VipA binds actin in vitro and directly polymerizes microfilaments without the requirement of additional proteins, displaying properties distinct from other bacterial actin nucleators. Microscopy studies revealed that fluorescently tagged VipA variants localize to puncta in eukaryotic cells. In yeast these puncta are associated with actin-rich regions and components of the Multivesicular Body pathway such as endosomes and the MVB-associated protein Bro1. During macrophage infection, native translocated VipA associated with actin patches and early endosomes. When ectopically expressed in mammalian cells, VipA-GFP displayed a similar distribution ruling out the requirement of additional effectors for binding to its eukaryotic targets. Interestingly, a mutant form of VipA, VipA-1, that does not interfere with organelle trafficking is also defective in actin binding as well as association with early endosomes and shows a homogeneous cytosolic localization. These results show that the ability of VipA to bind actin is related to its association with a specific subcellular location as well as its role in modulating organelle trafficking pathways. VipA constitutes a novel type of actin nucleator that may contribute to the intracellular lifestyle of Legionella by altering cytoskeleton dynamics to target host cell pathways.

 

Live Legionella pneumophila induces MUC5AC production by airway epithelial cells independently of intracellular invasion

Morinaga Y, Yanagihara K, Araki N, Migiyama Y, Nagaoka K, Harada Y, Yamada K, Hasegawa H, Nishino T, Izumikawa K, Kakeya H, Yamamoto Y, Kohno S, Kamihira S.

Department of Laboratory Medicine, Nagasaki University Graduate School of Biomedical Sciences, 1-7-1, Sakamoto, Nagasaki, Japan. k-yanagi@nagasaki-u.ac.jp

Can J Microbiol. 2012 Feb;58(2):151-7.

ABSTRACT: The airway epithelium is the initial barrier against airborne pathogens, and it plays many roles in host airway defense. Legionella pneumophila is an intracellular pathogen that causes rapidly advancing pneumonia and is sometimes life-threatening. Here, we evaluated the role of the airway epithelial cells in the defense against L. pneumophila by examining mucus production in vitro. The production of MUC5AC, a major mucin protein, was not induced by formalin- or ultraviolet-killed L. pneumophila, but it was induced by live L. pneumophila. Similarly, nuclear factor-kappaB (NF-κB) was activated only by live L. pneumophila. Inhibitors of ERK and JNK, but not p38, dose-dependently inhibited the induction of MUC5AC by live L. pneumophila. Inhibition of intracellular invasion by cytochalasin D did not affect MUC5AC production. Taken together, the results suggest that live L. pneumophila induces MUC5AC production via the ERK-JNK and NF-κB pathways without internalization of bacteria and that the airway epithelium produces mucin as part of the immune response against L. pneumophila.

 

Characteristic morphology of intracellular microcolonies of Legionella oakridgensis OR-10

Takekawa Y, Saito M, Wang C, Qin T, Ogawa M, Kanemaru T, Yoshida S.

Department of Bacteriology, Faculty of Medical Sciences, Kyushu University, Fukuoka, Japan. msaito@bact.med.kyushu-u.ac.jp

Can J Microbiol. 2012 Feb;58(2):179-83.

ABSTRACT: Legionella oakridgensis occasionally causes pneumonia in humans. We report here the characteristic morphology of intracellular microcolonies of L. oakridgensis OR-10 in infected epithelial cells. By light microscopy after Gimenez staining, the bacteria showed serpentine-like chain, disk-like conglomerate, and granular forms when they grew intracellularly in Vero cells, HeLa cells, and A549 cells. In a time-lapse study, we observed the progressive change from a serpentine-like chain form to a conglomerate form in Vero cells. Transmission electron microscopy showed that L. oakridgensis OR-10 proliferated both inside membrane structures and in the cytoplasm. Such highly serpentine chain growth has not been reported in any intracellular bacteria. Furthermore, these results imply that L. oakridgensis OR-10 may be proliferating inside the endoplasmic reticulum.

 

Population variation in NAIP functional copy number confers increased cell death upon Legionella pneumophila infection

Boniotto M, Tailleux L, Lomma M, Gicquel B, Buchrieser C, Garcia S, Quintana-Murci L.

Unit of Human Evolutionary Genetics, Institut Pasteur, F-75015 Paris, France. quintana@pasteur.fr

Hum Immunol. 2012 Feb;73(2):196-200.

ABSTRACT: The NAIP gene encodes an intracellular innate immunity receptor that senses flagellin. The genomic region containing NAIP presents a complex genomic organization and includes various NAIP paralogs. Here, we assessed the degree of copy number variation of the complete NAIP gene (NAIPFull) in various human populations and studied the functional impact of such variation on host cell fate using Legionella pneumophila as an infection model. We determined that African populations have a NAIPFull duplication at a higher frequency than Europeans and Asians, with an increased transcription of the gene. In addition, we demonstrated that a higher amount of the NAIPFull protein dramatically increases cell death upon infection by L. pneumophila, a mechanism that may account for increased host resistance to infection. We postulate that the NAIPFull gene duplication might have been evolutionary maintained, or even selected for, because it may confer an advantage to the host against flagellated bacteria.

nment and exploitation of host cell functions are critical for the success of these intracellular pathogens. The establishment and publication of the complete genome sequences of L. pneumophila and L. longbeachae isolates paved the way for major breakthroughs in understanding the biology of these organisms. In this review we present the knowledge gained from the analyses and comparison of the complete genome sequences of different L. pneumophila and L. longbeachae strains. Emphasis is given on putative virulence and Legionella life cycle related functions, such as the identification of an extended array of eukaryotic like proteins, many of which have been shown to modulate host cell functions to the pathogen's advantage. Surprisingly, many of the eukaryotic domain proteins identified in L. pneumophila as well as many substrates of the Dot/Icm type IV secretion system essential for intracellular replication are different between these two species, although they cause the same disease. Finally, evolutionary aspects regarding the eukaryotic like proteins in Legionella are discussed.

 

The overlapping host responses to bacterial cyclic dinucleotides

Abdul-Sater AA, Grajkowski A, Erdjument-Bromage H, Plumlee C, Levi A, Schreiber MT, Lee C, Shuman H, Beaucage SL, Schindler C.

Department of Microbiology & Immunology, Columbia University, New York, NY 10032, USA. cws4@columbia.edu

Microbes Infect. 2012 Feb;14(2):188-97.

ABSTRACT:Macrophages respond to infection with Legionella pneumophila by the induction of inflammatory mediators, including type I Interferons (IFN-Is). To explore whether the bacterial second messenger cyclic 3'-5' diguanylate (c-diGMP) activates some of these mediators, macrophages were infected with L. pneumophila strains in which the levels of bacterial c-diGMP had been altered. Intriguingly, there was a positive correlation between c-diGMP levels and IFN-I expression. Subsequent studies with synthetic derivatives of c-diGMP, and newly described cyclic 3'-5' diadenylate (c-diAMP), determined that these molecules activate overlapping inflammatory responses in human and murine macrophages. Moreover, UV crosslinking studies determined that both dinucleotides physically associate with a shared set of host proteins. Fractionation of macrophage extracts on a biotin-c-diGMP affinity matrix led to the identification of a set of candidate host binding proteins. These studies suggest that mammalian macrophages can sense and mount a specific inflammatory response to bacterial dinucleotides.

 

Legionella pneumophila secretes a mitochondrial carrier protein during infection

Dolezal P, Aili M, Tong J, Jiang JH, Marobbio CM, Lee SF, Schuelein R, Belluzzo S, Binova E, Mousnier A, Frankel G, Giannuzzi G, Palmieri F, Gabriel K, Naderer T, Hartland EL, Lithgow T.

Department of Biochemistry and Molecular Biology, Monash University, Clayton, Australia. trevor.lithgow@monash.edu

PLoS Pathog. 2012 Jan;8(1):e1002459.

ABSTRACT: The Mitochondrial Carrier Family (MCF) is a signature group of integral membrane proteins that transport metabolites across the mitochondrial inner membrane in eukaryotes. MCF proteins are characterized by six transmembrane segments that assemble to form a highly-selective channel for metabolite transport. We discovered a novel MCF member, termed Legionellanucleotide carrier Protein (LncP), encoded in the genome of Legionella pneumophila, the causative agent of Legionnaire's disease. LncP was secreted via the bacterial Dot/Icm type IV secretion system into macrophages and assembled in the mitochondrial inner membrane. In a yeast cellular system, LncP induced a dominant-negative phenotype that was rescued by deleting an endogenous ATP carrier. Substrate transport studies on purified LncP reconstituted in liposomes revealed that it catalyzes unidirectional transport and exchange of ATP transport across membranes, thereby supporting a role for LncP as an ATP transporter. A hidden Markov model revealed further MCF proteins in the intracellular pathogens, Legionella longbeachae and Neorickettsia sennetsu, thereby challenging the notion that MCF proteins exist exclusively in eukaryotic organisms.

 

The Legionella pneumophila effector DrrA is sufficient to stimulate SNARE-dependent membrane fusion

Arasaki K, Toomre DK, Roy CR.

Section of Microbial Pathogenesis, Yale University School of Medicine, New Haven, CT 06526, USA. craig.roy@yale.edu

Cell Host Microbe. 2012 Jan 19;11(1):46-57.

ABSTRACT: The intracellular bacterial pathogen Legionella pneumophila subverts host membrane transport pathways to promote fusion of vesicles exiting the endoplasmic reticulum (ER) with the pathogen-containing vacuole. During infection there is noncanonical pairing of the SNARE protein Sec22b on ER-derived vesicles with plasma membrane (PM)-localized syntaxin proteins on the vacuole. We show that the L. pneumophila Rab1-targeting effector DrrA is sufficient to stimulate this noncanonical SNARE association and promote membrane fusion. DrrA activation of the Rab1 GTPase on PM-derived organelles stimulated the tethering of ER-derived vesicles with the PM-derived organelle, resulting in vesicle fusion through the pairing of Sec22b with the PM syntaxin proteins. Thus, the effector protein DrrA stimulates a host membrane transport pathway that enables ER-derived vesicles to remodel a PM-derived organelle, suggesting that Rab1 activation at the PM is sufficient to promote the recruitment and fusion of ER-derived vesicles.

 

Legionella secreted effectors and innate immune responses

Luo ZQ.

Department of Biological Sciences, Purdue University, West Lafayette, IN 47907, USA. luoz@purdue.edu

Cell Microbiol. 2012 Jan;14(1):19-27

ABSTRACT:Legionella pneumophila is a facultative intracellular pathogen capable of replicating in a wide spectrum of cells. Successful infection by Legionella requires the Dot/Icm type IV secretion system, which translocates a large number of effector proteins into infected cells. By co-opting numerous host cellular processes, these proteins function to establish a specialized organelle that allows bacterial survival and proliferation. Even within the vacuole, L. pneumophila triggers robust immune responses. Recent studies reveal that a subset of Legionella effectors directly target some basic components of the host innate immunity systems such as phagosome maturation. Others play essential roles in engaging the host innate immune surveillance system. This review will highlight recent progress in our understanding of these interactions and discuss implications for the study of the immune detection mechanisms.

 

Legionella pneumophila regulates the small GTPase Rab1 activity by reversible phosphorylcholination

Tan Y, Arnold RJ, Luo ZQ.

Department of Biological Sciences, Purdue University, West Lafayette, IN 47907, USA. Luoz@purdue.edu

Proc Natl Acad Sci USA. 2011 Dec 27;108(52):21212-7.

ABSTRACT: Effectors delivered into host cells by the Legionella pneumophila Dot/Icm type IV transporter are essential for the biogenesis of the specialized vacuole that permits its intracellular growth. The biochemical function of most of these effectors is unknown, making it difficult to assign their roles in the establishment of successful infection. We found that several yeast genes involved in membrane trafficking, including the small GTPase Ypt1, strongly suppress the cytotoxicity of Lpg0695(AnkX), a protein known to interfere severely with host vesicle trafficking when overexpressed. Mass spectrometry analysis of Rab1 purified from a yeast strain inducibly expressing AnkX revealed that this small GTPase is modified posttranslationally at Ser(76) by a phosphorylcholine moiety. Using cytidine diphosphate-choline as the donor for phosphorylcholine, AnkX catalyzes the transfer of phosphorylcholine to Rab1 in a filamentation-induced by cAMP(Fic) domain-dependent manner. Further, we found that the activity of AnkX is regulated by the Dot/Icm substrate Lpg0696(Lem3), which functions as a dephosphorylcholinase to reverse AnkX-mediated modification on Rab1. Phosphorylcholination interfered with Rab1 activity by making it less accessible to the bacterial GTPase activation protein LepB; this interference can be alleviated fully by Lem3. Our results reveal reversible phosphorylcholination as a mechanism for balanced modulation of host cellular processes by a bacterial pathogen.

 

The N-acylneuraminate cytidyltransferase gene, neuA, is heterogenous in Legionella pneumophila strains but can be used as a marker for epidemiological typing in the consensus sequence-based typing scheme

Farhat C, Mentasti M, Jacobs E, Fry NK, Lück C.

Institut für Medizinische Mikrobiologie und Hygiene, TU Dresden, Fiedlerstrasse 42, D-01307 Dresden, Germany. Christian.Lueck@tu-dresden.de

J Clin Microbiol. 2011 Dec;49(12):4052-8.

ABSTRACT:Sequence-based typing (SBT) is the internationally recognized standard method for genotyping Legionella pneumophila. To date all strains of serogroup 1 (SG1) and some of SGs 2 to 14 yield a seven-allele profile and can be assigned a sequence type (ST). However, for some strains belonging to SGs 2 to 14, the targeted region of the neuA gene could not be amplified using the published standard primers. We determined the DNA sequence of a neuA gene homolog located in the lipopolysaccharide synthesis locus of strain Dallas-1E. By using newly designed degenerate consensus primers based on the neuA homolog in strains Dallas-1E, Philadelphia-1, Paris, Lens, and Corby, we were able to obtain DNA sequences for all 48 non-SG1 strains which were untypeable by the standard method. Our data show that the neuA gene is present in all L. pneumophila strains but differs significantly in some non-SG1 strains at both the DNA and amino acid levels. The new primers can be used to amplify and sequence the neuA gene in all strains and can substitute for the standard primers. This offers the possibility of assigning an ST to all strains of L. pneumophila.

 

Manipulation of host vesicular trafficking and innate immune defence by Legionella Dot/Icm effectors

Ge J, Shao F.

National Institute of Biological Sciences, Zhongguancun Life Science Park, Beijing 102206, China. shaofeng@nibs.ac.cn

Cell Microbiol. 2011 Dec;13(12):1870-80.

ABSTRACT:Legionella pneumophila, the causative agent of Legionnaires' disease, infects and replicates in macrophages and amoebas. Following internalization, L. pneumophila resides in a vacuole structure called Legionella-containing vacuole (LCV). The LCV escapes from the endocytic maturation process and avoids fusion with the lysosome, a hallmark of Legionella pathogenesis. Interference with the secretory vesicle transport and avoiding lysosomal targeting render the LCV permissive for L. pneumophila intracellular replication. Central to L. pneumophila pathogenesis is a defect in the organelle trafficking/intracellular multiplication (Dot/Icm) type IV secretion system that translocates a large number of effector proteins into host cells. Many of the Dot/Icm effectors employ diverse and sophisticated biochemical strategies to manipulate the host vesicular transport system, playing an important role in LCV biogenesis and trafficking. Similar to other bacterial pathogens, L. pneumophila also delivers effector proteins to modulate or counteract host innate immune defence pathways such as the NF-κB and apoptotic signalling. This review summarizes the known functions and mechanisms of Dot/Icm effectors that target host membrane trafficking and innate immune defence pathways.

 

Activation of NLRC4 by flagellated bacteria triggers caspase-1-dependent and -independent responses to restrict Legionella pneumophila replication in macrophages and in vivo

Pereira MS, Morgantetti GF, Massis LM, Horta CV, Hori JI, Zamboni DS.

Department of Cell Biology, School of Medicine of Ribeirão Preto, University of São Paulo, Ribeirão Preto, São Paulo 14049-900, Brazil. dszamboni@fmrp.usp.br

J Immunol. 2011 Dec 15;187(12):6447-55.

ABSTRACT: Although NLRC4/IPAF activation by flagellin has been extensively investigated, the downstream signaling pathways and the mechanisms responsible for infection clearance remain unclear. In this study, we used mice deficient for the inflammasome components in addition to wild-type (WT) Legionella pneumophila or bacteria deficient for flagellin (flaA) or motility (fliI) to assess the pathways responsible for NLRC4-dependent growth restriction in vivo and ex vivo. By comparing infections with WT L. pneumophila, fliI, and flaA, we found that flagellin and motility are important for the colonization of the protozoan host Acanthamoeba castellanii. However, in macrophages and mammalian lungs, flagellin expression abrogated bacterial replication. The flagellin-mediated growth restriction was dependent on NLRC4, and although it was recently demonstrated that NLRC4 is able to recognize bacteria independent of flagellin, we found that the NLRC4-dependent restriction of L. pneumophila multiplication was fully dependent on flagellin. By examining infected caspase-1(-/-) mice and macrophages with flaA, fliI, and WT L. pneumophila, we could detect greater replication of flaA, which suggests that caspase-1 only partially accounted for flagellin-dependent growth restriction. Conversely, WT L. pneumophila multiplied better in macrophages and mice deficient for NLRC4 compared with that in macrophages and mice deficient for caspase-1, supporting the existence of a novel caspase-1-independent response downstream of NLRC4. This response operated early after macrophage infection and accounted for the restriction of bacterial replication within bacteria-containing vacuoles. Collectively, our data indicate that flagellin is required for NLRC4-dependent responses to L. pneumophila and that NLRC4 triggers caspase-1-dependent and -independent responses for bacterial growth restriction in macrophages and in vivo.

 

Passage through Tetrahymena tropicalis enhances the resistance to stress and the infectivity of Legionella pneumophila

Koubar M, Rodier MH, Garduño RA, Frère J.

Laboratoire de Chimie et Microbiologie de l'Eau, UMR CNRS 6008, Poitiers University, Poitiers, France. jacques.frere@univ-poitiers.fr

FEMS Microbiol Lett. 2011 Dec;325(1):10-5.

ABSTRACT: Legionella pneumophila is a gram-negative bacterium prevalent in fresh water which accidentally infects humans and is responsible for the disease called legionellosis. Intracellular growth of L. pneumophila in Tetrahymena is inconsistent; in the species Tetrahymena tropicalis stationary-phase forms (SPFs) of L. pneumophila differentiate into mature intracellular forms (MIFs) without apparent bacterial replication and are expelled from the ciliate as pellets containing numerous MIFS. In the present work, we tested the impact of L. pneumophila passage through T. tropicalis. We observed that MIFs released from T. tropicalis are more resistant to various stresses than SPFs. Under our conditions, MIFs harboured a higher gentamicin resistance, maintained even after 3 months as pellets. Long-term survival essays revealed that MIFs survived better in a nutrient-poor environment than SFPs, as a reduction of only about 3 logs was observed after 4 months in the MIF population, whereas no cultivable SPFs were detected after 3 months in the same medium, corresponding to a loss of about 7 logs. We have also observed that MIFs are significantly more infectious in human pneumocyte cells compared with SPFs. These results strongly suggest a potential role of ciliates in increasing the risk of legionellosis.

 

Characterisation of Legionella pneumophila phospholipases and their impact on host cells

Lang C, Flieger A.

Division of Bacterial Infections, Robert Koch Institute, Burgstr. 37, 38855 Wernigerode, Germany.

Eur J Cell Biol. 2011 Nov;90(11):903-12. fliegera@rki.de

ABSTRACT: Phospholipases are a diverse class of enzymes produced both by eukaryotic hosts and their pathogens. Major insights into action pathways of bacterial phospholipases have been provided during the last years. On the one hand bacterial phospholipases act as potent membrane destructors and on the other hand they manipulate and initiate host signalling paths, such as chemokine expression or the inflammatory cascade. Reaction products of bacterial phospholipases may potentially influence many more host cell processes, such as cell respreading, lamellopodia formation, cell migration and membrane traffic. Phospholipases play a dominant role in the biology of the lung pathogen Legionella pneumophila. So far, 15 different phospholipase A-encoding genes have been identified in the L. pneumophila genome. These phospholipases can be divided into three major groups, the GDSL, the patatin-like and the PlaB-like enzymes. The first two lipase families are also found in higher plants (such as flowering plants) and the second family shows similarities to eukaryotic cytosolic phospholipases A. Therefore, when those enzymes are injected or secreted by the bacterium into the host cell they may mimic eukaryotic phospholipases. The current knowledge on L. pneumophila phospholipases is summarised here with emphasis on their activity, mode of secretion, localisation, expression and importance for host cell infections.

 

Protein LidA from Legionella is a Rab GTPase supereffector

Schoebel S, Cichy AL, Goody RS, Itzen A.

Department of Physical Biochemistry, Max-Planck-Institute of Molecular Physiology, Otto-Hahn-Strasse 11, Dortmund 44227, Germany. roger.goody@mpi-dortmund.mpg.de

 

Proc Natl Acad Sci U S A. 2011 Nov 1;108(44):17945-50.

ABSTRACT: The causative agent of Legionnaires disease, Legionella pneumophila, injects several hundred proteins into the cell it infects, many of which interfere with or exploit vesicular transport processes. One of these proteins, LidA, has been described as a Rab effector (i.e., a molecule that interacts preferentially with the GTP-bound form of Rab). We describe here the structure and biochemistry of a complex between the Rab-binding domain of LidA and active Rab8a. LidA displays structural peculiarities in binding to Rab8a, forming a considerably extended interface in comparison to known mammalian Rab effectors, and involving regions of the GTPase that are not seen in other Rab:effector complexes. In keeping with this extended binding interface, which involves four α-helices and two pillar-like structures of LidA, the stability of LidA-Rab interactions is dramatically greater than for other such complexes. For Rab1b and Rab8a, these affinities are extraordinarily high, but for the more weakly bound Rab6a, K(d) values of 4 nM for the inactive and 30 pM for the active form were found. Rab1b and Rab8a appear to bind LidA with K(d) values in the low picomolar range, making LidA a Rab supereffector.

 

Extensive recombination events and horizontal gene transfer shaped the Legionella pneumophila genomes

Gomez-Valero L, Rusniok C, Jarraud S, Vacherie B, Rouy Z, Barbe V, Medigue C, Etienne J, Buchrieser C.

Institut Pasteur, Biologie des Bactéries Intracellulaires, 75724, Paris, France. lgomez@pasteur.fr

BMC Genomics. 2011 Nov 1;12:536.

ABSTRACT: Background: Legionella pneumophila is an intracellular pathogen of environmental protozoa. When humans inhale contaminated aerosols this bacterium may cause a severe pneumonia called Legionnaires' disease. Despite the abundance of dozens of Legionella species in aquatic reservoirs, the vast majority of human disease is caused by a single serogroup (Sg) of a single species, namely L. pneumophila Sg1. To get further insights into genome dynamics and evolution of Sg1 strains, we sequenced strains Lorraine and HL 0604 1035 (Sg1) and compared them to the available sequences of Sg1 strains Paris, Lens, Corby and Philadelphia, resulting in a comprehensive multigenome analysis. Results: We show that L. pneumophila Sg1 has a highly conserved and syntenic core genome that comprises the many eukaryotic like proteins and a conserved repertoire of over 200 Dot/Icm type IV secreted substrates. However, recombination events and horizontal gene transfer are frequent. In particular the analyses of the distribution of nucleotide polymorphisms suggests that large chromosomal fragments of over 200 kbs are exchanged between L. pneumophila strains and contribute to the genome dynamics in the natural population. The many secretion systems present might be implicated in exchange of these fragments by conjugal transfer. Plasmids also play a role in genome diversification and are exchanged among strains and circulate between different Legionella species. Conclusions: Horizontal gene transfer among bacteria and from eukaryotes to L. pneumophila as well as recombination between strains allows different clones to evolve into predominant disease clones and others to replace them subsequently within relatively short periods of time.

 

Kinetic, mutagenic, and structural homology analysis of L-serine dehydratase from Legionella pneumophila

Xu XL, Chen S, Grant GA.

Department of Developmental Biology, Washington University School of Medicine, 660 S. Euclid Avenue, St. Louis, MO 63110, USA. ggrant@wustl.edu

Arch Biochem Biophys. 2011 Nov;515(1-2):28-36.

ABSTRACT:A structural database search has revealed that the same fold found in the allosteric substrate binding (ASB) domain of Mycobacterium tuberculosis D-3-phosphoglycerate dehydrogenase (PGDH) is found in l-serine dehydratase from Legionella pneumophila. The M. tuberculosis PGDH ASB domain functions in the control of catalytic activity. Bacterial l-serine dehydratases are 4Fe-4S proteins that convert l-serine to pyruvate and ammonia. Sequence homology reveals two types depending on whether their α and β domains are on the same (Type 2) or separate (Type 1) polypeptides. The α domains contain the catalytic iron-sulfur center while the β domains do not yet have a described function, but the structural homology with PGDH suggests a regulatory role. Type 1 β domains also contain additional sequence homologous to PGDH ACT domains. A continuous assay for l-serine dehydratase is used to demonstrate homotropic cooperativity, a broad pH range, and essential irreversibility. Product inhibition analysis reveals a Uni-Bi ordered mechanism with ammonia dissociating before pyruvate. l-Threonine is a poor substrate and l-cysteine and d-serine are competitive inhibitors with K(i) values that differ by almost 10-fold from those reported for Escherichia colil-serine dehydratase. Mutagenesis identifies the three cysteine residues at the active site that anchor the iron-sulfur complex.

 

De-AMPylation unmasked: modulation of host membrane trafficking

Ham H, Orth K.

Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA. kim.orth@utsouthwestern.edu

Sci Signal. 2011 Oct 11;4(194):pe42.

ABSTRACT: AMPylation, a posttranslational modification in which adenosine monophosphate (AMP) is added to hydroxyl side chains of protein substrates, is employed by many bacterial pathogens to subvert host signaling pathways during infection. The Legionella pneumophila effector protein SidM is a multifunctional enzyme that targets the guanosine triphosphatase (GTPase) Rab1 to manipulate intracellular vesicular trafficking in the host cell. SidM recruits Rab1 to the membranes of Legionella-containing vacuoles and activates Rab1 through its guanine nucleotide exchange factor activity. SidM then AMPylates Rab1, converting it into a constitutively active form that cannot be accessed by LepB, a GTPase-activating protein that is secreted by L. pneumophila. However, the molecular event that eventually leads to Rab1 inactivation and subsequent removal from Legionella-containing vacuoles has remained unknown. New evidence has identified SidD as a de-AMPylase that removes AMP from Rab1, which enables its inactivation by LepB later during the infection process. This finding demonstrates a complete pathway of reversible modifications regulated by specific bacterial enzymes to modulate host membrane trafficking.

 

Collagen IV-derived peptide binds hydrophobic cavity of Legionella pneumophila Mip and interferes with bacterial epithelial transmigration

Ünal C, Schwedhelm KF, Thiele A, Weiwad M, Schweimer K, Frese F, Fischer G, Hacker J, Faber C, Steinert M.

Institut für Mikrobiologie, Technische Universität Braunschweig, 38106 Braunschweig, Germany.

Cell Microbiol. 2011 Oct;13(10):1558-72. m.steinert@tu-bs.de

ABSTRACT:The Legionella virulence factor Mip (macrophage infectivity potentiator) contributes to bacterial dissemination within infected lung tissue. The Mip protein, which belongs to the enzyme family of FK506-binding proteins (FKBP), binds specifically to collagen IV. We identified a surface-exposed Mip-binding sequence in the NC1 domain of human collagen IV α1. The corresponding collagen IV-derived peptide (P290) co-precipitated with Mip and competitively inhibited the Mip-collagen IV binding. Transmigration of Legionella pneumophila across a barrier of NCI-H292 lung epithelial cells and extracellular matrix was efficiently inhibited by P290. This significantly reduced transmigration was comparable to the inefficient transmigration of PPIase-negative Mip mutant or rapamycin-treated L. pneumophila. Based on NMR data and docking studies a model for the mode of interaction of P290 and Mip was developed. The amino acids of the hydrophobic cavity of Mip, D142 and to a lesser extent Y185 were identified to be part of the interaction surface. In the complex structure of Mip(77-213) and P290, both amino acid residues form hydrogen bonds to P290. Utilizing modelling, molecular dynamics (MD) simulations and structural data of human PPIase FKBP12, the most related human orthologue of Mip, we were able to propose optimized P290 variants with increased binding specificity and selectivity for the putative bacterial drug target Mip.

 

Minimization of the Legionella pneumophila genome reveals chromosomal regions involved in host range expansion

O'Connor TJ, Adepoju Y, Boyd D, Isberg RR.

Howard Hughes Medical Institute and Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, MA 02111. ralph.isberg@tufts.edu

Proc Natl Acad Sci U S A. 2011 Sep 6;108(36):14733-40.

ABSTRACT: Legionella pneumophila is a bacterial pathogen of amoebae and humans. Intracellular growth requires a type IVB secretion system that translocates at least 200 different proteins into host cells. To distinguish between proteins necessary for growth in culture and those specifically required for intracellular replication, a screen was performed to identify genes necessary for optimal growth in nutrient-rich medium. Mapping of these genes revealed that the L. pneumophila chromosome has a modular architecture consisting of several large genomic islands that are dispensable for growth in bacteriological culture. Strains lacking six of these regions, and thus 18.5% of the genome, were viable but required secondary point mutations for optimal growth. The simultaneous deletion of five of these genomic loci had no adverse effect on growth of the bacterium in nutrient-rich media. Remarkably, this minimal genome strain, which lacked 31% of the known substrates of the type IVB system, caused only marginal defects in intracellular growth within mouse macrophages. In contrast, deletion of single regions reduced growth within amoebae. The importance of individual islands, however, differed among amoebal species. The host-specific requirements of these genomic islands support a model in which the acquisition of foreign DNA has broadened the L. pneumophila host range.

  

Two signal models in innate immunity

Fontana MF, Vance RE.

Division of Immunology & Pathogenesis, Department of Molecular & Cell Biology, University of California, Berkeley, CA, USA. rvance@berkeley.edu

Immunol Rev. 2011 Sep;243(1):26-39.

ABSTRACT: Summary: Two-signal models have a rich history in immunology. In the classic two-signal model of T-cell activation, signal one consists of engagement of the T-cell receptor by antigen/major histocompatibility complex, whereas signal two arises from costimulatory ligands on antigen-presenting cells. A requirement for two independent signals helps to ensure that T-cell responses are initiated only in response to bona fide infectious threats. Our studies have led us to conclude that initiation of innate immune responses to pathogens also often requires two signals: signal one is initiated by a microbe-derived ligand, such as lipopolysaccharide (LPS) or flagellin, whereas signal two conveys additional contextual information that often accompanies infectious microbes. Although signal one alone is sufficient to initiate many innate responses, certain responses-particularly ones with the potential for self-damage-require two signals for activation. Many of our studies have employed the intracellular bacterial pathogen Legionella pneumophila, which has been established as a valuable model for understanding innate immune responses. In this review, we discuss how the innate immune system integrates multiple signals to generate an effective response to L. pneumophila and other bacterial pathogens.

  

Identification and modelling of a PPM protein phosphatase fold in the Legionella pneumophila deAMPylase SidD

Rigden DJ.

University of Liverpool, Institute of Integrative Biology, Liverpool, UK. drigden@liv.ac.uk

FEBS Lett. 2011 Sep 2;585(17):2749-54.

ABSTRACT: The intracellular parasitic bacterium Legionella pneumophila subverts host vesicle transport through reversible AMPylation of Rab1. The effector enzyme for deAMPylation is SidD. Here a complete PPM protein phosphatase fold catalytic domain in SidD is identified and modelled. The SidD model reveals insertions and deletions near the metal ion containing catalytic site which presumably determine its novel activity. It also sheds light on possible substrate binding residues and highlights the lack of an obvious group to act as general acid during reaction. Assignment of a PPM fold to SidD offers an important pointer towards identification of further deAMPylases.

 

The atypical two-component sensor kinase Lpl0330 from Legionella pneumophila controls the bifunctional diguanylate cyclase-phosphodiesterase Lpl0329 to modulate bis-(3'-5')-cyclic dimeric GMP synthesis

Levet-Paulo M, Lazzaroni JC, Gilbert C, Atlan D, Doublet P, Vianney A.

Université de Lyon, Université Lyon 1, CNRS UMR5240 Microbiologie, Adaptation et Pathogénie, 69622 Villeurbanne, France. anne.vianney@univ-lyon1.fr

J Biol Chem. 2011 Sep 9;286(36):31136-44.

ABSTRACT:A significant part of bacterial two-component system response regulators contains effector domains predicted to be involved in metabolism of bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP), a second messenger that plays a key role in many physiological processes. The intracellular level of c-di-GMP is controlled by diguanylate cyclase and phosphodiesterases activities associated with GGDEF and EAL domains, respectively. The Legionella pneumophila Lens genome displays 22 GGDEF/EAL domain-encoding genes. One of them, lpl0329, encodes a protein containing a two-component system receiver domain and both GGDEF and EAL domains. Here, we demonstrated that the GGDEF and EAL domains of Lpl0329 are both functional and lead to simultaneous synthesis and hydrolysis of c-di-GMP. Moreover, these two opposite activities are finely regulated by Lpl0329 phosphorylation due to the atypical histidine kinase Lpl0330. Indeed, Lpl0330 was found to autophosphorylate on a histidine residue in an atypical H box, which is conserved in various bacteria species and thus defines a new histidine kinase subfamily. Lpl0330 also catalyzes the phosphotransferase to Lpl0329, which results in a diguanylate cyclase activity decrease whereas phosphodiesterase activity remains efficient. Altogether, these data present (i) a new histidine kinase subfamily based on the conservation of an original H box that we named HGN H box, and (ii) the first example of a bifunctional enzyme that modulates synthesis and turnover of c-di-GMP in response to phosphorylation of its receiver domain.

 

Cell mediated immunity in Legionnaires' disease

Hoffman PS, Edelstein PH.

Departments of Medicine and Microbiology, Division of Infectious Diseases and International Health, University of Virginia Health System, Charlottesville, United States. psh2n@virginia.edu

Vaccine. 2011 Sep 2;29(38):6437-8.

Letter to the editor.

 

Asc modulates the function of NLRC4 in response to infection of macrophages by Legionella pneumophila

Case CL, Roy CR.

Section of Microbial Pathogenesis, Yale University School of Medicine, New Haven, Connecticut, USA. craig.roy@yale.edu

MBio. 2011 Sep 1;2(4). pii: e00117-11.

ABSTRACT: Nucleotide-binding domain, leucine-rich repeat containing proteins (NLRs) activate caspase-1 in response to a variety of bacterium-derived signals in macrophages. NLR-mediated activation of caspase-1 by Legionella pneumophila occurs through both an NLRC4/NAIP5-dependent pathway and a pathway requiring the adapter protein Asc. Both pathways are needed for maximal activation of caspase-1 and for the release of the cytokines interleukin-1β (IL-1β) and IL-18. Asc is not required for caspase-1-dependent pore formation and cell death induced upon infection of macrophages by L. pneumophila. Here, temporal and spatial localization of caspase-1-dependent processes was examined to better define the roles of Asc and NLRC4 during infection. Imaging studies revealed that caspase-1 localized to a single punctate structure in infected cells containing Asc but not in cells lacking this adapter. Both endogenous Asc and ectopically produced NLRC4 tagged with green fluorescent protein (GFP) were found to localize to caspase-1 puncta following L. pneumophila infection, suggesting that NLRC4 and Asc coordinate signaling through this complex during caspase-1 activation. Formation of caspase-1-containing puncta correlated with caspase-1 processing, suggesting a role for the Asc/NLRC4/caspase-1 complex in caspase-1 cleavage. In cells deficient for Asc, NLRC4 did not assemble into discrete puncta, and pyroptosis occurred at an accelerated rate. These data indicate that Asc mediates integration of NLR components into caspase-1 processing platforms and that recruitment of NLR components into an Asc complex can dampen pyroptotic responses. Thus, a negative feedback role of complexes containing Asc may be important for regulating caspase-1-mediated responses during microbial infection. IMPORTANCE: Caspase-1 is a protease activated during infection that is central to the regulation of several innate immune pathways. Studies examining the macromolecular complexes containing this protein, known as inflammasomes, have provided insight into the regulation of this protease. This work demonstrates that the intracellular bacterium Legionella pneumophila induces formation of complexes containing caspase-1 by multiple mechanisms and illustrates that an adapter molecule called Asc integrates signals from multiple independent upstream caspase-1 activators in order to assemble a spatially distinct complex in the macrophage. There were caspase-1-associated activities such as cytokine processing and secretion that were controlled by Asc. Importantly, this work uncovered a new role for Asc in dampening a caspase-1-dependent cell death pathway called pyroptosis. These findings suggest that Asc plays a central role in controlling a distinct subset of caspase-1-dependent activities by both assembling complexes that are important for cytokine processing and suppressing processes that mediate pyroptosis.

 

Legionella pneumophila requires polyamines for optimal intracellular growth

Nasrallah GK, Riveroll AL, Chong A, Murray LE, Lewis PJ, Garduño RA.

Department of Microbiology and Immunology, Dalhousie University, Halifax, Nova Scotia, Canada. Rafael.Garduno@dal.ca

J Bacteriol. 2011 Sep;193(17):4346-60.

ABSTRACT: The Gram-negative intracellular pathogen Legionella pneumophila replicates in a membrane-bound compartment known as the Legionella-containing vacuole (LCV), into which it abundantly releases its chaperonin, HtpB. To determine whether HtpB remains within the LCV or reaches the host cell cytoplasm, we infected U937 human macrophages and CHO cells with L. pneumophila expressing a translocation reporter consisting of the Bordetella pertussis adenylate cyclase fused to HtpB. These infections led to increased cyclic AMP levels, suggesting that HtpB reaches the host cell cytoplasm. To identify potential functions of cytoplasmic HtpB, we expressed it in the yeast Saccharomyces cerevisiae, where HtpB induced pseudohyphal growth. A yeast-two-hybrid screen showed that HtpB interacted with S-adenosylmethionine decarboxylase (SAMDC), an essential yeast enzyme (encoded by SPE2) that is required for polyamine biosynthesis. Increasing the copy number of SPE2 induced pseudohyphal growth in S. cerevisiae; thus, we speculated that (i) HtpB induces pseudohyphal growth by activating polyamine synthesis and (ii) L. pneumophila may require exogenous polyamines for growth. A pharmacological inhibitor of SAMDC significantly reduced L. pneumophila replication in L929 mouse cells and U937 macrophages, whereas exogenously added polyamines moderately favored intracellular growth, confirming that polyamines and host SAMDC activity promote L. pneumophila proliferation. Bioinformatic analysis revealed that most known enzymes required for polyamine biosynthesis in bacteria (including SAMDC) are absent in L. pneumophila, further suggesting a need for exogenous polyamines. We hypothesize that HtpB may function to ensure a supply of polyamines in host cells, which are required for the optimal intracellular growth of L. pneumophila.

 

Covalent coercion by Legionella pneumophila

Itzen A, Goody RS.

Department of Physical Biochemistry, Max-Planck-Institute of Molecular Physiology, Otto-Hahn-Strasse, Dortmund, Germany. aymelt.itzen@mpi-dortmund.mpg.de

Cell Host Microbe. 2011 Aug 18;10(2):89-91.

ABSTRACT: Adenylylation of Rab proteins appears to be an intriguing mechanism that Legionella pneumophila uses to modulate their activity during infection. Now the reverse reaction (deadenylylation) (Neunuebel et al., 2011; Tan and Luo, 2011) and a new posttranslational modification (phosphocholination) of Rab1 (Mukherjee et al., 2011) have been reported.

 

Modulation of Rab GTPase function by a protein phosphocholine transferase

Mukherjee S, Liu X, Arasaki K, McDonough J, Galán JE, Roy CR.

Section of Microbial Pathogenesis, Yale University School of Medicine, Boyer Center for Molecular Medicine, Yale University, New Haven, Connecticut, CT 06536, USA. craig.roy@yale.edu

Nature. 2011 Aug 7;477(7362):103-6..

ABSTRACT: The intracellular pathogen Legionella pneumophila modulates the activity of host GTPases to direct the transport and assembly of the membrane-bound compartment in which it resides. In vitro studies have indicated that the Legionella protein DrrA post-translationally modifies the GTPase Rab1 by a process called AMPylation. Here we used mass spectrometry to investigate post-translational modifications to Rab1 that occur during infection of host cells by Legionella. Consistent with in vitro studies, DrrA-mediated AMPylation of a conserved tyrosine residue in the switch II region of Rab1 was detected during infection. In addition, a modification to an adjacent serine residue in Rab1 was discovered, which was independent of DrrA. The Legionella effector protein AnkX was required for this modification. Biochemical studies determined that AnkX directly mediates the covalent attachment of a phosphocholine moiety to Rab1. This phosphocholine transferase activity used CDP-choline as a substrate and required a conserved histidine residue located in the FIC domain of the AnkX protein. During infection, AnkX modified both Rab1 and Rab35, which explains how this protein modulates membrane transport through both the endocytic and exocytic pathways of the host cell. Thus, phosphocholination of Rab GTPases represents a mechanism by which bacterial FIC-domain-containing proteins can alter host-cell functions.

 

Catch and release: Rab1 exploitation by Legionella pneumophila

Machner MP, Chen Y.

Cell Biology and Metabolism Program; Eunice Kennedy Shriver National Institute of Child Health and Human Development; National Institutes of Health; Bethesda, MD USA. machnerm@mail.nih.gov

Cell Logist. 2011 Jul;1(4):133-138.

ABSTRACT: The intracellular pathogen Legionella pneumophila exploits host cell vesicular transport by manipulating the activity of the small GTPase Rab1. Bacterial proteins, so called effectors, that are delivered into the infected cell play a key role in this process. Here, we summarize recent developments in our quest to understand the molecular function of these effectors, and describe how L. pneumophila employs post-translational modification in a reversible manner to manipulate the activity of Rab1 on its vacuole.

 

De-AMPylation of the small GTPase Rab1 by the pathogen Legionella pneumophila

Neunuebel MR, Chen Y, Gaspar AH, Backlund PS Jr, Yergey A, Machner MP.

Cell Biology and Metabolism Program, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA. machnerm@mail.nih.gov

Science. 2011 Jul 22;333(6041):453-6.

ABSTRACT: The bacterial pathogen Legionella pneumophila exploits host cell vesicle transport by transiently manipulating the activity of the small guanosine triphosphatase (GTPase) Rab1. The effector protein SidM recruits Rab1 to the Legionella-containing vacuole (LCV), where it activates Rab1 and then AMPylates it by covalently adding adenosine monophosphate (AMP). L. pneumophila GTPase-activating protein LepB inactivates Rab1 before its removal from LCVs. Because LepB cannot bind AMPylated Rab1, the molecular events leading to Rab1 inactivation are unknown. We found that the effector protein SidD from L. pneumophila catalyzed AMP release from Rab1, generating de-AMPylated Rab1 accessible for inactivation by LepB. L. pneumophila mutants lacking SidD were defective for Rab1 removal from LCVs, identifying SidD as the missing link connecting the processes of early Rab1 accumulation and subsequent Rab1 removal during infection.

 

Legionella pneumophila SidD is a deAMPylase that modifies Rab1

Tan Y, Luo ZQ.

Department of Biological Sciences, Purdue University, 915 West State Street, West Lafayette, Indiana 47907, USA.

Nature. 2011 Jul 6;475(7357):506-9.

ABSTRACT: Legionella pneumophila actively modulates host vesicle trafficking pathways to facilitate its intracellular replication with effectors translocated by the Dot/Icm type IV secretion system (T4SS). The SidM/DrrA protein functions by locking the small GTPase Rab1 into an active form by its guanine nucleotide exchange factor (GEF) and AMPylation activity. Here we demonstrate that the L. pneumophila protein SidD preferably deAMPylates Rab1. We found that the deAMPylation activity of SidD could suppress the toxicity of SidM to yeast and is required to release Rab1 from bacterial phagosomes efficiently. A molecular mechanism for the temporal control of Rab1 activity in different phases of L. pneumophila infection is thus established. These observations indicate that AMPylation-mediated signal transduction is a reversible process regulated by specific enzymes.

 

The Legionella HtrA homologue DegQ is a self-compartmentizing protease that forms large 12-meric assemblies

Wrase R, Scott H, Hilgenfeld R, Hansen G.

Institute of Biochemistry, Center for Structural and Cell Biology in Medicine, University of Lübeck, Ratzeburger Allee 160, 23538 Lübeck, Germany. hilgenfeld@biochem.uni-luebeck.de

Proc Natl Acad Sci USA. 2011 Jun 28;108(26):10490-5.

ABSTRACT: Proteases of the HtrA family are key factors dealing with folding stress in the periplasmatic compartment of prokaryotes. In Escherichia coli, the well-characterized HtrA family members DegS and DegP counteract the accumulation of unfolded outer-membrane proteins under stress conditions. Whereas DegS serves as a folding-stress sensor, DegP is a chaperone-protease facilitating refolding or degradation of defective outer-membrane proteins. Here, we report the 2.15 A-resolution crystal structure of the second major chaperone-protease of the periplasm, DegQ from Legionella fallonii. DegQ assembles into large, cage-like 12-mers that form independently of unfolded substrate proteins. We provide evidence that 12-mer formation is essential for the degradation of substrate proteins but not for the chaperone activity of DegQ. In the current model for the regulation of periplasmatic chaperone-proteases, 6-meric assemblies represent important protease-resting states. However, DegQ is unable to form such 6-mers, suggesting divergent regulatory mechanisms for DegQ and DegP. To understand how the protease activity of DegQ is controlled, we probed its functional properties employing designed protein variants. Combining crystallographic, biochemical, and mutagenic data, we present a mechanistic model that suggests how protease activity of DegQ 12-mers is intrinsically regulated and how deleterious proteolysis by free DegQ 3-mers is prevented. Our study sheds light on a previously uncharacterized component of the prokaryotic stress-response system with implications for other members of the HtrA family.

 

Crystal structure of a copper-transporting PIB-type ATPase

Gourdon P, Liu XY, Skjørringe T, Morth JP, Møller LB, Pedersen BP, Nissen P.

Centre for Membrane Pumps in Cells and Disease-PUMPKIN, Danish National Research Foundation, Aarhus University, Department of Molecular Biology, Gustav Wieds Vej 10C, DK-8000 Aarhus C, Denmark.

Nature. 2011 Jun 29;475(7354):59-64.

ABSTRACT:Heavy-metal homeostasis and detoxification is crucial for cell viability. P-type ATPases of the class IB (PIB) are essential in these processes, actively extruding heavy metals from the cytoplasm of cells. Here we present the structure of a PIB-ATPase, a Legionella pneumophila CopA Cu(+)-ATPase, in a copper-free form, as determined by X-ray crystallography at 3.2 Å resolution. The structure indicates a three-stage copper transport pathway involving several conserved residues. A PIB-specific transmembrane helix kinks at a double-glycine motif displaying an amphipathic helix that lines a putative copper entry point at the intracellular interface. Comparisons to Ca(2+)-ATPase suggest an ATPase-coupled copper release mechanism from the binding sites in the membrane via an extracellular exit site. The structure also provides a framework to analyse missense mutations in the human ATP7A and ATP7B proteins associated with Menkes' and Wilson's diseases.

 

Lcl of Legionella pneumophila is an immunogenic GAG binding adhesin that promotes interactions with lung epithelial cells and plays a crucial role in biofilm formation

Duncan C, Prashar A, So J, Tang P, Low DE, Terebiznik M, Guyard C.

Ontario Agency for Health Protection and Promotion, 81 Resources Road, Toronto, ON M9P 3T1, Canada.

Infect Immun. 2011 Jun;79(6):2168-81.

ABSTRACT: Legionellosis is mostly caused by Legionella pneumophila and is defined by a severe respiratory illness with a case fatality rate ranging from 5 to 80%. In vitro and in vivo, interactions of L. pneumophila with lung epithelial cells are mediated by the sulfated glycosaminoglycans (GAGs) of the host extracellular matrix. In this study, we have identified several Legionella heparin binding proteins. We have shown that one of these proteins, designated Lcl, is a polymorphic adhesin of L. pneumophila that is produced during legionellosis. Homologues of Lcl are ubiquitous in L. pneumophila serogroups but are undetected in other Legionella species. Recombinant Lcl binds to GAGs, and a Δlpg2644 mutant demonstrated reduced binding to GAGs and human lung epithelial cells. Importantly, we showed that the Δlpg2644 strain is dramatically impaired in biofilm formation. These data delineate the role of Lcl in the GAG binding properties of L. pneumophila and provide molecular evidence regarding its role in L. pneumophila adherence and biofilm formation.

 

A New Twist in the Regulation of Legionella Replication through ASC and Caspase-1

Franchi L, Núñez G.

Department of Pathology and Comprehensive Cancer Center, University of Michigan Medical School Ann Arbor, MI, USA. bclx@umich.edu

Front Microbiol. 2011;2:57

NO ABSTRACT

 

Effector glycosyltransferases in legionella

Belyi Y, Jank T, Aktories K.

Gamaleya Research Institute Moscow, Russia. klaus.aktories@pharmakol.uni-freiburg.de

Front Microbiol. 2011;2:76.

ABSTRACT: Legionella causes severe pneumonia in humans. The pathogen produces an array of effectors, which interfere with host cell functions. Among them are the glucosyltransferases Lgt1, Lgt2 and Lgt3 from L. pneumophila. Lgt1 and Lgt2 are produced predominately in the post-exponential phase of bacterial growth, while synthesis of Lgt3 is induced mainly in the lag-phase before intracellular replication of bacteria starts. Lgt glucosyltransferases are structurally similar to clostridial glucosylating toxins. The enzymes use UDP-glucose as a donor substrate and modify eukaryotic elongation factor eEF1A at serine-53. This modification results in inhibition of protein synthesis and death of target cells.In addition to Lgts, Legionella genomes disclose several genes, coding for effector proteins likely to possess glycosyltransferase activities, including SetA (subversion of eukaryotic vesicle trafficking A), which influences vesicular trafficking in the yeast model system and displays tropism for late endosomal/lysosomal compartments of mammalian cells. This review mainly discusses recent results on the structure-function relationship of Lgt glucosyltransferases.

 

Anchors for effectors: subversion of phosphoinositide lipids by legionella

Hilbi H, Weber S, Finsel I.

Max von Pettenkofer Institute, Ludwig-Maximilians University Munich, Germany. hilbi@mvp.uni-muenchen.de

Front Microbiol. 2011;2:91.

ABSTRACT: The facultative intracellular pathogen Legionella pneumophila replicates in free-living amoebae and macrophages within a distinct compartment, the "Legionella-containing vacuole" (LCV). LCV formation involves phosphoinositide (PI) glycerolipids, which are key factors controlling vesicle trafficking pathways and membrane dynamics of eukaryotic cells. To govern the interactions with host cells, L. pneumophila employs the Icm/Dot type IV secretion system and more than 250 translocated "effector proteins" that presumably subvert host signaling and vesicle trafficking pathways. Some of the effector proteins anchor through distinct PIs to the cytosolic face of LCVs and promote the interaction with host vesicles and organelles, catalyze guanine nucleotide exchange of small GTPases, or bind to PI-metabolizing enzymes, such as OCRL1. The PI 5-phosphatase OCRL1 and its Dictyostelium homologue Dd5P4 restrict intracellular growth of L. pneumophila. Moreover, OCRL1/Dd5P4, PI 3-kinases (PI3Ks), and PI4KIIIβ regulate LCV formation and localization of the effector protein SidC, which selectively decorates the LCV membrane. SidC and its 20-kDa "P4C" fragment are robust and specific probes for phosphatidylinositol-4-phosphate, and SidC can be targeted to purify intact LCVs by immuno-magnetic separation. Taken together, bacterial PI-binding effectors as well as host PIs and PI-modulating enzymes play a pivotal role for intracellular replication of L. pneumophila, and the PI-binding effectors are valuable tools for the analysis of eukaryotic PI lipids.

 

Small Regulatory RNA and Legionella pneumophila

Faucher SP, Shuman HA.

Complex Traits Group, Department of Microbiology, McGill University Montreal, QC, Canada. sebastien.faucher@gmail.com

Front Microbiol. 2011;2:98.

ABSTRACT: Legionella pneumophila is a gram-negative bacterial species that is ubiquitous in almost any aqueous environment. It is the agent of Legionnaires' disease, an acute and often under-reported form of pneumonia. In mammals, L. pneumophila replicates inside macrophages within a modified vacuole. Many protein regulators have been identified that control virulence-related properties, including RpoS, LetA/LetS, and PmrA/PmrB. In the past few years, the importance of regulation of virulence factors by small regulatory RNA (sRNAs) has been increasingly appreciated. This is also the case in L. pneumophila where three sRNAs (RsmY, RsmZ, and 6S RNA) were recently shown to be important determinants of virulence regulation and 79 actively transcribed sRNAs were identified. In this review we describe current knowledge about sRNAs and their regulatory properties and how this relates to the known regulatory systems of L. pneumophila. We also provide a model for sRNA-mediated control of gene expression that serves as a framework for understanding the regulation of virulence-related properties of L. pneumophila.

 

Innate immunity to legionella pneumophila

Massis LM, Zamboni DS.

Department of Cell Biology, School of Medicine of Ribeirão Preto, University of São Paulo Ribeirão Preto, SP, Brazil. dszamboni@fmrp.usp.br

Front Microbiol. 2011;2:109.

ABSTRACT: Innate immune cells, such as macrophages, are highly adapted to rapidly recognize infections by distinct pathogens, including viruses, bacteria, fungi, and protozoa. This recognition is mediated by pattern recognition receptors (PRRs), which are found in host cell surface membranes and the host cell cytoplasm. PRRs include protein families such as the toll-like receptors, nod-like receptors, RIG-I-like receptors, and sensors of cytosolic DNA. The activation of these PRRs by pathogen-associated molecular patterns leads to transcriptional responses and specific forms of cell death. These processes effectively contribute to host resistance to infection either via cell-autonomous processes that lead to the intracellular restriction of microbial replication and/or by activating pathogen-specific adaptive immune responses. Legionella pneumophila, the causative agent of Legionnaires' disease, is a Gram-negative bacterium that triggers responses by multiple PRRs. Here, we review a set of studies that have contributed to our specific understanding of the molecular mechanisms by which innate immune cells recognize and respond to L. pneumophila and the importance of these processes to the outcome of infection.

 

The Nlrc4 Inflammasome Contributes to Restriction of Pulmonary Infection by Flagellated Legionella spp. that Trigger Pyroptosis

Pereira MS, Marques GG, Dellama JE, Zamboni DS.

Department of Cell Biology, School of Medicine of Ribeirão Preto, Universidade de São Paulo Ribeirão Preto, São Paulo, Brazil. szamboni@fmrp.usp.br

Front Microbiol. 2011;2:33

ABSTRACT: The Nlrc4 inflammasome is triggered in response to contamination of the host cell cytoplasm with bacterial flagellin, which induces pyroptosis, a form of cell death that accounts for restriction of bacterial infections. Although induction of pyroptosis has been extensively investigated in response to Salmonella typhimurium and Legionella pneumophila, little is known regarding the role of the inflammasome for restriction of non-pneumophila Legionella species. Here, we used five species of the Legionella genus to investigate the importance of the inflammasome for restriction of bacterial infection in vivo. By infecting mice deficient for inflammasome components, we demonstrated that caspase-1 and Nlrc4, but not Asc, contribute to restriction of pulmonary infection with L. micdadei, L. bozemanii, L. gratiana, and L. rubrilucens. L. longbeachae, a non-flagellated bacterium that fails to trigger pyroptosis, was not restricted by the inflammasome and induced death in the infected mice. In contrast to L. longbeachae, flagellin mutants of L. pneumophila did not induce mice death; therefore, besides bypassing the Nlrc4 inflammasome, L. longbeachae may employ additional virulence strategies to replicate in mammalian hosts. Collectively, our data indicate that the Nlrc4 inflammasome plays an important role in host protection against opportunistic pathogenic bacteria that express flagellin.

 

Striking a balance: modulation of host cell death pathways by legionella pneumophila

Luo ZQ.

Department of Biological Sciences, Purdue University West Lafayette, IN, USA. luoz@purdue.edu

Front Microbiol. 2011;2:36

ABSTRACT: Programmed cell death is considered the ultimate solution for the host to eliminate infected cells, leading to the abolishment of the niche for microbial replication and the ablation of infection. Thus, it is not surprising that successful pathogens have evolved diverse strategies to reprogram the cell death pathways for their proliferation. Using effector proteins translocated by the Dot/Icm type IV secretion system, the facultative intracellular pathogen Legionella pneumophila manipulates multiple host cellular processes to create a niche within host cells to support its replication. Investigation in the past decade has established that in mammalian cells this bacterium actively modulates two host cell death pathways, namely the canonical apoptotic pathway controlled by the mitochondrion and the pyroptotic pathway controlled by the Nod-like receptor Naip5 and the Ipaf inflammasome. In this review, I will discuss the recent progress in understanding the mechanisms the bacterium employs to interfere with these host cell death pathways and how such modulation contribute to the intracellular life cycle of the pathogen.

 

Immune Control of Legionella Infection: An in vivo Perspective

Schuelein R, Ang DK, van Driel IR, Hartland EL.

Department of Microbiology and Immunology, University of Melbourne Parkville, Victoria, Australia. hartland@unimelb.edu.au

Front Microbiol. 2011;2:126.

ABSTRACT: Legionella pneumophila is an intracellular pathogen that replicates within alveolar macrophages. Through its ability to activate multiple host innate immune components, L. pneumophila has emerged as a useful tool to dissect inflammatory signaling pathways in macrophages. However the resolution of L. pneumophila infection in the lung requires multiple cell types and abundant cross talk between immune cells. Few studies have examined the coordination of events that lead to effective immune control of the pathogen. Here we discuss L. pneumophila interactions with macrophages and dendritic cell subsets and highlight the paucity of knowledge around how these interactions recruit and activate other immune effector cells in the lung.

 

The Legionella pneumophila Chaperonin - An Unusual Multifunctional Protein in Unusual Locations

Garduño RA, Chong A, Nasrallah GK, Allan DS.

Department of Microbiology and Immunology, Dalhousie University Halifax, NS, Canada. rafael.garduno@dal.ca

Front Microbiol. 2011;2:122.

ABSTRACT: The Legionella pneumophila chaperonin, high temperature protein B (HtpB), was discovered as a highly immunogenic antigen, only a few years after the identification of L. pneumophila as the causative agent of Legionnaires' disease. As its counterparts in other bacterial pathogens, HtpB did not initially receive further attention, particularly because research was focused on a few model chaperonins that were used to demonstrate that chaperonins are essential stress proteins, present in all cellular forms of life and involved in helping other proteins to fold. However, chaperonins have recently attracted increasing interest, particularly after several reports confirmed their multifunctional nature and the presence of multiple chaperonin genes in numerous bacterial species. It is now accepted that bacterial chaperonins are capable of playing a variety of protein folding-independent roles. HtpB is clearly a multifunctional chaperonin that according to its location in the bacterial cell, or in the L. pneumophila-infected cell, plays different roles. HtpB exposed on the bacterial cell surface can act as an invasion factor for non-phagocytic cells, whereas the HtpB released in the host cell can act as an effector capable of altering organelle trafficking, the organization of actin microfilaments and cell signaling pathways. The road to discover the multifunctional nature of HtpB has been exciting and here we provide a historical perspective of the key findings linked to such discovery, as well as a summary of the experimental work (old and new) performed in our laboratory. Our current understanding has led us to propose that HtpB is an ancient protein that L. pneumophila uses as a key molecular tool important to the intracellular establishment of this fascinating pathogen.

 

Type IVB Secretion Systems of Legionella and Other Gram-Negative Bacteria

Nagai H, Kubori T.

Research Institute for Microbial Diseases, Osaka University Osaka, Japan. hnagai@biken.osaka-u.ac.jp

Front Microbiol. 2011;2:136.

ABSTRACT:Type IV secretion systems (T4SSs) play a central role in the pathogenicity of many important pathogens, including Agrobacterium tumefaciens, Helicobacter pylori, and Legionella pneumophila. The T4SSs are related to bacterial conjugation systems, and are classified into two subgroups, type IVA (T4ASS) and type IVB (T4BSS). The T4BSS, which is closely related to conjugation systems of IncI plasmids, was originally found in human pathogen L. pneumophila; pathogenesis by L. pneumophila infection requires functional Dot/Icm T4BSS. A zoonotic pathogen, Coxiella burnetii, and an arthropod pathogen, Rickettsiella grylli - both of which carry T4BSSs highly similar to the Legionella Dot/Icm system - are evolutionarily closely related and comprise a monophyletic group. A growing body of bacterial genomic information now suggests that T4BSSs are not limited to Legionella and related bacteria and IncI plasmids. Here, we review the current knowledge on T4BSS apparatus and component proteins, gained mainly from studies on L. pneumophila Dot/Icm T4BSS. Recent structural studies, along with previous findings, suggest that the Dot/Icm T4BSS contains components with primary or higher-order structures similar to those in other types of secretion systems - types II, III, IVA, and VI, thus highlighting the mosaic nature of T4BSS architecture.

 

Secrets of a successful pathogen: legionella resistance to progression along the autophagic pathway

Joshi AD, Swanson MS.

Department of Microbiology and Immunology, University of Michigan Medical School Ann Arbor, MI, USA. mswanson@umich.edu

Front Microbiol. 2011;2:138.

ABSTRACT: To proliferate within phagocytes, Legionella pneumophila relies on Type IV secretion to modulate host cellular pathways. Autophagy is an evolutionarily conserved degradative pathway that captures and transfers a variety of microbes to lysosomes. Biogenesis of L. pneumophila-containing vacuoles and autophagosomes share several features, including endoplasmic reticulum (ER)-derived membranes, contributions by the host GTPases Rab1, Arf1 and Sar1, and a final destiny in lysosomes. We discuss morphological, molecular genetic, and immunological data that support the model that, although A/J mouse macrophages efficiently engulf L. pneumophila within autophagosomal membranes, the Type IV effector proteins DrrA/SidM, LidA, and RalF prolong association with the ER. By inhibiting immediately delivery to lysosomes, the bacteria persist in immature autophagosomal vacuoles for a period sufficient to differentiate into an acid-resistant, replicative form. Subsequent secretion of the Type IV effector LepB releases the block to autophagosome maturation, and the adapted progeny continue to replicate within autophagolysosomes. Accordingly, L. pneumophila can be exploited as a genetic tool to analyze the recruitment and function of the macrophage autophagy pathway.

 

Legionella Pneumophila Transcriptome during Intracellular Multiplication in Human Macrophages

Faucher SP, Mueller CA, Shuman HA.

Department of Microbiology and Immunology, Columbia University Medical Center New York, NY, USA. hshuman@bsd.uchicago.edu

Front Microbiol. 2011;2:60.

ABSTRACT: Legionella pneumophila is the causative agent of Legionnaires' disease, an acute pulmonary infection. L. pneumophila is able to infect and multiply in both phagocytic protozoa, such as Acanthamoeba castellanii, and mammalian professional phagocytes. The best-known L. pneumophila virulence determinant is the Icm/Dot type IVB secretion system, which is used to translocate more than 150 effector proteins into host cells. While the transcriptional response of Legionella to the intracellular environment of A. castellanii has been investigated, much less is known about the Legionella transcriptional response inside human macrophages. In this study, the transcriptome of L. pneumophila was monitored during exponential and post-exponential phase in rich AYE broth as well as during infection of human cultured macrophages. This was accomplished with microarrays and an RNA amplification procedure called selective capture of transcribed sequences to detect small amounts of mRNA from low numbers of intracellular bacteria. Among the genes induced intracellularly are those involved in amino acid biosynthetic pathways leading to l-arginine, l-histidine, and l-proline as well as many transport systems involved in amino acid and iron uptake. Genes involved in catabolism of glycerol are also induced during intracellular growth, suggesting that glycerol could be used as a carbon source. The genes encoding the Icm/Dot system are not differentially expressed inside cells compared to control bacteria grown in rich broth, but the genes encoding several translocated effectors are strongly induced. Moreover, we used the transcriptome data to predict previously unrecognized Icm/Dot effector genes based on their expression pattern and confirmed translocation for three candidates. This study provides a comprehensive view of how L. pneumophila responds to the human macrophage intracellular environment.

 

Control of host cell phosphorylation by legionella pneumophila

Haenssler E, Isberg RR.

Department of Molecular Biology and Microbiology, Tufts University School of Medicine Boston, MA, USA. ralph.isberg@tufts.edu

Front Microbiol. 2011;2:64.

ABSTRACT: Phosphorylation is one of the most frequent modifications in intracellular signaling and is implicated in many processes ranging from transcriptional control to signal transduction in innate immunity. Many pathogens modulate host cell phosphorylation pathways to promote growth and establish an infectious disease. The intracellular pathogen Legionella pneumophila targets and exploits the host phosphorylation system throughout the infection cycle as part of its strategy to establish an environment beneficial for replication. Key to this manipulation is the L. pneumophila Icm/Dot type IV secretion system, which translocates bacterial proteins into the host cytosol that can act directly on phosphorylation cascades. This review will focus on the different stages of L. pneumophila infection, in which host kinases and phosphatases contribute to infection of the host cell and promote intracellular survival of the pathogen. This includes the involvement of phosphatidylinositol 3-kinases during phagocytosis as well as the role of phosphoinositide metabolism during the establishment of the replication vacuole. Furthermore, L. pneumophila infection modulates the NF-κB and mitogen-activated protein kinase pathways, two signaling pathways that are central to the host innate immune response and involved in regulation of host cell survival. Therefore, L. pneumophila infection manipulates host cell signal transduction by phosphorylation at multiple levels.

 

Virulence properties of the legionella pneumophila cell envelope

Shevchuk O, Jäger J, Steinert M.

Institut für Mikrobiologie, Technische Universität Braunschweig Braunschweig, Germany. m.steinert@tu-bs.de

Front Microbiol. 2011;2:74. review.

ABSTRACT: The bacterial envelope plays a crucial role in the pathogenesis of infectious diseases. In this review, we summarize the current knowledge of the structure and molecular composition of the Legionella pneumophila cell envelope. We describe lipopolysaccharides biosynthesis and the biological activities of membrane and periplasmic proteins and discuss their decisive functions during the pathogen-host interaction. In addition to adherence, invasion, and intracellular survival of L. pneumophila, special emphasis is laid on iron acquisition, detoxification, key elicitors of the immune response and the diverse functions of outer membrane vesicles. The critical analysis of the literature reveals that the dynamics and phenotypic plasticity of the Legionella cell surface during the different metabolic stages require more attention in the future.

 

Comparative and functional genomics of legionella identified eukaryotic like proteins as key players in host-pathogen interactions

Gomez-Valero L, Rusniok C, Cazalet C, Buchrieser C.

Institut Pasteur, Unité de Biologie des Bactéries Intracellulaires Paris, France. carmen.buchrieser@pasteur.fr

Front Microbiol. 2011;2:208. review

ABSTRACT: Although best known for its ability to cause severe pneumonia in people whose immune defenses are weakened, Legionellapneumophila and Legionellalongbeachae are two species of a large genus of bacteria that are ubiquitous in nature, where they parasitize protozoa. Adaptation to the host environment and exploitation of host cell functions are critical for the success of these intracellular pathogens. The establishment and publication of the complete genome sequences of L. pneumophila and L. longbeachae isolates paved the way for major breakthroughs in understanding the biology of these organisms. In this review we present the knowledge gained from the analyses and comparison of the complete genome sequences of different L. pneumophila and L. longbeachae strains. Emphasis is given on putative virulence and Legionella life cycle related functions, such as the identification of an extended array of eukaryotic like proteins, many of which have been shown to modulate host cell functions to the pathogen's advantage. Surprisingly, many of the eukaryotic domain proteins identified in L. pneumophila as well as many substrates of the Dot/Icm type IV secretion system essential for intracellular replication are different between these two species, although they cause the same disease. Finally, evolutionary aspects regarding the eukaryotic like proteins in Legionella are discussed.

 

Aminoacyl-tRNA-charged eukaryotic elongation factor 1A is the bona fide substrate for Legionella pneumophila effector glucosyltransferases

Tzivelekidis T, Jank T, Pohl C, Schlosser A, Rospert S, Knudsen CR, Rodnina MV, Belyi Y, Aktories K.

Institut für Experimentelle und Klinische Pharmakologie und Toxikologie, Albert-Ludwigs-Universität, Freiburg, Germany. klaus.aktories@pharmakol.uni-freiburg.de

PLoS One. 2011;6(12):e29525.

ABSTRACT: Legionella pneumophila, which is the causative organism of Legionnaireś disease, translocates numerous effector proteins into the host cell cytosol by a type IV secretion system during infection. Among the most potent effector proteins of Legionella are glucosyltransferases (lgt's), which selectively modify eukaryotic elongation factor (eEF) 1A at Ser-53 in the GTP binding domain. Glucosylation results in inhibition of protein synthesis. Here we show that in vitro glucosylation of yeast and mouse eEF1A by Lgt3 in the presence of the factors Phe-tRNA(Phe) and GTP was enhanced 150 and 590-fold, respectively. The glucosylation of eEF1A catalyzed by Lgt1 and 2 was increased about 70-fold. By comparison of uncharged tRNA with two distinct aminoacyl-tRNAs (His-tRNA(His) and Phe-tRNA(Phe)) we could show that aminoacylation is crucial for Lgt-catalyzed glucosylation. Aminoacyl-tRNA had no effect on the enzymatic properties of lgt's and did not enhance the glucosylation rate of eEF1A truncation mutants, consisting of the GTPase domain only or of a 5 kDa peptide covering Ser-53 of eEF1A. Furthermore, binding of aminoacyl-tRNA to eEF1A was not altered by glucosylation. Taken together, our data suggest that the ternary complex, consisting of eEF1A, aminoacyl-tRNA and GTP, is the bona fide substrate for lgt's.

  

Evaluation of the protective immunity of the Legionella pneumophila recombinant protein FlaA/MompS/PilE in an A/J mouse model

Xu Y, Guan W, Xu JN, Cao DP, Yang BB, Chen DL, Chen JP.

Department of Parasitology, West China School of Preclinical and Forensic Medicine, Sichuan University, Chengdu 610041, Sichuan, China. jpchen007@163.com

Vaccine. 2011 May 23;29(23):4051-7.

ABSTRACT: To investigate the protect effects of the recombinant protein FlaA/MompS/PilE against Legionella pneumophila (L. pneumophila), the coding sequences of the three proteins were optimized by DNA Star software firstly, cloned, expressed by Escherichia coli BL21, and purified. To give an enhanced the immunological response, the proteins were linked together with (Linker) or without a linker insert (NLinker) and were purified from E. coli BL21. The A/J mouse model was used to determine the level of the induction of protective immunity from the purified proteins. Our results showed that the IgG titer, which was measured by ELISA, was increased after the administration of the five proteins. Compared to the administration of the individual proteins, the chimeric Linker and NLinker proteins displayed lasting immunity to a lethal dose of L. pneumophila challenge. The Linker protein protected the A/J mouse against a higher dose of L. pneumonia compared to the other proteins used in this study, as it contained a more effective immunogen. The work presented here demonstrates that the bioinformatics software, DNA Star, is a valid tool to analyse the epitopes of proteins and was useful in the optimization of proteins that could induce the protective immune response to L. pneumophila. The cross-immunity of recombinant proteins, such as the Linker and the NLinker chimera, have higher generates a greater immune than the single proteins.

 

Protein expression, crystallization and preliminary X-ray crystallographic studies of LidA from Legionella pneumophila

Zhu W, Meng G, Liu Y, Zhang F, Zheng X.

State Key Lab of Protein and Plant Gene Research, Peking University, Beijing 100871, People's Republic of China. xiaofengz@pku.edu.cn

Acta Crystallogr Sect F Struct Biol Cryst Commun. 2011 May 1;67(Pt 5):637-9.

ABSTRACT: LidA, a translocated substrate of the Legionella pneumophila Dot/Icm type IV secretion system, is associated with maintenance of bacterial integrity and interferes with the early secretory pathway. However, the precise mechanism of LidA in these processes remains elusive. To further investigate the structure and function of LidA, the full-length protein was successfully expressed in Escherichia coli and purified. LidA was crystallized using sitting-drop vapour diffusion and diffracted to a resolution of 2.75 Å. The crystal belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 57.5, b = 64.5, c = 167.3 Å, α = β = γ = 90°. There is one molecule per asymmetric unit.

 

Legionella pneumophila type II secretion dampens the cytokine response of infected macrophages and epithelia

McCoy-Simandle K, Stewart CR, Dao J, DebRoy S, Rossier O, Bryce PJ, Cianciotto NP.

Department of Microbiology and Immunology, Northwestern University Medical School, 320 East Superior St., Chicago, IL 60611, USA. n-cianciotto@northwestern.edu.

Infect Immun. 2011 May;79(5):1984-97.

ABSTRACT: The type II secretion (T2S) system of Legionella pneumophila is required for the ability of the bacterium to grow within the lungs of A/J mice. By utilizing mutants lacking T2S (lsp), we now document that T2S promotes the intracellular infection of both multiple types of macrophages and lung epithelia. Following infection of macrophages, lsp mutants (but not a complemented mutant) elicited significantly higher levels of interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-α), IL-10, IL-8, IL-1β, and MCP-1 within tissue culture supernatants. A similar result was obtained with infected lung epithelial cell lines and the lungs of infected A/J mice. Infection with a mutant specifically lacking the T2S-dependent ProA protease (but not a complemented proA mutant) resulted in partial elevation of cytokine levels. These data demonstrate that the T2S system of L. pneumophila dampens the cytokine/chemokine output of infected host cells. Upon quantitative reverse transcription (RT)-PCR analysis of infected host cells, an lspF mutant, but not the proA mutant, produced significantly higher levels of cytokine transcripts, implying that some T2S-dependent effectors dampen signal transduction and transcription but that others, such as ProA, act at a posttranscriptional step in cytokine expression. In summary, the impact of T2S on lung infection is a combination of at least three factors: the promotion of growth in macrophages, the facilitation of growth in epithelia, and the dampening of the chemokine and cytokine output from infected host cells. To our knowledge, these data are the first to identify a link between a T2S system and the modulation of immune factors following intracellular infection.

  

Protein kinase LegK2 is a type IV secretion system effector involved in endoplasmic reticulum recruitment and intracellular replication of Legionella pneumophila

Hervet E, Charpentier X, Vianney A, Lazzaroni JC, Gilbert C, Atlan D, Doublet P.

Université de Lyon, Université Lyon 1, CNRS UMR 5240 Microbiologie, Adaptation et Pathogénie, 69622 Villeurbanne, France. patricia.doublet@univ-lyon1.fr

Infect Immun. 2011 May;79(5):1936-50.

ABSTRACT: Legionella pneumophila is the etiological agent of Legionnaires' disease. Crucial to the pathogenesis of this intracellular pathogen is its ability to subvert host cell defenses, permitting intracellular replication in specialized vacuoles within host cells. The Dot/Icm type IV secretion system (T4SS), which translocates a large number of bacterial effectors into host cell, is absolutely required for rerouting the Legionella phagosome. Many Legionella effectors display distinctive eukaryotic domains, among which are protein kinase domains. In silico analysis and in vitro phosphorylation assays identified five functional protein kinases, LegK1 to LegK5, encoded by the epidemic L. pneumophila Lens strain. Except for LegK5, the Legionella protein kinases are all T4SS effectors. LegK2 plays a key role in bacterial virulence, as demonstrated by gene inactivation. The legK2 mutant containing vacuoles displays less-efficient recruitment of endoplasmic reticulum markers, which results in delayed intracellular replication. Considering that a kinase-dead substitution mutant of legK2 exhibits the same virulence defects, we highlight here a new molecular mechanism, namely, protein phosphorylation, developed by L. pneumophila to establish a replicative niche and evade host cell defenses.

 

DsbA2 (27 kDa Com1-like protein) of Legionella pneumophila catalyses extracytoplasmic disulphide-bond formation in proteins including the Dot/Icm type IV secretion system

Jameson-Lee M, Garduño RA, Hoffman PS.

Department of Medicine, Division of Infectious Diseases and International Health, University of Virginia School of Medicine, Charlottesville, VA 22908, USA. psh2n@virginia.edu

Mol Microbiol. 2011 May;80(3):835-52.

ABSTRACT: In Gram-negative bacteria, thiol oxidoreductases catalyse the formation of disulphide bonds (DSB) in extracytoplasmic proteins. In this study, we sought to identify DSB-forming proteins required for assembly of macromolecular structures in Legionella pneumophila. Here we describe two DSB-forming proteins, one annotated as dsbA1 and the other annotated as a 27 kDa outer membrane protein similar to Com1 of Coxiella burnetii, which we designate as dsbA2. Both proteins are predicted to be periplasmic, and while dsbA1 mutants were readily isolated and without phenotype, dsbA2 mutants were not obtained. To advance studies of DsbA2, a cis-proline residue at position 198 was replaced with threonine that enables formation of stable disulphide-bond complexes with substrate proteins. Expression of DsbA2 P198T mutant protein from an inducible promoter produced dominant-negative effects on DsbA2 function that resulted in loss of infectivity for amoeba and HeLa cells and loss of Dot/Icm T4SS-mediated contact haemolysis of erythrocytes. Analysis of captured DsbA2 P198T-substrate complexes from L. pneumophila by mass spectrometry identified periplasmic and outer membrane proteins that included components of the Dot/Icm T4SS. More broadly, our studies establish a DSB oxidoreductase function for the Com1 lineage of DsbA2-like proteins which appear to be conserved among those bacteria also expressing T4SS.

 

Hyperoxia accelerates Fas-mediated signaling and apoptosis in the lungs of Legionella pneumophila pneumonia

Maeda T, Kimura S, Matsumoto T, Tanabe Y, Gejyo F, Yamaguchi K.

Department of Microbiology and Infectious Diseases, Toho University Faculty of Medicine, Tokyo 143-8540, Japan. kimsou@med.toho-u.ac.jp

BMC Res Notes. 2011 Apr 6;4:107.

ABSTRACT: BACKGROUND: Oxygen supplementation is commonly given to the patients with severe pneumonia including Legionella disease. Recent data suggested that apoptosis may play an important role, not only in the pathogenesis of Legionella pneumonia, but also in oxygen-induced tissue damage. In the present study, the lethal sensitivity to Legionella pneumonia were compared in the setting of hyperoxia between wild-type and Fas-deficient mice.

FINDINGS: C57BL/6 mice and B6.MRL-Faslpr mice characterized with Fas-deficiency were used in this study. After intratracheal administration of L. pneumophila, mice were kept in hyperoxic conditions (85-90% O2 conc.) in an airtight chamber for 3 days. Bone-marrow derived macrophages infected with L. pneumophila were also kept in hyperoxic conditions. Caspase activity and cytokine production were determined by using commercially available kits. Smaller increases of several apoptosis markers, such as caspase-3 and -8, were demonstrated in Fas-deficient mice, even though the bacterial burdens in Fas-deficient and wild type mice were similar. Bone-marrow derived macrophages from Fas-deficient mice were shown to be more resistant to Legionella-induced cytotoxicity than those from wild-type mice under hyperoxia.

CONCLUSIONS: These results demonstrated that Fas-mediated signaling and apoptosis may be a crucial factor in the pathogenesis of Legionella pneumonia in the setting of hyperoxia.

 

The E Block motif is associated with Legionella pneumophila translocated substrates

Huang L, Boyd D, Amyot WM, Hempstead AD, Luo ZQ, O'Connor TJ, Chen C, Machner M, Montminy T, Isberg RR.

Department of Molecular Biology and Microbiology, Tufts University School of Medicine, 150 Harrison Avenue, Boston, MA 02111, USA. ralph.isberg@tufts.edu

Cell Microbiol. 2011 Feb;13(2):227-45.

ABSTRACT: Legionella pneumophila promotes intracellular growth by moving bacterial proteins across membranes via the Icm/Dot system. A strategy was devised to identify large numbers of Icm/Dot translocated proteins, and the resulting pool was used to identify common motifs that operate as recognition signals. The 3' end of the sidC gene, which encodes a known translocated substrate, was replaced with DNA encoding 200 codons from the 3' end of 442 potential substrate-encoding genes. The resulting hybrid proteins were then tested in a high throughput assay, in which translocated SidC antigen was detected by indirect immunofluorescence. Among translocated substrates, regions of 6-8 residues called E Blocks were identified that were rich in glutamates. Analysis of SidM/DrrA revealed that loss of three Glu residues, arrayed in a triangle on an α-helical surface, totally eliminated translocation of a reporter protein. Based on this result, a second strategy was employed to identify Icm/Dot substrates having carboxyl terminal glutamates. From the fusion assay and the bioinformatic queries, carboxyl terminal sequences from 49 previously unidentified proteins were shown to promote translocation into target cells. These studies indicate that by analysing subsets of translocated substrates, patterns can be found that allow predictions of important motifs recognized by Icm/Dot.

 

Impact of the virulence-associated MAb3/1 epitope on the physicochemical surface properties of Legionella pneumophila sg1: An issue to explain infection potential?

Gosselin F, Duval JF, Simonet J, Ginevra C, Gaboriaud F, Jarraud S, Mathieu L.

Ecole Pratique des Hautes Etudes, UMR 7564 CNRS/Nancy Université, Pôle de l'Eau, F-54505 Vandoeuvre-lès-Nancy, France. laurence.mathieu@pharma.uhp-nancy.fr

Colloids Surf B Biointerfaces. 2011 Feb 1;82(2):283-90.

ABSTRACT: The relationship between the presence/absence of the virulence-associated MAb3/1 epitope of sixteen Legionella pneumophila serogroup 1 strains and their respective surface physicochemical properties is evidenced from electrokinetic measurements (microelectrophoresis) performed as a function of KNO(3) electrolyte concentration (range 1-100mM, pH6.5). Among the bacteria selected, nine original strains constitute the Dresden reference panel and differ according to the presence/absence of the virulence-associated monoclonal antibody MAb3/1 of the O-specific chain of the lipopolysaccharides (LPS). Five isogenic Lens strains, also investigated in the current study, present the epitope MAb3/1 of their LPS and were involved to some extent in the outbreak that stroke the Nord Pas-de-Calais region (France) in 2004. All bacteria exhibit the typical electrokinetic features of soft (permeable) particles. On the basis of Ohshima's model, analysis of the electrophoretic mobility data allows evaluating the intraparticular flow penetration length 1/λ(0) and the (negative) volume charge density ρ(0) that both reflect the structure and chemical composition of the soft bacterial component. Our results show that the virulent MAb3/1 positive strains are characterized on average by 1/λ(0) and ǀρ(0)ǀ values that are about 1.5 times larger and 5 times lower, respectively, than those derived for lesser virulent (MAb3/1 negative) strains. In other words, on average the soft surface layer of MAb3/1 positive strains is significantly less charged and more permeable than those of MAb3/1 negative strains. The intimate correlation between virulence-associated MAb3/1 epitope and charge density carried by the bacterial envelop was further confirmed by lower 1/λ(0) and greater ǀρ(0)ǀ values for lag-1 mutant CS332 strain, lacking the MAb3/1 epitope, compared to the parental strain AM511. A closer inspection of the dispersion in 1/λ(0) and ǀρ(0)ǀ data over the ensemble of analysed bacteria together with the reported number of Legionnaires' disease cases they are responsible for, points out the charge density ǀρ(0)ǀ as the parameter that is most suitable for discriminating highly virulent (MAb3/1 positive) from less virulent (MAb3/1 negative) strains. Although short-range interaction determines infection process, our results suggest that the infection potential of Legionella pneumophila serogroup 1 may be also controlled significantly by non-specific long-range electrostatic repulsion the bacteria undergo when approaching negatively charged host cells to be infected.

 

Enzymatic synthesis and properties of glycoconjugates with legionaminic acid as a replacement for neuraminic acid

Watson DC, Leclerc S, Wakarchuk WW, Young NM.

Institute for Biological Sciences, National Research Council of Canada, 100 Sussex Drive, Ottawa, Ontario, Canada. martin.young@nrc-cnrc.gc.ca

Glycobiology. 2011 Jan;21(1):99-108.

ABSTRACT: In addition to sialic acid, bacteria produce several other nonulosonic acids, including legionaminic acid (Leg). This has exactly the same stereochemistry as sialic acid, with the added features of 9-deoxy and 7-amino groups. In order to explore the biological effects of replacing sialic acid residues (Neu5Ac) in glycoconjugates with Leg in its diacetylated form, diacetyllegionaminic acid (Leg5Ac7Ac), we tested CMP-Leg5Ac7Ac as a donor substrate with a selection of bacterial and mammalian sialyltransferases. The CMP-Leg5Ac7Ac was synthesized in vitro by means of cloned enzymes from the bacillosamine portion of the Campylobacter jejuni N-glycan pathway and from the Leg pathway of Legionella pneumophila. Using fluorescent derivatives of lactose, Galβ1,4GlcNAcβ and T-antigen (Galβ1,3GalNAcα) as acceptors, we tested eight different sialyltransferases and found that the Pasteurella multocida PM0188h and porcine ST3Gal1 sialyltransferases were significantly active with CMP-Leg5Ac7Ac, showing 60% activity when compared with CMP-Neu5Ac. The Photobacterium α2,6 sialyltransferase was weakly active, with 6% relative activity. The Leg5Ac7Ac-α-2,3-lactose product was then tested as a substrate with six sialidases of viral, bacterial and mammalian origin. All showed much lower activities than with the corresponding sialic acid substrate, with the influenza virus N1 being the most active and human NEU2 being the least active. These results show the feasibility of producing glycoconjugates with Leg5Ac7Ac residues as the terminal sugars, which should display novel biological properties.

 

Insertion Sequences as Highly Resolutive Genomic Markers for Sequence Type 1 Legionella pneumophila Paris

Vergnes M, Ginevra C, Kay E, Normand P, Thioulouse J, Jarraud S, Maurin M, Schneider D.

Laboratoire Adaptation et Pathogénie des Microorganismes, CNRS UMR5163, Université Joseph Fourier, Institut Jean Roget, Campus Santé, Domaine de la Merci, BP170, 38042 Grenoble Cedex 9, France. dominique.schneider@ujf-grenoble.fr.

J Clin Microbiol. 2011 Jan;49(1):315-24.

ABSTRACT: The causative agent of legionellosis, Legionella pneumophila, colonizes all natural and human-made water networks, thus constituting the source of contaminated aerosols responsible for airborne human infections. Efficient control of infections, especially during epidemics, necessitates the fastest and most resolutive identification possible of the bacterial source for subsequent disinfection of reservoirs. We thus compared recognized typing approaches for Legionella with a method based on characterization of insertion sequence (IS) content. A total of 86 clinical or environmental isolates of L. pneumophila, including 84 Paris isolates, sampled from 25 clinical investigations in France between 2001 and 2007, were obtained from the Legionella National Reference Center. All strains were typed by monoclonal antibody subgrouping, sequence-based typing, pulsed-field gel electrophoresis, and restriction fragment length polymorphism based on the presence or absence of IS elements. We identified six different types of IS elements in L. pneumophila Paris and used them as genomic markers in hybridization experiments. One IS type, ISLpn11, revealed a high discriminatory power. Simpson's index of discrimination, calculated from the distribution of IS elements, was higher than that obtained with the other typing methods used for L. pneumophila Paris. Moreover, specific ISLpn11 copies were found only in strains isolated from particular cities. In more than half of the cases, each clinical isolate had an ISLpn11 profile that was recovered in at least one environmental isolate from the same geographical location, suggesting that our method could identify the infection source. Phylogenetic analysis suggests a clonal expansion for the L. pneumophila Paris strain.

 

Legionella metaeffector exploits host proteasome to temporally regulate cognate effector

Kubori T, Shinzawa N, Kanuka H, Nagai H.

Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, Japan. hnagai@biken.osaka-u.ac.jp

PLoS Pathog. 2010 Dec 2;6(12):e1001216.

ABSTRACT: Pathogen-associated secretion systems translocate numerous effector proteins into eukaryotic host cells to coordinate cellular processes important for infection. Spatiotemporal regulation is therefore important for modulating distinct activities of effectors at different stages of infection. Here we provide the first evidence of "metaeffector," a designation for an effector protein that regulates the function of another effector within the host cell. Legionella LubX protein functions as an E3 ubiquitin ligase that hijacks the host proteasome to specifically target the bacterial effector protein SidH for degradation. Delayed delivery of LubX to the host cytoplasm leads to the shutdown of SidH within the host cells at later stages of infection. This demonstrates a sophisticated level of coevolution between eukaryotic cells and L. pneumophila involving an effector that functions as a key regulator to temporally coordinate the function of a cognate effector protein.

 

NOD1 and NOD2 regulation of pulmonary innate immunity to Legionella pneumophila

Berrington WR, Iyer R, Wells RD, Smith KD, Skerrett SJ, Hawn TR.

University of Washington School of Medicine, Seattle, WA 98195-6523, USA. berring@uw.edu

Eur J Immunol. 2010 Dec;40(12):3519-27.

ABSTRACT: The role of nucleotide-binding oligomerization domain-1 (NOD1) and nucleotide-binding oligomerization domain-2 (NOD2), cytoplasmic receptors which detect bacterial cell wall molecules, in pulmonary innate immune responses is poorly understood. We determined that both NOD1 and NOD2 detect heat-killed Legionella and stimulate NF-κb and IFN-β promoter activity using an in vitro luciferase reporter system. We next infected NOD1- and NOD2-deficient animals with aerosolized Legionella pneumophila. At 3 days post infection, Nod1(-/-) mice had impaired bacterial clearance compared to WT controls. In addition, at 4h and 24h, Nod1(-/-) mice had impaired neutrophil recruitment to the alveolar space. In contrast, increased lung neutrophils were seen in the Nod2(-/-) animals at 24h. Analysis of cytokine production at 4h post infection revealed a significant decrease in proinflammatory cytokines in the Nod1(-/-) animals when compared to WT animals. In contrast, increased 4-h proinflammatory cytokines were seen in the Nod2(-/-) animals. Furthermore, the lungs of both Nod1(-/-) and Nod2(-/-) mice had significantly increased pro-inflammatory cytokine levels at 24h, suggesting possible suppressive roles for later stages of infection. Together, our data suggest that although both NOD1 and NOD2 can detect Legionella, these receptors modulate the in vivo pulmonary immune response differently.

 

Caspase-1-induced pyroptosis is an innate immune effector mechanism against intracellular bacteria

Miao EA, Leaf IA, Treuting PM, Mao DP, Dors M, Sarkar A, Warren SE, Wewers MD, Aderem A.

Institute for Systems Biology, Seattle, Washington, USA. emiao@systemsbiology.org

Nat Immunol. 2010 Dec;11(12):1136-42.

ABSTRACT: Macrophages mediate crucial innate immune responses via caspase-1-dependent processing and secretion of interleukin 1β (IL-1β) and IL-18. Although infection with wild-type Salmonella typhimurium is lethal to mice, we show here that a strain that persistently expresses flagellin was cleared by the cytosolic flagellin-detection pathway through the activation of caspase-1 by the NLRC4 inflammasome; however, this clearance was independent of IL-1β and IL-18. Instead, caspase-1-induced pyroptotic cell death released bacteria from macrophages and exposed the bacteria to uptake and killing by reactive oxygen species in neutrophils. Similarly, activation of caspase-1 cleared unmanipulated Legionella pneumophila and Burkholderia thailandensis by cytokine-independent mechanisms. This demonstrates that activation of caspase-1 clears intracellular bacteria in vivo independently of IL-1β and IL-18 and establishes pyroptosis as an efficient mechanism of bacterial clearance by the innate immune system.

 

Antibodies protect against intracellular bacteria by Fc receptor-mediated lysosomal targeting

Joller N, Weber SS, Müller AJ, Spörri R, Selchow P, Sander P, Hilbi H, Oxenius A.

Institute for Microbiology, Eidgenössiche Technische Hochschule Zürich, 8093 Zurich, Switzerland. oxenius@micro.biol.ethz.ch

Proc Natl Acad Sci U S A. 2010 Nov 23;107(47):20441-6.

ABSTRACT: The protective effect of antibodies (Abs) is generally attributed to neutralization or complement activation. Using Legionella pneumophila and Mycobacterium bovis bacillus Calmette-Guérin as a model, we discovered an additional mechanism of Ab-mediated protection effective against intracellular pathogens that normally evade lysosomal fusion. We show that Fc receptor (FcR) engagement by Abs, which can be temporally and spatially separated from bacterial infection, renders the host cell nonpermissive for bacterial replication and targets the pathogens to lysosomes. This process is strictly dependent on kinases involved in FcR signaling but not on host cell protein synthesis or protease activation. Based on these findings, we propose a mechanism whereby Abs and FcR engagement subverts the strategies by which intracellular bacterial pathogens evade lysosomal degradation.

 

Legionella pneumophila induces cathepsin B-dependent necrotic cell death with releasing high mobility group box1 in macrophages

Morinaga Y, Yanagihara K, Nakamura S, Hasegawa H, Seki M, Izumikawa K, Kakeya H, Yamamoto Y, Yamada Y, Kohno S, Kamihira S.

Department of Laboratory Medicine, Nagasaki University Graduate School of Biomedical Sciences, 1-7-1 Sakamoto, Nagasaki, 851-2128, Japan. kamihira@net.nagasaki-u.ac.jp

Respir Res. 2010 Nov 22;11:158.

ABSTRACT: BACKGROUND: Legionella pneumophila (LPN) can cause a lethal infectious disease with a marked inflammatory response in humans. However, the mechanism of this severe inflammation remains poorly understood. Since necrosis is known to induce inflammation, we investigated whether LPN induces necrosis in macrophages. We also analyzed the involvement of lysosomal cathepsin B in LPN-induced cell death.

METHODS: The human monocytic cell line THP-1 was infected with LPN, NUL1 strain. MG132-treated cells were used as apoptotic control cells. After infection, the type of cell death was analyzed by using microscopy, LDH release and flow cytometry. As a proinflammatory mediator, high-mobility group box 1 (HMGB-1), was measured. Cathepsin B activity was also measured and the inhibitory effects of cathepsin B on LPN-induced cell death were analyzed.

RESULTS: THP-1 cells after treatment with high dose of LPN showed necrotic features with releasing HMGB-1. This necrosis and the HMGB-1 release were inhibited by a specific lysosomal cathepsin B inhibitor and were characterized by a rapid and high activation of cathepsin B that was not observed in apoptotic control cells. The necrosis was also accompanied by cathepsin B-dependent poly(ADP-ribose) polymerase (PARP) cleavage.

CONCLUSIONS: We demonstrate here that L. pneumophila rapidly induces cathepsin B-dependent necrosis in a dose-dependent manner and releases a proinflammatory mediator, HMGB-1, from macrophages. This report describes a novel aspect of the pathogenesis of Legionnaires' disease and provides a possible therapeutic target for the regulation of inflammation.

 

Modulation of host cell function by Legionella pneumophila type IV effectors

Hubber A, Roy CR.

Section of Microbial Pathogenesis, School of Medicine, Yale University, New Haven, Connecticut 06536, USA. andree.hubber@yale.edu

Annu Rev Cell Dev Biol. 2010 Nov 10;26:261-83.

ABSTRACT: Macrophages and protozoa ingest bacteria by phagocytosis and destroy these microbes using a conserved pathway that mediates fusion of the phagosome with lysosomes. To survive within phagocytic host cells, bacterial pathogens have evolved a variety of strategies to avoid fusion with lysosomes. A virulence strategy used by the intracellular pathogen Legionella pneumophila is to manipulate host cellular processes using bacterial proteins that are delivered into the cytosolic compartment of the host cell by a specialized secretion system called Dot/Icm. The proteins delivered by the Dot/Icm system target host factors that play evolutionarily conserved roles in controlling membrane transport in eukaryotic cells, which enables L. pneumophila to create an endoplasmic reticulum-like vacuole that supports intracellular replication in both protozoan and mammalian host cells. This review focuses on intracellular trafficking of L. pneumophila and describes how bacterial proteins contribute to modulation of host processes required for survival within host cells.

 

Lipidation by the host prenyltransferase machinery facilitates membrane localization of Legionella pneumophila effector proteins

Ivanov SS, Charron G, Hang HC, Roy CR.

Section of Microbial Pathogenesis, Yale University School of Medicine, New Haven, Connecticut 06536, USA. craig.roy@yale.edu.

J Biol Chem. 2010 Nov 5;285(45):34686-98.

ABSTRACT: The intracellular human pathogen Legionella pneumophila translocates multiple proteins in the host cytosol known as effectors, which subvert host cellular processes to create a membrane-bound organelle that supports bacterial replication. It was observed that several Legionella effectors encode a prototypical eukaryotic prenylation CAAX motif (where C represents a cysteine residue and A denotes an aliphatic amino acid). These bacterial motifs mediated posttranslational modification of effector proteins resulting in the addition of either a farnesyl or geranylgeranyl isoprenyl lipid moiety to the cysteine residue of the CAAX tetrapeptide. Lipidation enhanced membrane affinity for most Legionella CAAX motif proteins and facilitated the localization of these effector proteins to host organelles. Host farnesyltransferase and class I geranylgeranyltransferase were both involved in the lipidation of the Legionella CAAX motif proteins. Perturbation of the host prenylation machinery during infection adversely affected the remodeling of the Legionella-containing vacuole. Thus, these data indicate that Legionella utilize the host prenylation machinery to facilitate targeting of effector proteins to membrane-bound organelles during intracellular infection.

  

3-Deoxy-D-manno-octulosonic acid (Kdo) hydrolase identified in Francisella tularensis, Helicobacter pylori, and Legionella pneumophila

Chalabaev S, Kim TH, Ross R, Derian A, Kasper DL.

Department of Microbiology and Molecular Genetics, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA. dennis_kasper@hms.harvard.edu.

J Biol Chem. 2010 Nov 5;285(45):34330-6.

ABSTRACT: 3-Deoxy-D-manno-octulosonic acid (Kdo) is an eight-carbon sugar ubiquitous in Gram-negative bacterial lipopolysaccharides (LPS). Although its biosynthesis is well described, no protein has yet been identified as a Kdo hydrolase. However, Kdo hydrolase enzymatic activity has been detected in membranes of Helicobacter pylori and Francisella tularensis and may be responsible for the removal of side-chain Kdo from the LPS core saccharides. We now report the identification of genes encoding a Kdo hydrolase in F. tularensis Schu S4 and live vaccine strain strains, in H. pylori 26695 strain and in Legionella pneumophila Philadelphia 1 strain. We have renamed the genes kdhA for keto-deoxyoctulosonate hydrolase A. Deletion of kdhA abolished Kdo hydrolase activity in membranes of F. tularensis live vaccine strain. The F. tularensis kdhA mutant synthesized a core oligosaccharide containing a Kdo disaccharide with one of the Kdo residues being a terminal side chain. This side-chain Kdo monosaccharide was absent in the wild-type core oligosaccharide. Expression in Escherichia coli of recombinant KdhA from F. tularensis, H. pylori, and L. pneumophila resulted in a reduction of membrane-associated side-chain Kdo. The identification of this previously faceless enzyme will accelerate study of the biosynthetic basis and biologic impact for postbiosynthetic LPS structural modification.

 

Inhibition of pathogen-induced apoptosis by a Coxiella burnetii type IV effector protein

Lührmann A, Nogueira CV, Carey KL, Roy CR.

Section of Microbial Pathogenesis, Yale University School of Medicine, New Haven, CT 06536, USA. craig.roy@yale.edu

Proc Natl Acad Sci U S A. 2010 Nov 2;107(44):18997-9001.

ABSTRACT: Coxiella burnetii and Legionella pneumophila are evolutionarily related pathogens with different intracellular infection strategies. C. burnetii persists within and is transmitted by mammalian hosts, whereas, L. pneumophila is found primarily in the environment associated with protozoan hosts. Although a type IV secretion system encoded by the defect in organelle trafficking (dot) and intracellular multiplication (icm) genes is a virulence determinant that remains highly conserved in both bacteria, the two pathogens encode a different array of effector proteins that are delivered into host cells by the Dot/Icm machinery. This difference suggests that adaptations to evolutionarily distinct hosts may be reflected in the effector protein repertoires displayed by these two pathogens. Here we provide evidence in support of this hypothesis. We show that a unique C. burnetii effector from the ankyrin repeat (Ank) family called AnkG interferes with the mammalian apoptosis pathway. AnkG was found to interact with the host protein gC1qR (p32). Either the addition of AnkG to the repertoire of L. pneumophila effector proteins or the silencing of p32 in mouse dendritic cells resulted in a gain of function that allowed intracellular replication of L. pneumophila in these normally restrictive mammalian host cells by preventing rapid pathogen-induced apoptosis. These data indicate that p32 regulates pathogen-induced apoptosis and that AnkG functions to block this pathway. Thus, emergence of an effector protein that interferes with a proapoptotic signaling pathway directed against intracellular bacteria correlates with adaptation of a pathogen to mammalian hosts.

 

The origins of eukaryotic-like proteins in Legionella pneumophila

Lurie-Weinberger MN, Gomez-Valero L, Merault N, Glöckner G, Buchrieser C, Gophna U.

Department of Molecular Microbiology and Biotechnology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, 69978, Israel. urigo@post.tau.ac.il

Int J Med Microbiol. 2010 Nov;300(7):470-81.

ABSTRACT: Legionella pneumophila, the causative agent of Legionnaires' disease, is known to be an intracellular pathogen of multiple species of protozoa and is assumed to have co-evolved with these organisms for millions of years. Genome sequencing of L. pneumophila strains has revealed an abundance of eukaryotic-like proteins (ELPs). Here, we study the evolution of these ELPs, in order to investigate their origin. Thirty-four new ELPs were identified, based on a higher similarity to eukaryotic proteins than to bacterial ones. Phylogenetic analyses demonstrated that both lateral gene transfer from eukaryotic hosts and bacterial genes that became eukaryotic-like by gradual adaptation to the intracellular milieu or gene fragment acquisition, contributed to the existing repertoire of ELPs, which comprise over 3% of the putative proteome of L. pneumophila strains. A PCR survey of 72 L. pneumophila strains showed that most ELPs were conserved in nearly all of these strains, indicating that they are likely to play important roles in this species. Genes of different evolutionary origin have distinct patterns of selection, as reflected by their ratio of a synonymous vs. synonymous mutations. One ELP is common to several strains of Legionella, but outside this genus has homologs only in Acanthamoeba polyphaga mimivirus, indicating that gene exchange involving eukaryotic viruses and intracellular bacterial pathogens may also contribute to the evolution of virulence in either or both of these groups of organisms. Information on selection patterns and eukaryotic-like status was combined as a novel approach to predict type IV secretion system effectors of Legionella, which represent promising targets for future study.

 

Legionella pneumophila strain 130b possesses a unique combination of type IV secretion systems and novel Dot/Icm secretion system effector proteins

Schroeder GN, Petty NK, Mousnier A, Harding CR, Vogrin AJ, Wee B, Fry NK, Harrison TG, Newton HJ, Thomson NR, Beatson SA, Dougan G, Hartland EL, Frankel G.

Centre for Molecular Microbiology and Infection, Division of Cell and Molecular Biology, Imperial College, London, United Kingdom. g.frankel@imperial.ac.uk

J Bacteriol. 2010 Nov;192(22):6001-16.

ABSTRACT: Legionella pneumophila is a ubiquitous inhabitant of environmental water reservoirs. The bacteria infect a wide variety of protozoa and, after accidental inhalation, human alveolar macrophages, which can lead to severe pneumonia. The capability to thrive in phagocytic hosts is dependent on the Dot/Icm type IV secretion system (T4SS), which translocates multiple effector proteins into the host cell. In this study, we determined the draft genome sequence of L. pneumophila strain 130b (Wadsworth). We found that the 130b genome encodes a unique set of T4SSs, namely, the Dot/Icm T4SS, a Trb-1-like T4SS, and two Lvh T4SS gene clusters. Sequence analysis substantiated that a core set of 107 Dot/Icm T4SS effectors was conserved among the sequenced L. pneumophila strains Philadelphia-1, Lens, Paris, Corby, Alcoy, and 130b. We also identified new effector candidates and validated the translocation of 10 novel Dot/Icm T4SS effectors that are not present in L. pneumophila strain Philadelphia-1. We examined the prevalence of the new effector genes among 87 environmental and clinical L. pneumophila isolates. Five of the new effectors were identified in 34 to 62% of the isolates, while less than 15% of the strains tested positive for the other five genes. Collectively, our data show that the core set of conserved Dot/Icm T4SS effector proteins is supplemented by a variable repertoire of accessory effectors that may partly account for differences in the virulences and prevalences of particular L. pneumophila strains.

 

The Legionella pneumophila F-box protein Lpp2082 (AnkB) modulates ubiquitination of the host protein parvin B and promotes intracellular replication

Lomma M, Dervins-Ravault D, Rolando M, Nora T, Newton HJ, Sansom FM, Sahr T, Gomez-Valero L, Jules M, Hartland EL, Buchrieser C.

Institut Pasteur, Biologie des Bactéries Intracellulaires, Departement Genomes et Génétique, F-75015 Paris, France. carmen.buchrieser@pasteur.fr

Cell Microbiol. 2010 Sep 1;12(9):1272-91.

ABSTRACT: The environmental pathogen Legionella pneumophila encodes three proteins containing F-box domains and additional protein-protein interaction domains, reminiscent of eukaryotic SCF ubiquitin-protein ligases. Here we show that the F-box proteins of L. pneumophila strain Paris are Dot/Icm effectors involved in the accumulation of ubiquitinated proteins associated with the Legionella-containing vacuole. Single, double and triple mutants of the F-box protein encoding genes were impaired in infection of Acanthamoeba castellanii, THP-1 macrophages and human lung epithelial cells. Lpp2082/AnkB was essential for infection of the lungs of A/J mice in vivo, and bound Skp1, the interaction partner of the SCF complex in mammalian cells, similar to AnkB from strain AA100/130b. Using a yeast two-hybrid screen and co-immunoprecipitation analysis we identified ParvB a protein present in focal adhesions and in lamellipodia, as a target. Immunofluorescence analysis confirmed that ectopically expressed Lpp2082/AnkB colocalized with ParvB at the periphery of lamellipodia. Unexpectedly, ubiquitination tests revealed that Lpp2082/AnkB diminishes endogenous ubiquitination of ParvB. Based on these results we propose that L. pneumophila modulates ubiquitination of ParvB by competing with eukaryotic E3 ligases for the specific protein-protein interaction site of ParvB, thereby revealing a new mechanism by which L. pneumophila may employ translocated effector proteins to promote bacterial survival.

 

E3 ubiquitin ligase activity and targeting of BAT3 by multiple Legionella pneumophila translocated substrates

Ensminger AW, Isberg RR.

Howard Hughes Medical Institute, Department of Molecular Biology and Microbiology, Tufts University School of Medicine, 150 Harrison Ave. J424, Boston, MA 02111, USA. Ralph.Isberg@tufts.edu

Infect Immun. 2010 Sep;78(9):3905-19.

ABSTRACT: The intracellular bacterial pathogen Legionella pneumophila modulates a number of host processes during intracellular growth, including the eukaryotic ubiquitination machinery, which dictates the stability, activity, and/or localization of a large number of proteins. A number of L. pneumophila proteins contain eukaryotic-like motifs typically associated with ubiquitination. Central among these is a family of five F-box-domain-containing proteins of Legionella pneumophila. Each of these five proteins is translocated to the host cytosol by the Dot/Icm type IV protein translocation system during infection. We show that three of these proteins, LegU1, LegAU13, and LicA, interact with components of the host ubiquitination machinery in vivo. In addition, LegU1 and LegAU13 are integrated into functional Skp-Cullin-F-box (SCF) complexes that confer E3 ubiquitin ligase activity. LegU1 specifically interacts with and can direct the ubiquitination of the host chaperone protein BAT3. In a screen for additional L. pneumophila proteins that associate with LegU1 in mammalian cells, we identified the bacterial protein Lpg2160. We demonstrate that Lpg2160 also associates with BAT3 independently of LegU1. We show that Lpg2160 is a translocated substrate of the Dot/Icm system and contains a C-terminal translocation signal. We propose a model in which LegU1 and Lpg2160 may function redundantly or in concert to modulate BAT3 activity during the course of infection.

 

The Legionella effector protein DrrA AMPylates the membrane traffic regulator Rab1b

Müller MP, Peters H, Blümer J, Blankenfeldt W, Goody RS, Itzen A.

Department of Physical Biochemistry, Max Planck Institute of Molecular Physiology, Dortmund, NRW, 44227, Germany. aymelt.itzen@mpi-dortmund.mpg.de

Science. 2010 Aug 20;329(5994):946-9.

ABSTRACT: In the course of Legionnaires' disease, the bacterium Legionella pneumophila affects the intracellular vesicular trafficking of infected eukaryotic cells by recruiting the small guanosine triphosphatase (GTPase) Rab1 to the cytosolic face of the Legionella-containing vacuole. In order to accomplish this, the Legionella protein DrrA contains a specific guanine nucleotide exchange activity for Rab1 activation that exchanges guanosine triphosphate (GTP) for guanosine diphosphate on Rab1. We found that the amino-terminal domain of DrrA possesses adenosine monophosphorylation (AMPylation) activity toward the switch II region of Rab1b, leading to posttranslational covalent modification of tyrosine 77. AMPylation of switch II by DrrA restricts the access of GTPase activating proteins, thereby rendering Rab1b constitutively active.

 

Exploitation of conserved eukaryotic host cell farnesylation machinery by an F-box effector of Legionella pneumophila

Price CT, Al-Quadan T, Santic M, Jones SC, Abu Kwaik Y.

Department of Microbiology and Immunology, School of Medicine and 2 Department of Biology, University of Louisville, Louisville, KY 40292, USA. abukwaik@louisville.edu

J Exp Med. 2010 Aug 2;207(8):1713-26.

ABSTRACT: Farnesylation involves covalent linkage of eukaryotic proteins to a lipid moiety to anchor them into membranes, which is essential for the biological function of Ras and other proteins. A large cadre of bacterial effectors is injected into host cells by intravacuolar pathogens through elaborate type III-VII translocation machineries, and many of these effectors are incorporated into the pathogen-containing vacuolar membrane by unknown mechanisms. The Dot/Icm type IV secretion system of Legionella pneumophila injects into host cells the F-box effector Ankyrin B (AnkB), which functions as platforms for the docking of polyubiquitinated proteins to the Legionella-containing vacuole (LCV) to enable intravacuolar proliferation in macrophages and amoeba. We show that farnesylation of AnkB is indispensable for its anchoring to the cytosolic face of the LCV membrane, for its biological function within macrophages and Dictyostelium discoideum, and for intrapulmonary proliferation in mice. Remarkably, the protein farnesyltransferase, RCE-1 (Ras-converting enzyme-1), and isoprenyl cysteine carboxyl methyltransferase host farnesylation enzymes are recruited to the LCV in a Dot/Icm-dependent manner and are essential for the biological function of AnkB. In conclusion, this study shows novel localized recruitment of the host farnesylation machinery and its anchoring of an F-box effector to the LCV membrane, and this is essential for biological function in vitro and in vivo.

 

 The role of fimV and the importance of its tandem repeat copy number in twitching motility, pigment production, and morphology in Legionella pneumophila

Coil DA, Anné J.

Laboratory of Bacteriology, Rega Institute for Medical Research, Katholieke Universiteit Leuven, Minderbroedersstraat 10, 3000 Leuven, Belgium. jozef.anne@rega.kuleuven.be

Arch Microbiol. 2010 Aug;192(8):625-31.

ABSTRACT: Twitching motility, a flagella-independent type of translocation of bacteria over moist surfaces, requires an array of proteins, including FimV. To investigate the role of this protein in twitching motility in Legionella pneumophila, we have generated a knockout mutant of fimV and characterized its phenotypic effects. In addition to a major reduction in twitching motility, deletion of the fimV gene caused a number of other phenotypic effects including decreased protective pigment formation, and it also affected cell morphology. Since fimV contains a variable number of tandem repeats, which can vary according to the origin of a given strain, we have examined the importance of this variability found within the coding region of this gene. By complementing the knockout strain with constructs containing a different number of this tandem repeat, we have been able to also show that repeat copy number is important in the functioning of this gene.

 

LnaB: a Legionella pneumophila activator of NF-kappaB

Losick VP, Haenssler E, Moy MY, Isberg RR.

Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, MA 02111, USA. Ralph.Isberg@tufts.edu

Cell Microbiol. 2010 Aug;12(8):1083-97.

ABSTRACT: Legionella pneumophila possesses a large arsenal of type IV translocated substrates. Over 100 such proteins have been identified, but the functions of most are unknown. Previous studies have demonstrated that L. pneumophila activates NF-kappaB, a master transcriptional regulator of the mammalian innate immune response. Activation of NF-kappaB is dependent on the Legionella Icm/Dot type IV protein translocation system, consistent with the possibility that translocated bacterial proteins contribute to this response. To test this hypothesis, an expression library of 159 known and putative translocated substrates was created to evaluate whether ectopic production of a single L. pneumophila protein could activate NF-kappaB in mammalian cells. Expression of two of these proteins, LnaB (Legionella NF-kappaB activator B) and LegK1, resulted in approximately 150-fold induction of NF-kappaB activity in HEK293T cells, levels similar to the strong induction that occurs with ectopic expression of the known activator Nod1. LnaB is a substrate of the Icm/Dot system, and in the absence of this protein, a partial reduction of NF-kappaB activation in host cells occurs after challenge by post-exponential phase bacteria. These data indicate that LnaB is an Icm/Dot substrate that contributes to NF-kappaB activation during L. pneumophila infection in host cells.

 

High-affinity binding of phosphatidylinositol 4-phosphate by Legionella pneumophila DrrA.

Schoebel S, Blankenfeldt W, Goody RS, Itzen A.

Department of Physical Biochemistry, Max Planck Institute of Molecular Physiology, Dortmund, North Rhine-Westphalia, Germany. aymelt.itzen@mpi-dortmund.mpg.de

EMBO Rep. 2010 Aug;11(8):598-604.

ABSTRACT: The DrrA protein of Legionella pneumophila is involved in mistargeting of endoplasmic reticulum-derived vesicles to Legionella-containing vacuoles through recruitment of the small GTPase Rab1. To this effect, DrrA binds specifically to phosphatidylinositol 4-phosphate (PtdIns(4)P) lipids on the cytosolic surface of the phagosomal membrane shortly after infection. In this study, we present the atomic structure of the PtdIns(4)P-binding domain of a protein (DrrA) from a human pathogen. A detailed kinetic investigation of its interaction with PtdIns(4)P reveals that DrrA binds to this phospholipid with, as yet unprecedented, high affinity, suggesting that DrrA can sense a very low abundance of the lipid.

 

Proteomic analysis of growth phase-dependent expression of Legionella pneumophila proteins which involves regulation of bacterial virulence traits

Hayashi T, Nakamichi M, Naitou H, Ohashi N, Imai Y, Miyake M.

Laboratory of Microbiology and Immunology, School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka, Japan. miyakem@ys7.u-shizuoka-ken.ac.jp

PLoS One. 2010 Jul 22;5(7):e11718.

ABSTRACT: Legionella pneumophila, which is a causative pathogen of Legionnaires' disease, expresses its virulent traits in response to growth conditions. In particular, it is known to become virulent at a post-exponential phase in vitro culture. In this study, we performed a proteomic analysis of differences in expression between the exponential phase and post-exponential phase to identify candidates associated with L. pneumophila virulence using 2-Dimentional Fluorescence Difference Gel Electrophoresis (2D-DIGE) combined with Matrix-Assisted Laser Desorption/Ionization-Mass Spectrometry (MALDI-TOF-MS). Of 68 identified proteins that significantly differed in expression between the two growth phases, 64 were up-regulated at a post-exponential phase. The up-regulated proteins included enzymes related to glycolysis, ketone body biogenesis and poly-3-hydroxybutyrate (PHB) biogenesis, suggesting that L. pneumophila may utilize sugars and lipids as energy sources, when amino acids become scarce. Proteins related to motility (flagella components and twitching motility-associated proteins) were also up-regulated, predicting that they enhance infectivity of the bacteria in host cells under certain conditions. Furthermore, 9 up-regulated proteins of unknown function were found. Two of them were identified as novel bacterial factors associated with hemolysis of sheep red blood cells (SRBCs). Another 2 were found to be translocated into macrophages via the Icm/Dot type IV secretion apparatus as effector candidates in a reporter assay with Bordetella pertussis adenylate cyclase. The study will be helpful for virulent analysis of L. pneumophila from the viewpoint of physiological or metabolic modulation dependent on growth phase.

 

Isotopologue profiling of Legionella pneumophila: role of serine and glucose as carbon substrates

Eylert E, Herrmann V, Jules M, Gillmaier N, Lautner M, Buchrieser C, Eisenreich W, Heuner K.

Lehrstuhl für Biochemie, Technische Universität München, Lichtenbergstrasse 4, 85747 Garching, Germany. heunerk@rki.de

J Biol Chem. 2010 Jul 16;285(29):22232-43.

ABSTRACT: Legionella pneumophila (Lp) is commonly found in freshwater habitats but is also the causative agent of Legionnaires' disease when infecting humans. Although various virulence factors have been reported, little is known about the nutrition and the metabolism of the bacterium. Here, we report the application of isotopologue profiling for analyzing the metabolism of L. pneumophila. Cultures of Lp were supplied with [U-(13)C(3)]serine, [U-(13)C(6)]glucose, or [1,2-(13)C(2)]glucose. After growth, (13)C enrichments and isotopologue patterns of protein-derived amino acids and poly-3-hydroxybutyrate were determined by mass spectrometry and/or NMR spectroscopy. The labeling patterns detected in the experiment with [U-(13)C(3)]serine showed major carbon flux from serine to pyruvate and from pyruvate to acetyl-CoA, which serves as a precursor of poly-3-hydroxybutyrate or as a substrate of a complete citrate cycle with Si specificity of the citrate synthase. Minor carbon flux was observed between pyruvate and oxaloacetate/malate by carboxylation and decarboxylation, respectively. The apparent lack of label in Val, Ile, Leu, Pro, Phe, Met, Arg, and Tyr confirmed that L. pneumophila is auxotrophic for these amino acids. Experiments with [(13)C]glucose showed that the carbohydrate is also used as a substrate to feed the central metabolism. The specific labeling patterns due to [1,2-(13)C(2)]glucose identified the Entner-Doudoroff pathway as the predominant route for glucose utilization. In line with these observations, a mutant lacking glucose-6-phosphate dehydrogenase (Delta zwf) did not incorporate label from glucose at significant levels and was slowly outcompeted by the wild type strain in successive rounds of infection in Acanthamoeba castellanii, indicating the importance of this enzyme and of carbohydrate usage in general for the life cycle of Lp.

 

Immunology taught by bacteria

Vance RE.

Division of Immunology & Pathogenesis, Department of Molecular & Cell Biology, University of California, Berkeley, 415 Life Science Addition, Berkeley, CA, 94720, USA.  rvance@berkeley.edu

J Clin Immunol. 2010 Jul;30(4):507-11.

ABSTRACT: INTRODUCTION: It has been proposed that the innate immune system might discriminate living and virulent pathogens from dead or harmless microbes, but the molecular mechanisms by which this discrimination could occur remain unclear. Although studies of model antigens and adjuvants have illuminated important principles underlying immune responses, the specific immune responses made to living, virulent pathogens can only be discovered by studies of the living, virulent pathogens themselves. METHODS AND FINDINGS: Here, I review what one particular bacterium, Legionella pneumophila, has taught us about the innate immune response. Pathogens differ greatly in the mechanisms they use to invade, replicate within, and transmit among their hosts. However, a theme that emerges is that the pathogenic activities sensed by host cells are conserved among multiple pathogenic bacteria. CONCLUSION: Thus, immunology taught by L. pneumophila may lead to a more general understanding of the host response to infection.

 

Bacterial gene regulation by alpha-hydroxyketone signaling

Tiaden A, Spirig T, Hilbi H.

Institute of Molecular Life Sciences, University of Zürich, Winterthurerstrasse 190, 8057 Zürich, Switzerland. hubert.hilbi@imls.uzh.ch

Trends Microbiol. 2010 Jul;18(7):288-97.

ABSTRACT: Bacteria produce diffusible, small signaling molecules termed autoinducers to promote cell-cell communication. Recently, a novel class of signaling molecules, the alpha-hydroxyketones (AHKs), was discovered in the facultative human pathogens Legionella pneumophila and Vibrio cholerae. In this review, we summarize and compare findings on AHK signaling in these bacteria. The L. pneumophila lqs (Legionella quorum sensing) and V. cholerae cqs (cholera quorum sensing) gene clusters synthesize and detect Legionella autoinducer 1 (3-hydroxypentadecan-4-one) or cholera autoinducer-1 (3-hydroxytridecan-4-one), respectively. In addition to the autoinducer synthase and cognate sensor kinase encoded in the cqs locus, the lqs cluster also harbors a prototypic response regulator. AHK signaling regulates pathogen-host cell interactions, bacterial virulence, formation of biofilms or extracellular filaments, and expression of a genomic island. The lqs/cqs gene cluster is present in several environmental bacteria, suggesting that AHKs are widely used for cell-cell signaling.

 

Bacterial proteases from the intracellular vacuole niche; protease conservation and adaptation for pathogenic advantage

Huston WM.

Institute of Health and Biomedical Innovation and School of Life Sciences, Queensland University of Technology, Brisbane, QLD, Australia. w.huston@qut.edu.au

FEMS Immunol Med Microbiol. 2010 Jun 1;59(1):1-10.

ABSTRACT: Proteases with important roles for bacterial pathogens that specifically reside within intracellular vacuoles are frequently homologous to those that have important virulence functions for other bacteria. Research has identified that some of these conserved proteases have evolved specialized functions for intracellular vacuole-residing bacteria. Unique proteases with pathogenic functions have also been described from Chlamydia, Mycobacteria, and Legionella. These findings suggest that there are further novel functions for proteases from these bacteria that remain to be described. This review summarizes the recent findings of novel protease functions from the intracellular human pathogenic bacteria that reside exclusively in vacuoles.

 

Cooperation between multiple microbial pattern recognition systems is important for host protection against the intracellular pathogen Legionella pneumophila

Archer KA, Ader F, Kobayashi KS, Flavell RA, Roy CR.

Section of Microbial Pathogenesis, Yale University School of Medicine, Boyer Center for Molecular Medicine, 295 Congress Avenue, New Haven, CT 06536, USA. craig.roy@yale.edu

Infect Immun. 2010 Jun;78(6):2477-87.

ABSTRACT: Multiple pattern recognition systems have been shown to initiate innate immune responses to microbial pathogens. The degree to which these detection systems cooperate with each other to provide host protection is unknown. Here, we investigated the importance of several immune surveillance pathways in protecting mice against lethal infection by the intracellular pathogen Legionella pneumophila, the causative agent of a severe pneumonia called Legionnaires' disease. Rip2 and Naip5/NLRC4 signaling was found to contribute to the innate immune response generated against L. pneumophila in the lung. Elimination of Rip2 or Naip5/NLRC4 signaling in MyD88-deficient mice resulted in increased replication and dissemination of L. pneumophila and higher rates of mortality. Irradiated wild-type mice receiving bone marrow cells from pattern recognition receptor-deficient mice displayed L. pneumophila infection phenotypes similar to those of donor mice. Rip2 and Naip5/NLRC4 signaling provided additive effects in protecting MyD88-deficient mice from lethal infection by L. pneumophila, with the contribution of Naip5/NLRC4 being slightly greater than that of Rip2. Thus, activation of the Rip2, MyD88, and Naip5/NLRC4 signaling pathways triggers a coordinated and synergistic response that protects the host against lethal infection by L. pneumophila. These data provide new insight into how different pattern recognition systems interact functionally to generate innate immune responses that protect the host from lethal infection by activating cellular pathways that restrict intracellular replication of L. pneumophila and by recruiting to the site of infection additional phagocytes that eliminate extracellular bacteria.

 

The Legionella pneumophila LetA/LetS two-component system exhibits rheostat-like behavior

Edwards RL, Jules M, Sahr T, Buchrieser C, Swanson MS.

University of Michigan Medical School, Ann Arbor, Michigan, USA. mswanson@umich.edu

Infect Immun. 2010 Jun;78(6):2571-83.

ABSTRACT: When confronted with metabolic stress, replicative Legionella pneumophila bacteria convert to resilient, infectious cells equipped for transmission. Differentiation is promoted by the LetA/LetS two-component system, which belongs to a family of signal-transducing proteins that employ a four-step phosphorelay to regulate gene expression. Histidine 307 of LetS was essential to switch on the transmission profile, but a threonine substitution at position 311 (T311M) suggested a rheostat-like function. The letS(T311M) bacteria resembled the wild type (WT) for some traits and letS null mutants for others, whereas they displayed intermediate levels of infectivity, cytotoxicity, and lysosome evasion. Although only 30 to 50% of letS(T311M) mutants became motile, flow cytometry determined that every cell eventually activated the flagellin promoter to WT levels, but expression was delayed. Likewise, letS(T311M) mutants exhibited delayed induction of RsmY and RsmZ, regulatory RNAs that relieve CsrA repression of transmission traits. Transcriptional profile analysis revealed that letS(T311M) mutants expressed the flagellar regulon and multiple other transmissive-phase loci at a higher cell density than the WT. Accordingly, we postulate that the letS(T311M) mutant may relay phosphate less efficiently than the WT LetS sensor protein, leading to sluggish gene expression and a variety of phenotypic profiles. Thus, as first described for BvgA/BvgS, rather than acting as on/off switches, this family of two-component systems exhibit rheostat activity that likely confers versatility as microbes adapt to fluctuating environments.

 

Nlrc4/Ipaf/CLAN/CARD12: more than a flagellin sensor

Abdelaziz DH, Amr K, Amer AO.

Division of Pulmonary, Allergy, Critical Care and Sleep Medicine, Center for Microbial Interface Biology and The Department of Internal Medicine, Ohio State University, Columbus, OH 43210, United States. amal.amer@osumc.edu

Int J Biochem Cell Biol. 2010 Jun;42(6):789-91.

ABSTRACT: Nlrc4 is a member of the Nod-like receptors (NLRs), a family of cytosolic receptors involved in sensing bacterial molecules. NLRs are a group of proteins containing spans of leucine-rich repeats that senses bacterial factors within the eukaryotic cytosol. The recognition of bacterial factors provokes the formation of the inflammasome complex which includes specific NLRs. The inflammasome is responsible for caspase-1 activation which leads to the cleavage and maturation of inflammatory cytokines such as IL-1beta and IL-18. Nlrc4 was considered to be a devoted flagellin sensor in eukaryotic cells. However, studies using a variety of pathogens such as Salmonella, Legionella, Shigella and Pseudomonas at high bacterial burdens revealed that Nlrc4 can mediate caspase-1 activation independent of bacterial flagellin. On the other hand, new reports showed that Nlrc4 can restrict bacterial infection independently of caspase-1. Therefore, Nlrc4 maybe involved in sensing more than one bacterial molecule and may participate in several immune complexes. Published by Elsevier Ltd.

 

Phospholipase PlaB is a new virulence factor of Legionella pneumophila

Schunder E, Adam P, Higa F, Remer KA, Lorenz U, Bender J, Schulz T, Flieger A, Steinert M, Heuner K.

Robert Koch-Institut, Research Group P26, Nosocomial Infections of the Elderly, Nordufer 20, 13353 Berlin, Germany. heunerk@rki.de

Int J Med Microbiol. 2010 Jun;300(5):313-23.

ABSTRACT: We previously identified Legionella pneumophila PlaB as the major cell-associated phospholipase A/lysophospholipase A with contact-dependent hemolytic activity. In this study, we further characterized this protein and found it to be involved in the virulence of L. pneumophila. PlaB was mainly expressed and active during exponential growth. Active PlaB was outer membrane-associated and at least in parts surface-exposed. Transport to the outer membrane was not dependent on the type I (T1SS), II (T2SS), IVB (T4BSS) or Tat secretion pathways. Furthermore, PlaB activity was not dependent on the presence of the macrophage infectivity potentiator (Mip) or the major secreted zinc metalloproteinase A (MspA). Despite the fact that PlaB is not essential for replication in protozoa or macrophage cell lines, we found that plaB mutants were impaired for replication in the lungs and dissemination to the spleen in the guinea pig infection model. Histological sections monitored less inflammation and destruction of the lung tissue after infection with the plaB mutants compared to L. pneumophila wild type. Taken together, PlaB is the first phospholipase A/lysophospholipase A with a confirmed role in the establishment of Legionnaires' disease. Copyright 2010 Elsevier GmbH. All rights reserved.

 

Glucose metabolism in Legionella pneumophila: dependence on the Entner-Doudoroff pathway and connection with intracellular bacterial growth

Harada E, Iida K, Shiota S, Nakayama H, Yoshida S.

Department of Bacteriology, Faculty of Medical Sciences, Kyushu University, Higashi-ku, Fukuoka 812-8582, Japan. harada-e@kokyu.med.kyushu-u.ac.jp

J Bacteriol. 2010 Jun;192(11):2892-9.

ABSTRACT: Glucose metabolism in Legionella pneumophila was studied by focusing on the Entner-Doudoroff (ED) pathway with a combined genetic and biochemical approach. The bacterium utilized exogenous glucose for synthesis of acid-insoluble cell components but manifested no discernible increase in the growth rate. Assays with permeabilized cell preparations revealed the activities of three enzymes involved in the pathway, i.e., glucokinase, phosphogluconate dehydratase, and 2-dehydro-3-deoxy-phosphogluconate aldolase, presumed to be encoded by the glk, edd, and eda genes, respectively. Gene-disrupted mutants for the three genes and the ywtG gene encoding a putative sugar transporter were devoid of the ability to metabolize exogenous glucose, indicating that the pathway is almost exclusively responsible for glucose metabolism and that the ywtG gene product is the glucose transporter. It was also established that these four genes formed part of an operon in which the gene order was edd-glk-eda-ywtG, as predicted by genomic information. Intriguingly, while the mutants exhibited no appreciable change in growth characteristics in vitro, they were defective in multiplication within eukaryotic cells, strongly indicating that the ED pathway must be functional for the intracellular growth of the bacterium to occur. Curiously, while the deficient glucose metabolism of the ywtG mutant was successfully complemented by the ywtG(+) gene supplied in trans via plasmid, its defect in intracellular growth was not. However, the latter defect was also manifested in wild-type cells when a plasmid carrying the mutant ywtG gene was introduced. This phenomenon, resembling so-called dominant negativity, awaits further investigation.

 

Cutting edge: Pulmonary Legionella pneumophila is controlled by plasmacytoid dendritic cells but not type I IFN

Ang DK, Oates CV, Schuelein R, Kelly M, Sansom FM, Bourges D, Boon L, Hertzog PJ, Hartland EL, van Driel IR.

Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, Australia. ivd@unimelb.edu.au

J Immunol. 2010 May 15;184(10):5429-33.

ABSTRACT: Plasmacytoid dendritic cells (pDCs) are well known as the major cell type that secretes type I IFN in response to viral infections. Their role in combating other classes of infectious organisms, including bacteria, and their mechanisms of action are poorly understood. We have found that pDCs play a significant role in the acute response to the intracellular bacterial pathogen Legionella pneumophila. pDCs were rapidly recruited to the lungs of L. pneumophila-infected mice, and depletion of pDCs resulted in increased bacterial load. The ability of pDCs to combat infection did not require type I IFN. This study points to an unappreciated role for pDCs in combating bacterial infections and indicates a novel mechanism of action for this cell type.

 

The autoinducer synthase LqsA and putative sensor kinase LqsS regulate phagocyte interactions, extracellular filaments and a genomic island of Legionella pneumophila

Tiaden A, Spirig T, Sahr T, Wälti MA, Boucke K, Buchrieser C, Hilbi H.

Institute of Molecular Life Sciences, University of Zürich, Zürich, Switzerland. hubert.hilbi@imls.uzh.ch

Environ Microbiol. 2010 May;12(5):1243-59.

ABSTRACT: The amoebae-resistant opportunistic pathogen Legionella pneumophila employs a biphasic life cycle to replicate in host cells and spread to new niches. Upon entering the stationary growth phase, the bacteria switch to a transmissive (virulent) state, which involves a complex regulatory network including the lqs gene cluster (lqsA-lqsR-hdeD-lqsS). LqsR is a putative response regulator that promotes host-pathogen interactions and represses replication. The autoinducer synthase LqsA catalyses the production of the diffusible signalling molecule 3-hydroxypentadecan-4-one (LAI-1) that is presumably recognized by the sensor kinase LqsS. Here, we analysed L. pneumophila strains lacking lqsA or lqsS. Compared with wild-type L. pneumophila, the DeltalqsS strain was more salt-resistant and impaired for the Icm/Dot type IV secretion system-dependent uptake by phagocytes. Legionella pneumophila strains lacking lqsS, lqsR or the alternative sigma factor rpoS sedimented more slowly and produced extracellular filaments. Deletion of lqsA moderately reduced the uptake of L. pneumophila by phagocytes, and the defect was complemented by expressing lqsA in trans. Unexpectedly, the overexpression of lqsA also restored the virulence defect and reduced filament production of L. pneumophila mutant strains lacking lqsS or lqsR, but not the phenotypes of strains lacking rpoS or icmT. These results suggest that LqsA products also signal through sensors not encoded by the lqs gene cluster. A transcriptome analysis of the DeltalqsA and DeltalqsS mutant strains revealed that under the conditions tested, lqsA regulated only few genes, whereas lqsS upregulated the expression of 93 genes at least twofold. These include 52 genes clustered in a 133 kb high plasticity genomic island, which is flanked by putative DNA-mobilizing genes and encodes multiple metal ion efflux pumps. Upon overexpression of lqsA, a cluster of 19 genes in the genomic island was also upregulated, suggesting that LqsA and LqsS participate in the same regulatory circuit.

  

The autoinducer synthase LqsA and putative sensor kinase LqsS regulate phagocyte interactions, extracellular filaments and a genomic island of Legionella pneumophila

Tiaden A, Spirig T, Sahr T, Wälti MA, Boucke K, Buchrieser C, Hilbi H.

Institute of Molecular Life Sciences, University of Zürich, Zürich, Switzerland. hubert.hilbi@imls.uzh.ch

Environ Microbiol. 2010 May;12(5):1243-59.

ABSTRACT: The amoebae-resistant opportunistic pathogen Legionella pneumophila employs a biphasic life cycle to replicate in host cells and spread to new niches. Upon entering the stationary growth phase, the bacteria switch to a transmissive (virulent) state, which involves a complex regulatory network including the lqs gene cluster (lqsA-lqsR-hdeD-lqsS). LqsR is a putative response regulator that promotes host-pathogen interactions and represses replication. The autoinducer synthase LqsA catalyses the production of the diffusible signalling molecule 3-hydroxypentadecan-4-one (LAI-1) that is presumably recognized by the sensor kinase LqsS. Here, we analysed L. pneumophila strains lacking lqsA or lqsS. Compared with wild-type L. pneumophila, the DeltalqsS strain was more salt-resistant and impaired for the Icm/Dot type IV secretion system-dependent uptake by phagocytes. Legionella pneumophila strains lacking lqsS, lqsR or the alternative sigma factor rpoS sedimented more slowly and produced extracellular filaments. Deletion of lqsA moderately reduced the uptake of L. pneumophila by phagocytes, and the defect was complemented by expressing lqsA in trans. Unexpectedly, the overexpression of lqsA also restored the virulence defect and reduced filament production of L. pneumophila mutant strains lacking lqsS or lqsR, but not the phenotypes of strains lacking rpoS or icmT. These results suggest that LqsA products also signal through sensors not encoded by the lqs gene cluster. A transcriptome analysis of the DeltalqsA and DeltalqsS mutant strains revealed that under the conditions tested, lqsA regulated only few genes, whereas lqsS upregulated the expression of 93 genes at least twofold. These include 52 genes clustered in a 133 kb high plasticity genomic island, which is flanked by putative DNA-mobilizing genes and encodes multiple metal ion efflux pumps. Upon overexpression of lqsA, a cluster of 19 genes in the genomic island was also upregulated, suggesting that LqsA and LqsS participate in the same regulatory circuit.

 

Indispensable role for the eukaryotic-like ankyrin domains of the ankyrin B effector of Legionella pneumophila within macrophages and amoebae

Price CT, Al-Khodor S, Al-Quadan T, Abu Kwaik Y.

Department of Microbiology and Immunology, College of Medicine, University of Louisville, Louisville, KY 40202, USA. abukwaik@louisville.edu

Infect Immun. 2010 May;78(5):2079-88.

ABSTRACT: The Dot/Icm-translocated ankyrin B (AnkB) effector of Legionella pneumophila exhibits molecular mimicry of eukaryotic F-box proteins and is essential for intracellular replication in macrophages and protozoa. In addition to two eukaryotic-like ankyrin (ANK) domains, AnkB harbors a conserved eukaryotic F-box domain, which is involved in polyubiquitination of proteins throughout the eukaryotic kingdom. We have recently shown that the F-box domain of the AnkB effector is essential for decoration of the Legionella-containing vacuole (LCV) with polyubiquitinated proteins within macrophages and protozoan hosts. To decipher the role of the two ANK domains in the function of AnkB, we have constructed in-frame deletion of either or both of the ANK domain-encoding regions (ankB Delta A1, ankB Delta A2, and ankB Delta A1A2) to trans-complement the ankB null mutant. Deletion of the ANK domains results in defects in intracellular proliferation and decoration of the LCV with polyubiquitinated proteins. Export of the truncated variants of AnkB was reduced, and this may account for the observed defects. However, while full-length AnkB ectopically expressed in mammalian cells trans-rescues the ankB null mutant for intracellular proliferation, ectopic expression of AnkB Delta A1, AnkB Delta A2, and AnkB Delta A1A2 fails to trans-rescue the ankB null mutant. Importantly, ectopically expressed full-length AnkB is targeted to the host cell plasma membrane, where it recruits polyubiquitinated proteins. In contrast, AnkB Delta A1, AnkB Delta A2, and AnkB Delta A1A2 are diffusely distributed throughout the cytosol and fail to recruit polyubiquitinated proteins. We conclude that the two eukaryotic-like ANK domains of AnkB are essential for intracellular proliferation, for targeting AnkB to the host membranes, and for decoration of the LCV with polyubiquitinated proteins.

 

Legionella pneumophila promotes functional interactions between plasma membrane syntaxins and Sec22b

Arasaki K, Roy CR.

Section of Microbial Pathogenesis, Yale University School of Medicine, Boyer Center for Molecular Medicine, New Haven, CT 06536, USA. craig.roy@yale.edu

Traffic. 2010 May;11(5):587-600.

ABSTRACT: Biogenesis of a specialized organelle that supports intracellular replication of Legionella pneumophila involves the fusion of secretory vesicles exiting the endoplasmic reticulum (ER) with phagosomes containing this bacterial pathogen. Here, we investigated host plasma membrane SNARE proteins to determine whether they play a role in trafficking of vacuoles containing L. pneumophila. Depletion of plasma membrane syntaxins by RNA interference resulted in delayed acquisition of the resident ER protein calnexin and enhanced retention of Rab1 on phagosomes containing virulent L. pneumophila, suggesting that these SNARE proteins are involved in vacuole biogenesis. Plasma membrane-localized SNARE proteins syntaxin 2, syntaxin 3, syntaxin 4 and SNAP23 localized to vacuoles containing L. pneumophila. The ER-localized SNARE protein Sec22b was found to interact with plasma membrane SNAREs on vacuoles containing virulent L. pneumophila, but not on vacuoles containing avirulent mutants of L. pneumophila. The addition of alpha-SNAP and N-ethylmaleimide-sensitive factor (NSF) to the plasma membrane SNARE complexes formed by virulent L. pneumophila resulted in the dissociation of Sec22b, indicating functional pairing between these SNAREs. Thus, L. pneumophila stimulates the non-canonical pairing of plasma membrane t-SNAREs with the v-SNARE Sec22b to promote fusion of the phagosome with ER-derived vesicles. The mechanism by which L. pneumophila promotes pairing of plasma membrane syntaxins and Sec22b could provide unique insight into how the secretory vesicles could provide an additional membrane reserve subverted during phagosome maturation.

 

Legionella pneumophila 6S RNA optimizes intracellular multiplication

Faucher SP, Friedlander G, Livny J, Margalit H, Shuman HA.

Department of Microbiology and Immunology, Columbia University Medical Center, New York, NY 10032, USA. sebastien.faucher@gmail.com

Proc Natl Acad Sci U S A. 2010 Apr 20;107(16):7533-8.

ABSTRACT: Legionella pneumophila is a Gram-negative opportunistic human pathogen that infects and multiplies in a broad range of phagocytic protozoan and mammalian phagocytes. Based on the observation that small regulatory RNAs (sRNAs) play an important role in controlling virulence-related genes in several pathogenic bacteria, we attempted to identify sRNAs expressed by L. pneumophila. We used computational prediction followed by experimental verification to identify and characterize sRNAs encoded in the L. pneumophila genome. A 50-mer probe microarray was constructed to test the expression of predicted sRNAs in bacteria grown under a variety of conditions. This strategy successfully identified 22 expressed RNAs, out of which 6 were confirmed by northern blot and RACE. One of the identified sRNAs is highly expressed in postexponential phase, and computational prediction of its secondary structure reveals a striking similarity to the structure of 6S RNA, a widely distributed prokaryotic sRNA, known to regulate the activity of sigma(70)-containing RNA polymerase. A 70-mer probe microarray was used to identify genes affected by L. pneumophila 6S RNA in stationary phase. The 6S RNA positively regulates expression of genes encoding type IVB secretion system effectors, stress response genes such as groES and recA, as well as many genes involved in acquisition of nutrients and genes with unknown or hypothetical functions. Deletion of 6S RNA significantly reduced L. pneumophila intracellular multiplication in both protist and mammalian host cells, but had no detectable effect on growth in rich media.

 

Molecular pathogenesis of infections caused by Legionella pneumophila

Newton HJ, Ang DK, van Driel IR, Hartland EL.

Department of Microbiology and Immunology, University of Melbourne, Grattan Street, Parkville, Victoria 3010, Australia. hartland@unimelb.edu.au

Clin Microbiol Rev. 2010 Apr;23(2):274-98.

ABSTRACT: The genus Legionella contains more than 50 species, of which at least 24 have been associated with human infection. The best-characterized member of the genus, Legionella pneumophila, is the major causative agent of Legionnaires' disease, a severe form of acute pneumonia. L. pneumophila is an intracellular pathogen, and as part of its pathogenesis, the bacteria avoid phagolysosome fusion and replicate within alveolar macrophages and epithelial cells in a vacuole that exhibits many characteristics of the endoplasmic reticulum (ER). The formation of the unusual L. pneumophila vacuole is a feature of its interaction with the host, yet the mechanisms by which the bacteria avoid classical endosome fusion and recruit markers of the ER are incompletely understood. Here we review the factors that contribute to the ability of L. pneumophila to infect and replicate in human cells and amoebae with an emphasis on proteins that are secreted by the bacteria into the Legionella vacuole and/or the host cell. Many of these factors undermine eukaryotic trafficking and signaling pathways by acting as functional and, in some cases, structural mimics of eukaryotic proteins. We discuss the consequences of this mimicry for the biology of the infected cell and also for immune responses to L. pneumophila infection.

 

Distinct roles of ppGpp and DksA in Legionella pneumophila differentiation

Dalebroux ZD, Yagi BF, Sahr T, Buchrieser C, Swanson MS.

Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI, USA. mswanson@umich.edu

Mol Microbiol. 2010 Apr;76(1):200-19.

ABSTRACT: To transit between hosts, intracellular Legionella pneumophila transform into a motile, infectious, transmissive state. Here we exploit the pathogen's life cycle to examine how guanosine tetraphosphate (ppGpp) and DksA cooperate to govern bacterial differentiation. Transcriptional profiling revealed that during transmission alarmone accumulation increases the mRNA for flagellar and Type IV-secretion components, secreted host effectors and regulators, and decreases transcripts for translation, membrane modification and ATP synthesis machinery. DksA is critical for differentiation, since mutants are defective for stationary phase survival, flagellar gene activation, lysosome avoidance and macrophage cytotoxicity. The roles of ppGpp and DksA depend on the context. For macrophage transmission, ppGpp is essential, whereas DksA is dispensable, indicating that ppGpp can act autonomously. In broth, DksA promotes differentiation when ppGpp levels increase, or during fatty acid stress, as judged by flaA expression and evasion of degradation by macrophages. For flagella morphogenesis, DksA is required for basal fliA (sigma(28)) promoter activity. When alarmone levels increase, DksA cooperates with ppGpp to generate a pulse of Class II rod RNA or to amplify the Class III sigma factor and Class IV flagellin RNAs. Thus, DksA responds to the level of ppGpp and other stress signals to co-ordinate L. pneumophila differentiation.

 

Modulation of caspases and their non-apoptotic functions by Legionella pneumophila

Amer AO.

Department of Internal Medicine, Division of Pulmonary, Allergy, Critical Care and Sleep Medicine and the Center for Microbial Interface Biology, Ohio State University, Columbus, OH 43210, USA. amal.amer@osumc.edu

Cell Microbiol. 2010 Feb;12(2):140-7.

ABSTRACT: Legionella pneumophila has become a model system to decipher the non-apoptotic functions of caspases and their role in immunity. In permissive cells, the L. pneumophila-containing vacuole evades endosomal traffic and is remodelled by the endoplasmic reticulum. Evasion of the endosomes is mediated by the Dot/Icm type IV secretion system. Upon L. pneumophila infection of genetically restrictive cells such as wild-type (WT) C57Bl/6J murine macrophages, flagellin is sensed by the NOD-like receptor Nlrc4 leading to caspase-1 activation by the inflammasome complex. Then, caspase-7 is activated downstream of the Nlrc4 inflammasome, promoting non-apoptotic functions such as L. pneumophila-containing phagosome maturation and bacterial degradation. Interestingly, caspase-3 is activated in permissive cells during early stages of infection. However, caspase-3 activation does not lead to apoptosis until late stages of infection because it is associated with potent Dot/Icm-mediated anti-apoptotic stimuli that render the infected cells resistant to external apoptotic inducers. Therefore, the role of caspase-1 and non-apoptotic functions of executioner caspases are temporally and spatially modulated during infection by L. pneumophila, which determine permissiveness to intracellular bacterial proliferation. This review will examine the novel activation pathways of caspases by L. pneumophila and discuss their role in genetic restriction and permissiveness to infection.

 

Mouse macrophages are permissive to motile Legionella species that fail to trigger pyroptosis

Whitfield NN, Byrne BG, Swanson MS.

Cellular and Molecular Biology Program, University of Michigan Medical School, Ann Arbor, Michigan, USA. mswanson@umich.edu

Infect Immun. 2010 Jan;78(1):423-32.

ABSTRACT: Legionella pneumophila, a motile opportunistic pathogen of humans, is restricted from replicating in the lungs of C57BL/6 mice. Resistance of mouse macrophages to L. pneumophila depends on recognition of cytosolic flagellin. Once detected by the NOD-like receptors Naip5 and Ipaf (Nlrc4), flagellin triggers pyroptosis, a proinflammatory cell death. In contrast, motile strains of L. parisiensis and L. tucsonensis replicate profusely within C57BL/6 macrophages, similar to flagellin-deficient L. pneumophila. To gain insight into how motile species escape innate defense mechanisms of mice, we compared their impacts on macrophages. L. parisiensis and L. tucsonensis do not induce proinflammatory cell death, as measured by lactate dehydrogenase (LDH) release and interleukin-1beta (IL-1beta) secretion. However, flagellin isolated from L. parisiensis and L. tucsonensis triggers cell death and IL-1beta secretion when transfected into the cytosol of macrophages. Neither strain displays three characteristics of the canonical L. pneumophila Dot/Icm type IV secretion system: sodium sensitivity, LAMP-1 evasion, and pore formation. Therefore, we postulate that when L. parisiensis and L. tucsonensis invade a mouse macrophage, flagellin is confined to the phagosome, protecting the bacteria from recognition by the cytosolic surveillance system and allowing Legionella to replicate. Despite their superior capacity to multiply in mouse macrophages, L. parisiensis and L. tucsonensis have been associated with only two cases of disease, both in renal transplant patients. These results point to the complexity of disease, a product of the pathogenic potential of the microbe, as defined in the laboratory, and the capacity of the host to mount a measured defense.

 

Mediators of lipid A modification, RNA degradation, and central intermediary metabolism facilitate the growth of Legionella pneumophila at low temperatures

Söderberg MA, Cianciotto NP.

Department of Microbiology and Immunology, Northwestern University Medical School, Chicago, IL 60611, USA. n-cianciotto@northwestern.edu

Curr Microbiol. 2010 Jan;60(1):59-65.

ABSTRACT: Legionella pneumophila is an aquatic bacterium that is also the agent of Legionnaires' disease pneumonia. Since L. pneumophila is transmitted directly from the environment to the lung, it is important to understand how legionellae survive at low temperatures. To identify genes that are needed for L. pneumophila growth at low temperature, we screened a population of mutagenized legionellae for strains that are specifically impaired for growth at 17 degrees C. From the 7,400 mutants tested, 11 displayed defects ranging from ca. 10-fold to a complete inability to grow at the low temperature. PCR and sequence analysis were then utilized to identify the genes whose loss had compromised growth. The proteins thereby implicated in low-temperature growth included components of the type II secretion system (LspE, LspG, LspH), a lipid A biosynthetic enzyme (LpxP), a ribonuclease (RNAse R), an RNA helicase (CsdA/DeaD), TCA cycle enzymes (citrate synthase), enzymes linked to fatty acid (FadB) or amino acid (aspartate aminotransferase) catabolism, and two putative membrane proteins that were, based upon their sequences, unlike previously characterized proteins. Given the magnitude of their mutant's defect, the aspartate aminotransferase, RNA helicase, and one of the putative membrane proteins were the factors most critical for L. pneumophila low-temperature growth. Thus, L. pneumophila not only employs some of the same processes and factors as other bacteria do in order to survive at low temperatures (e.g., LpxP, CsdA), but it also appears to possess novel modes of cold adaptation.

 

Control of flagellar gene regulation in Legionella pneumophila and its relation to growth phase

Albert-Weissenberger C, Sahr T, Sismeiro O, Hacker J, Heuner K, Buchrieser C.

Institut Pasteur, Biologie des Bactéries Intracellulaires, 75724 Paris Cedex 15, France. cbuch@pasteur.fr

J Bacteriol. 2010 Jan;192(2):446-55.

ABSTRACT: The bacterial pathogen Legionella pneumophila responds to environmental changes by differentiation. At least two forms are well described: replicative bacteria are avirulent; in contrast, transmissive bacteria express virulence traits and flagella. Phenotypic analysis, Western blotting, and electron microscopy of mutants of the regulatory genes encoding RpoN, FleQ, FleR, and FliA demonstrated that flagellin expression is strongly repressed and that the mutants are nonflagellated in the transmissive phase. Transcriptome analyses elucidated that RpoN, together with FleQ, enhances transcription of 14 out of 31 flagellar class II genes, which code for the basal body, hook, and regulatory proteins. Unexpectedly, FleQ independent of RpoN enhances the transcription of fliA encoding sigma 28. Expression analysis of a fliA mutant showed that FliA activates three out of the five remaining flagellar class III genes and the flagellar class IV genes. Surprisingly, FleR does not induce but inhibits expression of at least 14 flagellar class III genes on the transcriptional level. Thus, we propose that flagellar class II genes are controlled by FleQ and RpoN, whereas the transcription of the class III gene fliA is controlled in a FleQ-dependent but RpoN-independent manner. However, RpoN and FleR might influence flagellin synthesis on a posttranscriptional level. In contrast to the commonly accepted view that enhancer-binding proteins such as FleQ always interact with RpoN to fullfill their regulatory functions, our results strongly indicate that FleQ regulates gene expression that is RpoN dependent and RpoN independent. Finally, FliA induces expression of flagellar class III and IV genes leading to the complete synthesis of the flagellum.

 

Structural insights into the dual nucleotide exchange and GDI displacement activity of SidM/DrrA

Suh HY, Lee DW, Lee KH, Ku B, Choi SJ, Woo JS, Kim YG, Oh BH.

Department of Life Sciences and Center for Biomolecular Recognition, Pohang University of Science and Technology, Pohang, Kyungbuk, Korea. bhoh@postech.ac.kr

EMBO J. 2010 Jan 20;29(2):496-504.

ABSTRACT: GDP-bound prenylated Rabs, sequestered by GDI (GDP dissociation inhibitor) in the cytosol, are delivered to destined sub-cellular compartment and subsequently activated by GEFs (guanine nucleotide exchange factors) catalysing GDP-to-GTP exchange. The dissociation of GDI from Rabs is believed to require a GDF (GDI displacement factor). Only two RabGDFs, human PRA-1 and Legionella pneumophila SidM/DrrA, have been identified so far and the molecular mechanism of GDF is elusive. Here, we present the structure of a SidM/DrrA fragment possessing dual GEF and GDF activity in complex with Rab1. SidM/DrrA reconfigures the Switch regions of the GTPase domain of Rab1, as eukaryotic GEFs do toward cognate Rabs. Structure-based mutational analyses show that the surface of SidM/DrrA, catalysing nucleotide exchange, is involved in GDI1 displacement from prenylated Rab1:GDP. In comparison with an eukaryotic GEF TRAPP I, this bacterial GEF/GDF exhibits high binding affinity for Rab1 with GDP retained at the active site, which appears as the key feature for the GDF activity of the protein.

 

RabGDI displacement by DrrA from Legionella is a consequence of its guanine nucleotide exchange activity

Schoebel S, Oesterlin LK, Blankenfeldt W, Goody RS, Itzen A.

Department of Physical Biochemistry, Max Planck Institute of Molecular Physiology, Dortmund, NRW, Germany. aymelt.itzen@mpi-dortmund.mpg.de

Mol Cell. 2009 Dec 25;36(6):1060-72.

ABSTRACT: Prenylated Rab proteins exist in the cytosol as soluble, high-affinity complexes with GDI that need to be disrupted for membrane attachment and targeting of Rab proteins. The Legionella pneumophila protein DrrA displaces GDI from Rab1:GDI complexes, incorporating Rab1 into Legionella-containing vacuoles and activating Rab1 by exchanging GDP for GTP. Here, we present the crystal structure of a complex between the GEF domain of DrrA and Rab1 and a detailed kinetic analysis of this exchange. DrrA efficiently catalyzes nucleotide exchange and mimics the general nucleotide exchange mechanism of mammalian GEFs for Ras-like GTPases. We show that the GEF activity of DrrA is sufficient to displace prenylated Rab1 from the Rab1:GDI complex. Thus, apparent GDI displacement by DrrA is linked directly to nucleotide exchange, suggesting a basic model for GDI displacement and specificity of Rab localization that does not require discrete GDI displacement activity.

 

Molecular mimicry by an F-box effector of Legionella pneumophila hijacks a conserved polyubiquitination machinery within macrophages and protozoa

Price CT, Al-Khodor S, Al-Quadan T, Santic M, Habyarimana F, Kalian A, Kwaik YA.

Department of Microbiology and Immunology, College of Medicine, University of Louisville, Kentucky, USA. abukwaik@louisville.edu

PLoS Pathog. 2009 Dec;5(12):e1000704.

ABSTRACT: The ability of Legionella pneumophila to proliferate within various protozoa in the aquatic environment and in macrophages indicates a remarkable evolution and microbial exploitation of evolutionarily conserved eukaryotic processes. Ankyrin B (AnkB) of L. pneumophila is a non-canonical F-box-containing protein, and is the only known Dot/Icm-translocated effector of L. pneumophila essential for intra-vacuolar proliferation within both macrophages and protozoan hosts. We show that the F-box domain of AnkB and the (9)L(10)P conserved residues are essential for intracellular bacterial proliferation and for rapid acquisition of polyubiquitinated proteins by the Legionella-containing vacuole (LCV) within macrophages, Dictyostelium discoideum, and Acanthamoeba. Interestingly, translocation of AnkB and recruitment of polyubiquitinated proteins in macrophages and Acanthamoeba is rapidly triggered by extracellular bacteria within 5 min of bacterial attachment. Ectopically expressed AnkB within mammalian cells is localized to the periphery of the cell where it co-localizes with host SKP1 and recruits polyubiquitinated proteins, which results in restoration of intracellular growth to the ankB mutant similar to the parental strain. While an ectopically expressed AnkB-(9)L(10)P/AA variant is localized to the cell periphery, it does not recruit polyubiquitinated proteins and fails to trans-rescue the ankB mutant intracellular growth defect. Direct in vivo interaction of AnkB but not the AnkB-(9)L(10)P/AA variant with the host SKP1 is demonstrated. Importantly, RNAi-mediated silencing of expression of SKP1 renders the cells non-permissive for intracellular proliferation of L. pneumophila. The role of AnkB in exploitation of the polyubiquitination machinery is essential for intrapulmonary bacterial proliferation in the mouse model of Legionnaires' disease. Therefore, AnkB exhibits a novel molecular and functional mimicry of eukaryotic F-box proteins that exploits conserved polyubiquitination machinery for intracellular proliferation within evolutionarily distant hosts.

 

Identification of host cytosolic sensors and bacterial factors regulating the type I interferon response to Legionella pneumophila

Monroe KM, McWhirter SM, Vance RE.

Division of Immunology and Pathogenesis, Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA. rvance@berkeley.edu

PLoS Pathog. 2009 Nov;5(11):e1000665.

ABSTRACT: Legionella pneumophila is a gram-negative bacterial pathogen that replicates in host macrophages and causes a severe pneumonia called Legionnaires' Disease. The innate immune response to L. pneumophila remains poorly understood. Here we focused on identifying host and bacterial factors involved in the production of type I interferons (IFN) in response to L. pneumophila. It was previously suggested that the delivery of L. pneumophila DNA to the host cell cytosol is the primary signal that induces the type I IFN response. However, our data are not easily reconciled with this model. We provide genetic evidence that two RNA-sensing proteins, RIG-I and MDA5, participate in the IFN response to L. pneumophila. Importantly, these sensors do not seem to be required for the IFN response to L. pneumophila DNA, whereas we found that RIG-I was required for the response to L. pneumophila RNA. Thus, we hypothesize that bacterial RNA, or perhaps an induced host RNA, is the primary stimulus inducing the IFN response to L. pneumophila. Our study also identified a secreted effector protein, SdhA, as a key suppressor of the IFN response to L. pneumophila. Although viral suppressors of cytosolic RNA-sensing pathways have been previously identified, analogous bacterial factors have not been described. Thus, our results provide new insights into the molecular mechanisms by which an intracellular bacterial pathogen activates and also represses innate immune responses.

 

The TolC protein of Legionella pneumophila plays a major role in multi-drug resistance and the early steps of host invasion

Ferhat M, Atlan D, Vianney A, Lazzaroni JC, Doublet P, Gilbert C.

Université de Lyon, Lyon, France. gilbert@biomserv.univ-lyon1.fr

PLoS One. 2009 Nov 4;4(11):e7732.

ABSTRACT: Pneumonia associated with Iegionnaires's disease is initiated in humans after inhalation of contaminated aerosols. In the environment, Legionella pneumophila is thought to survive and multiply as an intracellular parasite within free-living amoeba. In the genome of L. pneumophila Lens, we identified a unique gene, tolC, encoding a protein that is highly homologous to the outer membrane protein TolC of Escherichia coli. Deletion of tolC by allelic exchange in L. pneumophila caused increased sensitivity to various drugs. The complementation of the tolC mutation in trans restored drug resistance, indicating that TolC is involved in multi-drug efflux machinery. In addition, deletion of tolC caused a significant attenuation of virulence towards both amoebae and macrophages. Thus, the TolC protein appears to play a crucial role in virulence which could be mediated by its involvement in efflux pump mechanisms. These findings will be helpful in unraveling the pathogenic mechanisms of L. pneumophila as well as in developing new therapeutic agents affecting the efflux of toxic compounds.

 

Temporal resolution of two-tracked NF-kappaB activation by Legionella pneumophila

Bartfeld S, Engels C, Bauer B, Aurass P, Flieger A, Brüggemann H, Meyer TF.

Max Planck Institute for Infection Biology, Department of Molecular Biology, Berlin, Germany. tfm@mpiib-berlin.mpg.de

Cell Microbiol. 2009 Nov;11(11):1638-51.

ABSTRACT: The intracellular pathogen Legionella pneumophila activates the transcription factor NF-kappaB in macrophages and human epithelial cells, contributing to cytokine production and anti-apoptosis. The former is important for the innate immune response to infection, the latter for intracellular replication by securing host cell survival. Here, we demonstrate biphasic activation of NF-kappaB by L. pneumophila in human epithelial cells, using a p65-GFP expressing variant of A549 cells. Early in infection, a strong but transient nuclear translocation of p65 was observed. Only flagellin-deficient (DeltafliA and DeltaflaA) mutants could not induce this first, TLR5 and MyD88-dependent activation. The second p65 translocation event, however, is a long-term activation, independent of flagellin, TLR5 and MyD88, and marked by permanent nuclear localization of p65-GFP without oscillation for 30 h. Persistent p65 translocation also involved degradation of IkappaBalpha and upregulation of anti-apoptotic genes. L. pneumophila mutants lacking a functional Dot/Icm secretion system (DeltadotA; DeltaicmB/dotO), Dot/Icm effectors (DeltasdbA; DeltalubX) and two bacterial effector mutants (DeltaenhC; DeltaptsP) could not induce persistent p65 translocation. Strikingly, all these mutants were deficient in intracellular replication in A549 cells. Our data underline the strong connection between NF-kappaB activation and intracellular replication and hints at an active interference of NF-kappaB signalling by L. pneumophila.

 

Legionella pneumophila - Host Interactions: Insights Gained from Comparative Genomics and Cell Biology

Lomma M, Gomez Valero L, Rusniok C, Buchrieser C.

Institut Pasteur, Unité Biologie des Bactéries Intracellulaires and CNRS URA 2171, Paris, France. carmen.buchrieser@pasteur.fr

Genome Dyn. 2009;6:170-186.

ABSTRACT: Legionella pneumophila is the etiological agent of Legionnaires' disease and of the less acute disease Pontiac fever. It is a Gram-negative bacterium present in fresh and artificial water environments that replicates in protozoan hosts and is also found in biofilms. Replication within protozoa is essential for the survival of the bacterium. The last years have seen a giant step forward in the genomics of L. pneumophila. The establishment and publication of the complete genome sequences of three clinical L. pneumophila isolates in 2004 and a fourth in 2007 has paved the way for major breakthroughs in understanding the biology of L. pneumophila in particular and Legionella in general. Sequence analysis identified several specific features of Legionella: (i) an extraordinary genetic diversity among the different isolates and (ii) the presence of an unexpected high number and variety of eukaryotic-like proteins, predicted to be involved in the exploitation of the host cellular processes by mimicking specific eukaryotic functions. In this chapter, we will first discuss the insights gained from genomics by highlighting the characteristic features and common traits of the four L. pneumophila genomes obtained through genome analysis and comparison and then we will focus on the newest results obtained by functional analysis of different eukaryotic-like proteins and describe their involvementin the pathogenicity of L. pneumophila.

 

A Legionella type IV effector activates the NF-kappaB pathway by phosphorylating the IkappaB family of inhibitors

Ge J, Xu H, Li T, Zhou Y, Zhang Z, Li S, Liu L, Shao F.

College of Life Sciences, Beijing Normal University, Beijing 100875, China. shaofeng@nibs.ac.cn

Proc Natl Acad Sci U S A. 2009 Aug 18;106(33):13725-30.

ABSTRACT: NF-kappaB is critical in innate immune defense responses against invading microbial pathogens. Legionella pneumophila infection of lung macrophages causes Legionnaire's disease with pneumonia symptoms. A set of NF-kappaB-controlled genes involved in inflammation and anti-apoptosis are up-regulated in macrophages upon L. pneumophila infection in a Legionella Dot/Icm type IV secretion system-dependent manner. Among approximately 100 Dot/Icm substrates screened, we identified LegK1 as the sole Legionella protein that harbors a highly potent NF-kappaB-stimulating activity. LegK1 does not affect MAPK and IFN pathways. Activation of the NF-kappaB pathway by LegK1 requires its eukaryotic-like Ser/Thr kinase activity and is independent of upstream components in the NF-kappaB pathway, including TRAFs, NIK, MEKK3, and TAK1. Cell-free reconstitution revealed that LegK1 stimulated NF-kappaB activation in the absence of IKKalpha and IKKbeta, and LegK1 efficiently phosphorylated IkappaBalpha on Ser-32 and Ser-36 both in vitro and in cells. LegK1 seems to mimic the host IKK as LegK1 also directly phosphorylated other IkappaB family of inhibitors including p100 in the noncanonical NF-kappaB pathway. Phosphorylation of p100 by LegK1 led to its maturation into p52. Thus, LegK1 is a bacterial effector that directly activates the host NF-kappaB signaling and likely plays important roles in modulating macrophage defense or inflammatory responses during L. pneumophila infection.

 

Legionella pneumophila secretes an endoglucanase that belongs to the family-5 of glycosyl hydrolases and is dependent upon type II secretion

Pearce MM, Cianciotto NP.

Department of Microbiology and Immunology, Northwestern University Medical School, Chicago, IL, USA.

n-cianciotto@northwestern.edu

FEMS Microbiol Lett. 2009 Nov;300(2):256-64.

ABSTRACT: Examination of cell-free culture supernatants revealed that Legionella pneumophila strains secrete an endoglucanase activity. Legionella pneumophila lspF mutants were deficient for this activity, indicating that the endoglucanase is secreted by the bacterium's type II protein secretion (T2S) system. Inactivation of celA, encoding a member of the family-5 of glycosyl hydrolases, abolished the endoglucanase activity in L. pneumophila culture supernatants. The cloned celA gene conferred activity upon recombinant Escherichia coli. Thus, CelA is the major secreted endoglucanase of L. pneumophila. Mutants inactivated for celA grew normally in protozoa and macrophage, indicating that CelA is not required for the intracellular phase of L. pneumophila. The CelA endoglucanase is one of at least 25 proteins secreted by the type II system of L. pneumophila and the 17th type of enzyme effector associated with this pathway. Only a subset of the other Legionella species tested expressed secreted endoglucanase activity, suggesting that the T2S output differs among the different legionellae. Overall, this study represents the first documentation of an endoglucanase (EC 3.2.1.4) being produced by a strain of Legionella.

 

Restriction of Legionella pneumophila replication in macrophages requires concerted action of the transcriptional regulators Irf1 and Irf8 and nod-like receptors Naip5 and Nlrc4

Fortier A, Doiron K, Saleh M, Grinstein S, Gros P.

Department of Biochemistry, McGill University, McIntyre Medical Building, 3655 Promenade Sir William Osler, Room 907, Montréal, Québec, Canada H3G 1Y6. philippe.gros@mcgill.ca

Infect Immun 2009 Nov;77(11):4794-805.

ABSTRACT: The unique permissiveness of A/J mouse macrophages for replication of Legionella pneumophila is caused by a deficiency in the Nod-like receptor (NLR) protein and intracellular sensor for L. pneumophila flagellin (Naip5). The signaling pathways and proteins activated by Naip5 sensing in macrophages were investigated. Transcript profiling of macrophages from susceptible A/J mice and from resistant A/J mice harboring a transgenic wild-type copy of Naip5 at 4 h following L. pneumophila infection suggested that two members of the Irf transcriptional regulator family, Irf1 and Irf8, are regulated in response to Naip5 sensing of L. pneumophila. We show that macrophages having defective alleles of either Irf1 (Irf1-/-) or its heterodimerization partner gene Irf8 (Irf8R294C) become permissive for L. pneumophila replication, indicating that both the Irf1 and Irf8 proteins are essential for macrophage defense against L. pneumophila. Moreover, macrophages doubly heterozygous (Naip5AJ/WT Irf8R294C/WT or Nlrc4-/+ Irf8R294C/WT) for combined loss-of-function mutations in Irf8 and in either Naip5 or Nlrc4 are highly susceptible to L. pneumophila, indicating that there is a strong genetic interaction between Irf8 and the NLR protein family in the macrophage response to L. pneumophila. Legionella-containing phagosomes (LCPs) formed in permissive Irf1-/- or Irf8R294C macrophages behave like LCPs formed in Naip5-insufficient and Nlrc4-deficient macrophages which fail to acidify. These results suggest that, in addition to Naip5 and Nlrc4, Irf1 and Irf8 play a critical role in the early response of macrophages to infection with L. pneumophila, including antagonizing the ability of L. pneumophila to block phagosome acidification. They also suggest that flagellin sensing by the NLR proteins Naip5 and Nlrc4 may be coupled to Irf1-Irf8-mediated transcriptional activation of key effector genes essential for macrophage resistance to L. pneumophila infection.

 

The purified and recombinant Legionella pneumophila chaperonin alters mitochondrial trafficking and microfilament organization

Chong A, Lima CA, Allan DS, Nasrallah GK, Garduño RA.

Infect Immun. 2009 Nov;77(11):4724-39.

Department of Microbiology and Immunology, 5850 College Street, Sir Charles Tupper Medical Building, 7th Floor, Dalhousie University, Halifax, Nova Scotia B3H 1X5, Canada. rafael.garduno@dal.ca

ABSTRACT: A portion of the total cellular pool of the Legionella pneumophila chaperonin, HtpB, is found on the bacterial cell surface, where it can mediate invasion of nonphagocytic cells. HtpB continues to be abundantly produced and released by internalized L. pneumophila and may thus have postinvasion functions. We used here two functional models (protein-coated beads and expression of recombinant proteins in CHO cells) to investigate the competence of HtpB in mimicking early intracellular trafficking events of L. pneumophila, including the recruitment of mitochondria, cytoskeletal alterations, the inhibition of phagosome-lysosome fusion, and association with the endoplasmic reticulum. Microscopy and flow cytometry studies indicated that HtpB-coated beads recruited mitochondria in CHO cells and U937-derived macrophages and induced transient changes in the organization of actin microfilaments in CHO cells. Ectopic expression of HtpB in the cytoplasm of transfected CHO cells also led to modifications in actin microfilaments similar to those produced by HtpB-coated beads but did not change the distribution of mitochondria. Association of phagosomes containing HtpB-coated beads with the endoplasmic reticulum was not consistently detected by either fluorescence or electron microscopy studies, and only a modest delay in the fusion of TrOv-labeled lysosomes with phagosomes containing HtpB-coated beads was observed. HtpB is the first Legionella protein and the first chaperonin shown to, by means of our functional models, induce mitochondrial recruitment and microfilament rearrangements, two postinternalization events that typify the early trafficking of virulent L. pneumophila.

 

Temporal resolution of two-tracked NF-kappaB activation by Legionella pneumophila.

Bartfeld S, Engels C, Bauer B, Aurass P, Flieger A, Brüggemann H, Meyer TF.

Max Planck Institute for Infection Biology, Department of Molecular Biology, Berlin, Germany. tfm@mpiib-berlin.mpg.de

Cell Microbiol. 2009 Nov;11(11 ):1638-51.

ABSTRACT: The intracellular pathogen Legionella pneumophila activates the transcription factor NF-kappaB in macrophages and human epithelial cells, contributing to cytokine production and anti-apoptosis. The former is important for the innate immune response to infection, the latter for intracellular replication by securing host cell survival. Here, we demonstrate biphasic activation of NF-kappaB by L. pneumophila in human epithelial cells, using a p65-GFP expressing variant of A549 cells. Early in infection, a strong but transient nuclear translocation of p65 was observed. Only flagellin-deficient (DeltafliA and DeltaflaA) mutants could not induce this first, TLR5 and MyD88-dependent activation. The second p65 translocation event, however, is a long-term activation, independent of flagellin, TLR5 and MyD88, and marked by permanent nuclear localization of p65-GFP without oscillation for 30 h. Persistent p65 translocation also involved degradation of IkappaBalpha and upregulation of anti-apoptotic genes. L. pneumophila mutants lacking a functional Dot/Icm secretion system (DeltadotA; DeltaicmB/dotO), Dot/Icm effectors (DeltasdbA; DeltalubX) and two bacterial effector mutants (DeltaenhC; DeltaptsP) could not induce persistent p65 translocation. Strikingly, all these mutants were deficient in intracellular replication in A549 cells. Our data underline the strong connection between NF-kappaB activation and intracellular replication and hints at an active interference of NF-kappaB signalling by L. pneumophila.

 

Interferons Direct an Effective Innate Response to Legionella pneumophila Infection

Plumlee CR, Lee C, Beg AA, Decker T, Shuman HA, Schindler C.

From the Departments of Biological Sciences. cws4@columbia.edu

J Biol Chem 2009 Oct 30;284(44):30058-66.

ABSTRACT: Legionella pneumophila remains an important opportunistic pathogen of human macrophages. Its more limited ability to replicate in murine macrophages has been attributed to redundant innate sensor systems that detect and effectively respond to this infection. The current studies evaluate the role of one of these innate response systems, the type I interferon (IFN-I) autocrine loop. The ability of L. pneumophila to induce IFN-I expression was found to be dependent on IRF-3, but not NF-kappaB. Secreted IFN-Is then in turn suppress the intracellular replication of L. pneumophila. Surprisingly, this suppression is mediated by a pathway that is independent of Stat1, Stat2, Stat3, but correlates with the polarization of macrophages toward the M1 or classically activated phenotype.

  

Phospholipase PlaB of Legionella pneumophila represents a novel lipase family: protein residues essential for lipolytic activity, substrate specificity, and hemolysis

Bender J, Rydzewski K, Broich M, Schunder E, Heuner K, Flieger A.

Division of Bacterial Infections, FG11, Robert Koch-Institut, Burgstrasse 37, Wernigerode 38855, Germany. fliegera@rki.de

J Biol Chem 2009 Oct 2;284(40):27185-94.

ABSTRACT: Legionella pneumophila possesses several phospholipases capable of host cell manipulation and lung damage. Recently, we discovered that the major cell-associated hemolytic phospholipase A (PlaB) shares no homology to described phospholipases and is dispensable for intracellular replication in vitro. Nevertheless, here we show that PlaB is the major lipolytic activity in L. pneumophila cell infections and that PlaB utilizes a typical catalytic triad of Ser-Asp-His for effective hydrolysis of phospholipid substrates. Crucial residues were found to be located within the N-terminal half of the protein, and amino acids embedding these active sites were unique for PlaB and homologs. We further showed that catalytic activity toward phosphatidylcholine but not phosphatidylglycerol is directly linked to hemolytic potential of PlaB. Although the function of the prolonged PlaB C terminus remains to be elucidated, it is essential for lipolysis, since the removal of 15 amino acids already abolishes enzyme activity. Additionally, we determined that PlaB preferentially hydrolyzes long-chain fatty acid substrates containing 12 or more carbon atoms. Since phospholipases play an important role as bacterial virulence factors, we examined cell-associated enzymatic activities among L. pneumophila clinical isolates and non-pneumophila species. All tested clinical isolates showed comparable activities, whereas of the non-pneumophila species, only Legionella gormanii and Legionella spiritensis possessed lipolytic activities similar to those of L. pneumophila and comprised plaB-like genes. Interestingly, phosphatidylcholine-specific phospholipase A activity and hemolytic potential were more pronounced in L. pneumophila. Therefore, hydrolysis of the eukaryotic membrane constituent phosphatidylcholine triggered by PlaB could be an important virulence tool for Legionella pathogenicity.

 

The perplexing functions and surprising origins of Legionella pneumophila type IV secretion effectors

Franco IS, Shuman HA, Charpentier X.

Department of Microbiology, Columbia University Medical Center, New York, NY 10032, USA. xc2121@columbia.edu

Cell Microbiol. 2009 Oct;11(10):1435-43.

ABSTRACT: Only a limited number of bacterial pathogens evade destruction by phagocytic cells such as macrophages. Legionella pneumophila is a Gram-negative gamma-proteobacterial species that can infect and replicate in alveolar macrophages, causing Legionnaires' disease, a severe pneumonia. L. pneumophila uses a complex secretion system to inject host cells with effector proteins capable of disrupting or altering the host cell processes. The L. pneumophila effectors target multiple processes but are essentially aimed at modifying the properties of the L. pneumophila phagosome by altering vesicular trafficking, gradually creating a specialized vacuole in which the bacteria replicate robustly. In nature, L. pneumophila is thought to parasitize free-living protists, which may have selected for traits that promote virulence of L. pneumophila in humans. Indeed, many effector genes encode proteins with eukaryotic domains and are likely to be of protozoan origin. Sustained horizontal gene transfer events within the protozoan niche may have allowed L. pneumophila to become a professional parasite of phagocytes, simultaneously giving rise to its ability to infect macrophages, cells that constitute the first line of cellular defence against bacterial infections.

 

Many substrates and functions of type II secretion: lessons learned from Legionella pneumophila

Cianciotto NP.

Department of Microbiology & Immunology, Northwestern University Medical School, 320 East Superior St., Chicago, IL 60611, USA. n-cianciotto@northwestern.edu

Future Microbiol 2009 Sep;4:797-805.

ABSTRACT: Type II secretion is one of six systems that exist in Gram-negative bacteria for the purpose of secreting proteins into the extracellular milieu and/or into host cells. This article will review the various recent studies of Legionella pneumophila that have increased our appreciation of the numbers, types and novelties of proteins that can be secreted via the type II system, as well as the many ways in which type II secretion can promote bacterial physiology, growth, ecology, intracellular infection and virulence. In this context, type II secretion represents a potentially important target for industrial and biomedical applications.

 

Cellular accumulation and pharmacodynamic evaluation of the intracellular activity of CEM-101, a novel fluoroketolide, against Staphylococcus aureus, Listeria monocytogenes, and Legionella pneumophila in human THP-1 macrophages

Lemaire S, Van Bambeke F, Tulkens PM.

Unité de Pharmacologie Cellulaire et Moléculaire and Louvain Drug Research Institute, Université Catholique de Louvain, Brussels, Belgium. tulkens@facm.ucl.ac.be

Antimicrob Agents Chemother. 2009 Sep;53(9):3734-43.

ABSTRACT: CEM-101 is a novel fluoroketolide with lower MICs than those of telithromycin and macrolides. Our aim was to assess the cellular accumulation and intracellular activity of CEM-101 using models developed for analyzing the pharmacokinetics and pharmacological properties of antibiotics against phagocytized bacteria. We used THP-1 macrophages and Staphylococcus aureus (ATCC 25923 [methicillin (meticillin) sensitive]), Listeria monocytogenes (strain EGD), and Legionella pneumophila (ATCC 33153). CEM-101 reached cellular-to-extracellular-concentration ratios of about 350 within 24 h (versus approximately 20, 30, and 160 for telithromycin, clarithromycin, and azithromycin, respectively). This intracellular accumulation was suppressed by incubation at a pH of < or = 6 and by monensin (proton ionophore) and was unaffected by verapamil (P-glycoprotein inhibitor; twofold accumulation increase for azithromycin) or gemfibrozil. While keeping with the general properties of the macrolide antibiotics in terms of maximal efficacy (Emax; approximately 1-log10-CFU decrease compared to the postphagocytosis inoculum after a 24-h incubation), CEM-101 showed significantly greater potency against phagocytized S. aureus than telithromycin, clarithromycin, and azithromycin (for which the 50% effective concentration [EC50] and static concentrations were about 3-, 6-, and 15-fold lower, respectively). CEM-101 was also about 50-fold and 100-fold more potent than azithromycin against phagocytized L. monocytogenes and L. pneumophila, respectively. These differences in EC50s and static concentrations between drugs were minimized when data were expressed as multiples of the MIC, demonstrating the critical role of intrinsic drug activity (MIC) in eliciting the antibacterial intracellular effects, whereas accumulation per se was unimportant. CEM-101 should show enhanced in vivo potency if used at doses similar to those of the comparators tested here.

 

Distribution of lag-1 alleles and sequence-based types among Legionella pneumophila serogroup 1 clinical and environmental isolates in the United States

Kozak NA, Benson RF, Brown E, Alexander NT, Taylor TH Jr, Shelton BG, Fields BS.

Centers for Disease Control and Prevention, Atlanta, GA 30033, USA. htv2@cdc.gov

J Clin Microbiol. 2009 Aug;47(8):2525-35.

ABSTRACT: Approximately 84% of legionellosis cases are due to Legionella pneumophila serogroup 1. Moreover, a majority of L. pneumophila serogroup 1 clinical isolates react positively with monoclonal antibody 2 (MAb2) of the international standard panel. Over 94% of the legionellosis outbreaks investigated by the Centers for Disease Control and Prevention are due to this subset of L. pneumophila serogroup 1. To date, there is no complete explanation for the enhanced ability of these strains to cause disease. To better characterize these organisms, we subtyped 100 clinical L. pneumophila serogroup 1 isolates and 50 environmental L. pneumophila serogroup 1 isolates from the United States by (i) reactivity with MAb2, (ii) presence of a lag-1 gene required for the MAb2 epitope, and (iii) sequence-based typing analysis. Our results showed that the MAb2 epitope and lag-1 gene are overrepresented in clinical L. pneumophila serogroup 1 isolates. MAb2 recognized 75% of clinical isolates but only 6% of environmental isolates. Similarly, 75% of clinical isolates but only 8% of environmental isolates harbored lag-1. We identified three distinct lag-1 alleles, referred to as Philadelphia, Arizona, and Lens alleles, among 79 isolates carrying this gene. The Arizona allele is described for the first time in this study. We identified 59 different sequence types (STs), and 34 STs (58%) were unique to the United States. Our results support the hypothesis that a select group of STs may have an enhanced ability to cause legionellosis. Combining sequence typing and lag-1 analysis shows that STs tend to associate with a single lag-1 allele type, suggesting a hierarchy of virulence genotypes. Further analysis of ST and lag-1 profiles may identify genotypes of L. pneumophila serogroup 1 that warrant immediate intervention.

 

Molecular mimicry: an important virulence strategy employed by Legionella pneumophila to subvert host functions

Nora T, Lomma M, Gomez-Valero L, Buchrieser C.

Institut Pasteur, Biologie des Bactéries Intracellulaires & CNRS URA 2171, 75724 Paris, France. tamara.nora@pasteur.fr

Future Microbiol 2009 Aug;4:691-701.

ABSTRACT: It is 32 years since Legionella pneumophila was identified and recognized as a human pathogen, causing the severe form of pneumonia termed Legionnaires' disease, or legionellosis. This bacterium is found in freshwater reservoirs where it replicates in aquatic protozoa and can invade man-made water distribution systems. Although the disease can be treated by antibiotherapy and prevented through surveillance and control measures, reported cases of Legionnaires' disease continue to rise across Europe and outbreaks of major public health significance still occur. Genome sequencing and analyses led to a giant step forward by suggesting new ways by which this intracellular bacterium might subvert host functions. One particular feature revealed was the presence of many eukaryotic-like proteins, possibly mimicking host proteins to allow intracellular replication of Legionella. Here, we describe the identification and analysis of these proteins and report on recent advances detailing the mechanisms by which these proteins function. Finally, comparative and evolutionary genomic aspects regarding the eukaryotic-like proteins are presented. Collectively, these data have shed new light on the virulence strategies of L. pneumophila, a major aspect of which is molecular mimicry. 

 

bdhA-patD operon as a virulence determinant, revealed by a novel large-scale approach for identification of Legionella pneumophila mutants defective for amoeba infection

Aurass P, Pless B, Rydzewski K, Holland G, Bannert N, Flieger A.

Robert Koch Institute, Berlin, Germany. fliegera@rki.de

Appl Environ Microbiol. 2009;75(13):4506-15.

ABSTRACT: Legionella pneumophila, the causative agent of Legionnaires' disease, is an intracellular parasite of eukaryotic cells. In the environment, it colonizes amoebae. After being inhaled into the human lung, the bacteria infect and damage alveolar cells in a way that is mechanistically similar to the amoeba infection. Several L. pneumophila traits, among those the Dot/Icm type IVB protein secretion machinery, are essential for exploiting host cells. In our search for novel Legionella virulence factors, we developed an agar plate assay, designated the scatter screen, which allowed screening for mutants deficient in infecting Acanthamoeba castellanii amoebae. Likewise, an L. pneumophila clone bank consisting of 23,000 transposon mutants was investigated here, and 19 different established Legionella virulence genes, for example, dot/icm genes, were identified. Importantly, 70 novel virulence-associated genes were found. One of those is L. pneumophila bdhA, coding for a protein with homology to established 3-hydroxybutyrate dehydrogenases involved in poly-3-hydroxybutyrate metabolism. Our study revealed that bdhA is cotranscribed with patD, encoding a patatin-like protein of L. pneumophila showing phospholipase A and lysophospholipase A activities. In addition to strongly reduced lipolytic activities and increased poly-3-hydroxybutyrate levels, the L. pneumophila bdhA-patD mutant showed a severe replication defect in amoebae and U937 macrophages. Our data suggest that the operon is involved in poly-3-hydroxybutyrate utilization and phospholipolysis and show that the bdhA-patD operon is a virulence determinant of L. pneumophila. In summary, the screen for amoeba-sensitive Legionella clones efficiently isolated mutants that do not grow in amoebae and, in the case of the bdhA-patD mutant, also human cells.

 

The CMP-legionaminic acid pathway in Campylobacter: biosynthesis involving novel GDP-linked precursors

Schoenhofen IC, Vinogradov E, Whitfield DM, Brisson JR, Logan SM.

Institute for Biological Sciences, National Research Council, Ottawa, Ontario, K1A 0R6 Canada. ian.schoenhofen@nrc-cnrc.gc.ca

Glycobiology. 2009 Jul;19(7):715-25. Epub 2009 Mar 12.

ABSTRACT: The sialic acid-like sugar 5,7-diacetamido-3,5,7,9-tetradeoxy-D-glycero-D-galacto-nonulosonic acid, or legion-aminic acid, is found as a virulence-associated cell-surface glycoconjugate in the Gram-negative bacteria Legionella pneumophila and Campylobacter coli. L. pneumophila serogroup 1 strains, causative agents of Legionnaire's disease, contain an alpha2,4-linked homopolymer of legionaminic acid within their lipopolysaccharide O-chains, whereas the gastrointestinal pathogen C. coli modifies its flagellin with this monosaccharide via O-linkage. In this work, we have purified and biochemically characterized 11 candidate biosynthetic enzymes from Campylobacter jejuni, thereby fully reconstituting the biosynthesis of legionaminic acid and its CMP-activated form, starting from fructose-6-P. This pathway involves unique GDP-linked intermediates, likely providing a cellular mechanism for differentiating between this and similar UDP-linked pathways, such as UDP-2,4-diacetamido-bacillosamine biosynthesis involved in N-linked protein glycosylation. Importantly, these findings provide a facile method for efficient large-scale synthesis of legionaminic acid, and since legionaminic acid and sialic acid share the same D-glycero-D-galacto absolute configuration, this sugar may now be evaluated for its potential as a sialic acid mimic.

 

Chemical genetics reveals bacterial and host cell functions critical for type IV effector translocation by Legionella pneumophila

Charpentier X, Gabay JE, Reyes M, Zhu JW, Weiss A, Shuman HA.

Department of Microbiology, Columbia University Medical Center, New York, NY, USA.

PLoS Pathog. 2009 Jul;5(7):e1000501.

ABSTRACT: Delivery of effector proteins is a process widely used by bacterial pathogens to subvert host cell functions and cause disease. Effector delivery is achieved by elaborate injection devices and can often be triggered by environmental stimuli. However, effector export by the L. pneumophila Icm/Dot Type IVB secretion system cannot be detected until the bacterium encounters a target host cell. We used chemical genetics, a perturbation strategy that utilizes small molecule inhibitors, to determine the mechanisms critical for L. pneumophila Icm/Dot activity. From a collection of more than 2,500 annotated molecules we identified specific inhibitors of effector translocation. We found that L. pneumophila effector translocation in macrophages requires host cell factors known to be involved in phagocytosis such as phosphoinositide 3-kinases, actin and tubulin. Moreover, we found that L. pneumophila phagocytosis and effector translocation also specifically require the receptor protein tyrosine phosphate phosphatases CD45 and CD148. We further show that phagocytosis is required to trigger effector delivery unless intimate contact between the bacteria and the host is artificially generated. In addition, real-time analysis of effector translocation suggests that effector export is rate-limited by phagocytosis. We propose a model in which L. pneumophila utilizes phagocytosis to initiate an intimate contact event required for the translocation of pre-synthesized effector molecules. We discuss the need for host cell participation in the initial step of the infection and its implications in the L. pneumophila lifestyle. Chemical genetic screening provides a novel approach to probe the host cell functions and factors involved in host-pathogen interactions.

 

Targeting eEF1A by a Legionella pneumophila effector leads to inhibition of protein synthesis and induction of host stress response

Shen X, Banga S, Liu Y, Xu L, Gao P, Shamovsky I, Nudler E, Luo ZQ.

Department of Biological Sciences, Purdue University, West Lafayette, IN 47907, USA. luoz@purdue.edu

Cell Microbiol. 2009 Jun;11(6):911-26.

ABSTRACT: The Legionella pneumophila Dot/Icm type IV secretion system is essential for the biogenesis of a phagosome that supports bacterial multiplication, most likely via the functions of its protein substrates. Recent studies indicate that fundamental cellular processes, such as vesicle trafficking, stress response, autophagy and cell death, are modulated by these effectors. However, how each translocated protein contributes to the modulation of these pathways is largely unknown. In a screen to search substrates of the Dot/Icm transporter that can cause host cell death, we identified a gene whose product is lethal to yeast and mammalian cells. We demonstrate that this protein, called SidI, is a substrate of the Dot/Icm type IV protein transporter that targets the host protein translation process. Our results indicate that SidI specifically interacts with eEF1A and eEF1Bgamma, two components of the eukaryotic protein translation elongation machinery and such interactions leads to inhibition of host protein synthesis. Furthermore, we have isolated two SidI substitution mutants that retain the target binding activity but have lost toxicity to eukaryotic cells, suggesting potential biochemical effect of SidI on eEF1A and eEF1Bgamma. We also show that infection by L. pneumophila leads to eEF1A-mediated activation of the heat shock regulatory protein HSF1 in a virulence-dependent manner and deletion of sidI affects such activation. Moreover, similar response occurred in cells transiently transfected to express SidI. Thus, inhibition of host protein synthesis by specific effectors contributes to the induction of stress response in L. pneumophila-infected cells.

 

Rapid pathogen-induced apoptosis: a mechanism used by dendritic cells to limit intracellular replication of Legionella pneumophila

Nogueira CV, Lindsten T, Jamieson AM, Case CL, Shin S, Thompson CB, Roy CR.

Section of Microbial Pathogenesis, Yale University School of Medicine, New Haven, CT, USA. roy@yale.edu

PLoS Pathog. 2009 Jun;5(6):e1000478.

ABSTRACT: Dendritic cells (DCs) are specialized phagocytes that internalize exogenous antigens and microbes at peripheral sites, and then migrate to lymphatic organs to display foreign peptides to naïve T cells. There are several examples where DCs have been shown to be more efficient at restricting the intracellular replication of pathogens compared to macrophages, a property that could prevent DCs from enhancing pathogen dissemination. To understand DC responses to pathogens, we investigated the mechanisms by which mouse DCs are able to restrict replication of the intracellular pathogen Legionella pneumophila. We show that both DCs and macrophages have the ability to interfere with L. pneumophila replication through a cell death pathway mediated by caspase-1 and Naip5. L. pneumophila that avoided Naip5-dependent responses, however, showed robust replication in macrophages but remained unable to replicate in DCs. Apoptotic cell death mediated by caspase-3 was found to occur much earlier in DCs following infection by L. pneumophila compared to macrophages infected similarly. Eliminating the pro-apoptotic proteins Bax and Bak or overproducing the anti-apoptotic protein Bcl-2 were both found to restore L. pneumophila replication in DCs. Thus, DCs have a microbial response pathway that rapidly activates apoptosis to limit pathogen replication.

 

 

Chemical structure and biological significance of lipopolysaccharide from legionella

Palusińska-Szysz M, Russa R.

Department of Genetics and Microbiology, Maria Curie-Skłodowska University, Lublin, Poland. marta.szysz@poczta.umcs.lublin.pl

Recent Pat Antiinfect Drug Discov. 2009 Jun;4(2):96-107.

ABSTRACT: Legionella are aerobic, gram-negative, motile, rod-shaped bacteria, which form a distinct taxonomic unit within the gamma - 2 subdivision of the Proteobacteria. The reservoirs of Legionella are natural or man-made water systems where the bacteria survive and disseminate as obligate intracellular parasites of free living protozoa. In the human lung, the bacteria invade alveolar macrophages inducing the potentially lethal pneumonia commonly known as Legionnaires' disease. Although all Legionella species are considered potentially pathogenic for humans, Legionella pneumophila is the aetiological agent responsible for most reported cases of community- and nosocomially-acquired legionellosis. The O-polysaccharide in the lipopolysaccharide of L. pneumophila is composed of a repeating homopolymer of alpha-(2-->4)-linked 5,7-diamino-3,5,7,9-tetradeoxy-D-glycero-D-galacto-non-2-ulosonic acid (legionaminic acid). The outer region of the core enriched with 6-deoxy sugars and N- and O- acetylated sugars as well as the highly N- and O-acylated O-chain contribute to a high hydrophobicity of the bacterial surface, which enables these bacteria to spread. Lipids A from Legionella contain a backbone with 2,3-diamino-2,3-dideoxy-D-glucose and unusual fatty acids. The present article indicates some patents useful in the diagnostics of Legionnaires' disease.

 

Autophagy induced by 2-deoxy-D-glucose suppresses intracellular multiplication of Legionella pneumophila in A/J mouse macrophages

Matsuda F, Fujii J, Yoshida S.

Department of Bacteriology, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, Japan. fmatsuda@bact.med.kyushu-u.ac.jp

Autophagy. 2009 May;5(4):484-93.

ABSTRACT: Legionella pneumophila Philadelphia-1 (Lp-1) can grow intracellularly in A/J mouse peritoneal macrophages (A/J Mphi). We previously reported that 2-deoxy-D-glucose (2dG), when added in macrophage culture medium, inhibited the intracellular multiplication of Lp-1 in A/J Mphi. We found that 1 mM of 2dG causes LC3-II-conversion that reflects an induction of autophagy and that 1 and 10 mM of 2dG induced apoptosis associated with caspase-4 activation. We therefore investigated whether 2dG-induced autophagy or apoptosis suppresses the replication ofLp-1 in 2dG-treated A/J Mphi. When the autophagy-related (Atg)gene Atg5 was knocked down by RNA interference, the Atg5-siRNA-transfected cells revealed an enhanced replication of Lp-1 in A/J Mphi compared with the non-targetting siRNA-transfected cells. However, caspase-4 inhibitor did not affect the 2dG-induced inhibition of intracellular multiplication of Lp-1 in A/J Mphi. These findings suggested that autophagy, not apoptosis, suppressed the intracellular growth of Lp-1 in A/J Mphi when 1 or 10 mM of 2dG were added to the culture media.

 

Asc and Ipaf Inflammasomes direct distinct pathways for caspase-1 activation in response to Legionella pneumophila

Case CL, Shin S, Roy CR.

Section of Microbial Pathogenesis, Yale University School of Medicine, Boyer Center for Molecular Medicine, 295 Congress Avenue, New Haven, CT 06536, USA. craig.roy@yale.edu

Infect Immun. 2009 May;77(5):1981-91.

ABSTRACT: Caspase-1 activation is a key feature of the innate immune response of macrophages elicited by pathogens and a variety of toxins. Here, we determined the requirement for different adapter proteins involved in regulating host processes mediated by caspase-1 after macrophage infection by Legionella pneumophila. The adapter protein Asc was found to be important for caspase-1 activation during L. pneumophila infection. Activation of caspase-1 through Asc did not require the flagellin-sensing pathway involving the host nucleotide-binding domain and leucine-rich repeat-containing protein Ipaf (NLRC4). Asc-dependent caspase-1 activation was inhibited by high extracellular potassium levels, whereas Ipaf-dependent activation was unaffected by potassium treatment. Activation of caspase-1 in macrophages occurred independently of Nalp3 and proteasome activity, suggesting that a previously uncharacterized mechanism for caspase-1 activation through Asc may be triggered by L. pneumophila. Rapid pore formation and pyroptosis induced by L. pneumophila required caspase-1, Ipaf, and bacterial flagellin but occurred independently of Asc. Equivalent levels of active interleukin-18 (IL-18) were detected in the lungs of mice infected with a flagellin-deficient strain of L. pneumophila and Asc-deficient mice infected with wild-type L. pneumophila. Active IL-18 was undetectable in the lungs of Asc-deficient mice infected with an L. pneumophila flagellin mutant, indicating independent roles for Ipaf and Asc in caspase-1-mediated processing and release of IL-18 in vivo. Ipaf-dependent activation of caspase-1 restricted bacterial replication in vivo, whereas Asc was dispensable for restriction of L. pneumophila replication in mice. Thus, L. pneumophila-mediated caspase-1 activation involves the coordinate activities of inflammasomes differentially regulated by Ipaf and Asc.

 

The LetA-RsmYZ-CsrA regulatory cascade, together with RpoS and PmrA, post-transcriptionally regulates stationary phase activation of Legionella pneumophila Icm/Dot effectors

Rasis M, Segal G.

Department of Molecular Microbiology and Biotechnology, George S. Wise Faculty of Life Sciences, Tel.Aviv University, Ramat-Aviv, Tel.Aviv 69978, Israel. gils@tauex.tau.ac.il

Mol Microbiol. 2009 May;72(4):995-1010.

ABSTRACT: Legionella pneumophila utilize the Icm/Dot type-IV secretion system to translocate effector proteins into host cells. Some of these effectors were shown before to be regulated at the transcriptional level by the PmrAB and CpxRA two-component systems. In addition, the stationary phase-related regulators LetA and CsrA, which are both members of the same post-transcriptional regulatory cascade, were shown to be involved in L. pneumophila virulence. In this report, we identified two small non-coding RNAs which are part of the LetA-CsrA regulatory cascade and three effector-encoding genes which are directly controlled by this regulatory system. We found that the small non-coding RNAs RsmY and RsmZ, were upregulated by LetA at stationary phase, and relieve the repression of CsrA from its target genes. The three effector-encoding genes were found to be post-transcriptionally upregulated at stationary phase and to contain CsrA regulatory elements that were found to be essential for their stationary phase activation. In addition, rsmY and rsmZ were found to be regulated by the RpoS sigma-factor and the csrA encoding gene was found to be regulated by PmrA. Our results demonstrate that L. pneumophila effectors are regulated at both the transcriptional and the post-transcriptional levels by a complicated regulatory network.

 

Structure and function of interacting IcmR-IcmQ domains from a type IVb secretion system in Legionella pneumophila

Raychaudhury S, Farelli JD, Montminy TP, Matthews M, Ménétret JF, Duménil G, Roy CR, Head JF, Isberg RR, Akey CW.

Department of Physiology and Biophysics, Boston University School of Medicine, Boston, MA 02118-2526, USA. cakey@bu.edu

Structure. 2009 Apr 15;17(4):590-601.

ABSTRACT: During infection, Legionella pneumophila creates a replication vacuole within eukaryotic cells and this requires a Type IVb secretion system (T4bSS). IcmQ plays a critical role in the translocase and associates with IcmR. In this paper, we show that the N-terminal domain of IcmQ (Qn) mediates self-dimerization, whereas the C-terminal domain with a basic linker promotes membrane association. In addition, the binding of IcmR to IcmQ prevents self-dimerization and also blocks membrane permeabilization. However, IcmR does not completely block membrane binding by IcmQ. We then determined crystal structures of Qn with the interacting region of IcmR. In this complex, each protein forms an alpha-helical hairpin within a parallel four-helix bundle. The amphipathic nature of helices in Qn suggests two possible models for membrane permeabilization by IcmQ. The Rm-Qn structure also suggests how IcmR-like proteins in other L. pneumophila species may interact with their IcmQ partners.

 

Reciprocal expression of integration host factor and HU in the developmental cycle and infectivity of Legionella pneumophila

Morash MG, Brassinga AK, Warthan M, Gourabathini P, Garduño RA, Goodman SD, Hoffman PS.

Department of Microbiology and Immunology, Faculty of Medicine, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4H7. psh2n@virginia.edu

Appl Environ Microbiol. 2009 Apr;75(7):1826-37.

ABSTRACT: Legionella pneumophila is an intracellular parasite of protozoa that differentiates late in infection into metabolically dormant cysts that are highly infectious. Regulation of this process is poorly understood. Here we report that the small DNA binding regulatory proteins integration host factor (IHF) and HU are reciprocally expressed over the developmental cycle, with HU expressed during exponential phase and IHF expressed postexponentially. To assess the role of these regulatory proteins in development, chromosomal deletions were constructed. Single (ihfA or ihfB) and double deletion (Deltaihf) IHF mutants failed to grow in Acanthamoeba castellanii unless complemented in trans when expressed temporally from the ihfA promoter but not under P(tac) (isopropyl-beta-d-thiogalactopyranoside). In contrast, IHF mutants were infectious for HeLa cells, though electron microscopic examination revealed defects in late-stage cyst morphogenesis (thickened cell wall, intracytoplasmic membranes, and inclusions of poly-beta-hydroxybutyrate), and were depressed for the developmental marker MagA. Green fluorescent protein promoter fusion assays indicated that IHF and the stationary-phase sigma factor RpoS were required for full postexponential expression of magA. Finally, defects in cyst morphogenesis noted for Deltaihf mutants in HeLa cells correlated with a loss of both detergent resistance and hyperinfectivity compared with results for wild-type cysts. These studies establish IHF and HU as markers of developmental stages and show that IHF function is required for both differentiation and full virulence of L. pneumophila in natural amoebic hosts.

 

SigmaS controls multiple pathways associated with intracellular multiplication of Legionella pneumophila

Hovel-Miner G, Pampou S, Faucher SP, Clarke M, Morozova I, Morozov P, Russo JJ, Shuman HA, Kalachikov S.

Department of Microbiology, Columbia University Medical Center, 701 West 168th Street, New York, NY 10032, USA. has7@columbia.edu

J Bacteriol. 2009 Apr;191(8):2461-73.

ABSTRACT: Legionella pneumophila is the causative agent of the severe and potentially fatal pneumonia Legionnaires' disease. L. pneumophila is able to replicate within macrophages and protozoa by establishing a replicative compartment in a process that requires the Icm/Dot type IVB secretion system. The signals and regulatory pathways required for Legionella infection and intracellular replication are poorly understood. Mutation of the rpoS gene, which encodes sigma(S), does not affect growth in rich medium but severely decreases L. pneumophila intracellular multiplication within protozoan hosts. To gain insight into the intracellular multiplication defect of an rpoS mutant, we examined its pattern of gene expression during exponential and postexponential growth. We found that sigma(S) affects distinct groups of genes that contribute to Legionella intracellular multiplication. We demonstrate that rpoS mutants have a functional Icm/Dot system yet are defective for the expression of many genes encoding Icm/Dot-translocated substrates. We also show that sigma(S) affects the transcription of the cpxR and pmrA genes, which encode two-component response regulators that directly affect the transcription of Icm/Dot substrates. Our characterization of the L. pneumophila small RNA csrB homologs, rsmY and rsmZ, introduces a link between sigma(S) and the posttranscriptional regulator CsrA. We analyzed the network of sigma(S)-controlled genes by mutational analysis of transcriptional regulators affected by sigma(S). One of these, encoding the L. pneumophila arginine repressor homolog gene, argR, is required for maximal intracellular growth in amoebae. These data show that sigma(S) is a key regulator of multiple pathways required for L. pneumophila intracellular multiplication.

 

Caspase-7 activation by the Nlrc4/Ipaf inflammasome restricts Legionella pneumophila infection

Akhter A, Gavrilin MA, Frantz L, Washington S, Ditty C, Limoli D, Day C, Sarkar A, Newland C, Butchar J, Marsh CB, Wewers MD, Tridandapani S, Kanneganti TD, Amer AO.

Division of Pulmonary, Allergy, Critical Care, and Sleep Medicine, Center for Microbial Interface Biology and the Department of Internal Medicine, Ohio State University, Columbus, OH, USA. Thirumala-Devi.Kanneganti@StJude.org

PLoS Pathog. 2009 Apr;5(4):e1000361.

ABSTRACT: Legionella pneumophila (L. pneumophila), the causative agent of a severe form of pneumonia called Legionnaires' disease, replicates in human monocytes and macrophages. Most inbred mouse strains are restrictive to L. pneumophila infection except for the A/J, Nlrc4(-/-) (Ipaf(-/-)), and caspase-1(-/-) derived macrophages. Particularly, caspase-1 activation is detected during L. pneumophila infection of murine macrophages while absent in human cells. Recent in vitro experiments demonstrate that caspase-7 is cleaved by caspase-1. However, the biological role for caspase-7 activation downstream of caspase-1 is not known. Furthermore, whether this reaction is pertinent to the apoptosis or to the inflammation pathway or whether it mediates a yet unidentified effect is unclear. Using the intracellular pathogen L. pneumophila, we show that, upon infection of murine macrophages, caspase-7 was activated downstream of the Nlrc4 inflammasome and required caspase-1 activation. Such activation of caspase-7 was mediated by flagellin and required a functional Naip5. Remarkably, mice lacking caspase-7 and its macrophages allowed substantial L. pneumophila replication. Permissiveness of caspase-7(-/-) macrophages to the intracellular pathogen was due to defective delivery of the organism to the lysosome and to delayed cell death during early stages of infection. These results reveal a new mechanism for caspase-7 activation downstream of the Nlrc4 inflammasome and present a novel biological role for caspase-7 in host defense against an intracellular bacterium.

 

 

Evidence that the intra-amoebal Legionella drancourtii acquired a sterol reductase gene from eukaryotes

Moliner C, Raoult D, Fournier PE.

URMITE CNRS-IRD UMR 6236, Faculté de Médecine, 27 boulevard Jean Moulin, 13385 Marseille, Cedex 05, France. Pierre-Edouard.Fournier@medecine.univ-mrs.fr.

BMC Res Notes. 2009 Mar 27;2:51.

ABSTRACT: BACKGROUND: Free-living amoebae serve as a natural reservoir for some bacteria that have evolved into <<amoeba-resistant>> bacteria. Among these, some are strictly intra-amoebal, such as Candidatus "Protochlamydia amoebophila" (Candidatus "P. amoebophila"), whose genomic sequence is available. We sequenced the genome of Legionella drancourtii (L. drancourtii), another recently described intra-amoebal bacterium. By comparing these two genomes with those of their closely related species, we were able to study the genetic characteristics specific to their amoebal lifestyle. FINDINGS: We identified a sterol delta-7 reductase-encoding gene common to these two bacteria and absent in their relatives. This gene encodes an enzyme which catalyses the last step of cholesterol biosynthesis in eukaryotes, and is probably functional within L. drancourtii since it is transcribed. The phylogenetic analysis of this protein suggests that it was acquired horizontally by a few bacteria from viridiplantae. This gene was also found in the Acanthamoeba polyphaga Mimivirus genome, a virus that grows in amoebae and possesses the largest viral genome known to date. CONCLUSION: L. drancourtii acquired a sterol delta-7 reductase-encoding gene of viridiplantae origin. The most parsimonious hypothesis is that this gene was initially acquired by a Chlamydiales ancestor parasite of plants. Subsequently, its descendents transmitted this gene in amoebae to other intra-amoebal microorganisms, including L. drancourtii and Coxiella burnetii. The role of the sterol delta-7 reductase in prokaryotes is as yet unknown but we speculate that it is involved in host cholesterol parasitism.

 

The inositol polyphosphate 5-phosphatase OCRL1 restricts intracellular growth of Legionella, localizes to the replicative vacuole and binds to the bacterial effector LpnE

Weber SS, Ragaz C, Hilbi H.

Institute of Microbiology, ETH Zürich, Zürich, Switzerland. hilbi@micro.biol.ethz.ch

Cell Microbiol. 2009 Mar;11(3):442-60.

ABSTRACT: Legionella pneumophila, the causative agent of Legionnaires' disease, replicates within a specific vacuole in amoebae and macrophages. To form these 'Legionella-containing vacuoles' (LCVs), the bacteria employ the Icm/Dot type IV secretion system and effector proteins, some of which anchor to the LCV membrane via the host glycolipid phosphatidylinositol 4-phosphate [PtdIns(4)P]. Here we analysed the role of inositol polyphosphate 5-phosphatases (IP5Ps) during L. pneumophila infections. Bacterial replication and LCV formation occurred more efficiently in Dictyostelium discoideum amoebae lacking the IP5P Dd5P4, a homologue of human OCRL1 (Oculocerebrorenal syndrome of Lowe), implicated in retrograde endosome to Golgi trafficking. The phenotype was complemented by Dd5P4 but not the catalytically inactive 5-phosphatase. Ectopically expressed Dd5P4 or OCRL1 localized to LCVs in D. discoideum via an N-terminal domain previously not implicated in membrane targeting, and OCRL1 was also identified on LCVs in macrophages. Dd5P4 was catalytically active on LCVs and accumulated on LCVs harbouring wild-type but not DeltaicmT mutant L. pneumophila. The N-terminal domain of OCRL1 bound L. pneumophila LpnE, a Sel1-like repeat protein involved in LCV formation, which localizes to LCVs and selectively binds PtdIns(3)P. Our results indicate that OCRL1 restricts intracellular growth of L. pneumophila and binds to LCVs in association with LpnE.

 

Surface translocation by Legionella pneumophila: a form of sliding motility that is dependent upon type II protein secretion

Stewart CR, Rossier O, Cianciotto NP.

Department of Microbiology and Immunology, Northwestern University Medical School, Chicago, IL 60611, USA. n-cianciotto@northwestern.edu

J Bacteriol. 2009 Mar;191(5):1537-46.

ABSTRACT: Legionella pneumophila exhibits surface translocation when it is grown on a buffered charcoal yeast extract (BCYE) containing 0.5 to 1.0% agar. After 7 to 22 days of incubation, spreading legionellae appear in an amorphous, lobed pattern that is most manifest at 25 to 30 degrees C. All nine L. pneumophila strains examined displayed the phenotype. Surface translocation was also exhibited by some, but not all, other Legionella species examined. L. pneumophila mutants that were lacking flagella and/or type IV pili behaved as the wild type did when plated on low-percentage agar, indicating that the surface translocation is not swarming or twitching motility. A translucent film was visible atop the BCYE agar, advancing ahead of the spreading legionellae. Based on its abilities to disperse water droplets and to promote the spreading of heterologous bacteria, the film appeared to manipulate surface tension and, as such, acted like a surfactant. Indeed, a sample obtained from the film rapidly dispersed when it was spotted onto a plastic surface. L. pneumophila type II secretion (Lsp) mutants, but not their complemented derivatives, were defective for both surface translocation and film production. In contrast, mutants defective for type IV secretion exhibited normal surface translocation. When lsp mutants were spotted onto film produced by the wild type, they were able to spread, suggesting that type II secretion promotes the elaboration of the Legionella surfactant. Together, these data indicate that L. pneumophila exhibits a form of surface translocation that is most akin to "sliding motility" and uniquely dependent upon type II secretion.

 

 

CB(1) and CB(2) cannabinoid receptors mediate different aspects of delta-9-tetrahydrocannabinol (THC)-induced T helper cell shift following immune activation by Legionella pneumophila infection

Newton CA, Chou PJ, Perkins I, Klein TW.

Department of Molecular Medicine, University of South Florida, Tampa, FL 33612, USA. cnewton@health.usf.edu

J Neuroimmune Pharmacol. 2009 Mar;4(1):92-102.

ABSTRACT: Legionella pneumophila infection of mice induces proinflammatory cytokines and Th1 immunity as well as rapid increases in serum levels of IL-12 and IFNgamma and splenic IL-12Rbeta2 expression. Delta-9-tetrahydrocannabinol (THC) treatment prior to infection causes a shift from Th1 to Th2 immunity and here we demonstrate that CB(1) and CB(2) cannabinoid receptors mediate different aspects of the shift. Using cannabinoid receptor antagonists and cannabinoid receptor gene deficient mice (CB(1) (-/-) and CB(2) (-/-)), we showed that both CB(1) and CB(2) receptors were involved in the THC-induced attenuation of serum IL-12 and IFNgamma. IFNgamma production is dependent upon signaling through IL-12Rbeta2 (beta2) and THC treatment suppressed splenic beta2 message; moreover, this effect was CB(1) but not CB(2)-dependent from studies with receptor antagonists and CB1(-/-) and CB2(-/-) mice. Furthermore, observed increases in IL-4 induced by THC, were not involved in the drug effect on beta2 from studies with IL-4 deficient mice. The GATA-3 transcription factor is necessary for IL-4 production and is selectively expressed in Th2 cells. GATA-3 message levels were elevated in spleens of THC-treated and L. pneumophila-infected mice and the effect was shown to be CB(2) but not CB(1)-dependent. Furthermore, GATA-3 regulatory factors were modulated in that Notch ligand Delta4 mRNA was decreased and Jagged1 increased by THC also in a CB2-dependent manner and splenic NFkappaB p65 was increased. Together, these results indicate that CB(1) and CB(2) mediate the THC-induced shift in T helper activity in L. pneumophila-infected mice, with CB(1) involved in suppressing IL-12Rbeta2 and CB(2) involved in enhancing GATA-3.

 

 

A type II secreted RNase of Legionella pneumophila facilitates optimal intracellular infection of Hartmannella vermiformis

Rossier O, Dao J, Cianciotto NP.

Department of Microbiology-Immunology, Northwestern University Medical School, Chicago, IL 60611, USA. n-cianciotto@northwestern.edu

Microbiology. 2009 Mar;155(Pt 3):882-90.

ABSTRACT: Type II protein secretion plays a role in a wide variety of functions that are important for the ecology and pathogenesis of Legionella pneumophila. Perhaps most dramatic is the critical role that this secretion pathway has in L. pneumophila intracellular infection of aquatic protozoa. Recently, we showed that virulent L. pneumophila strain 130b secretes RNase activity through its type II secretion system. We now report the cloning and mutational analysis of the gene (srnA) encoding that novel type of secreted activity. The SrnA protein was defined as being a member of the T2 family of secreted RNases. Supernatants from mutants inactivated for srnA completely lacked RNase activity, indicating that SrnA is the major secreted RNase of L. pneumophila. Although srnA mutants grew normally in bacteriological media and human U937 cell macrophages, they were impaired in their ability to grow within Hartmannella vermiformis amoebae. This finding represents the second identification of a L. pneumophila type II effector being necessary for optimal intracellular infection of amoebae, with the first being the ProA zinc metalloprotease. Newly constructed srnA proA double mutants displayed an even larger infection defect that appeared to be the additive result of losing both SrnA and ProA. Overall, these data represent the first demonstration of a secreted RNase promoting an intracellular infection event, and support our long-standing hypothesis that the infection defects of L. pneumophila type II secretion mutants are due to the loss of multiple secreted effectors.

 

Legionella pneumophila couples fatty acid flux to microbial differentiation and virulence

Edwards RL, Dalebroux ZD, Swanson MS.

Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI, USA. mswanson@umich.edu

Mol Microbiol. 2009 Mar;71(5):1190-1204.

ABSTRACT: During its life cycle, Legionella pneumophila alternates between at least two phenotypes: a resilient, infectious form equipped for transmission and a replicative cell type that grows in amoebae and macrophages. Considering its versatility, we postulated that multiple cues regulate L. pneumophila differentiation. Beginning with a Biolog Phenotype MicroArray screen, we demonstrate that excess short-chain fatty acids (SCFAs) trigger replicative cells to cease growth and activate their panel of transmissive traits. To co-ordinate their response to SCFAs, L. pneumophila utilizes the LetA/LetS two-component system, but not phosphotransacetylase or acetyl kinase, two enzymes that generate high-energy phosphate intermediates. Instead, the stringent response enzyme SpoT appears to monitor fatty acid biosynthesis to govern transmission trait expression, as an altered distribution of acylated acyl carrier proteins correlated with the SpoT-dependent differentiation of cells treated with either excess SCFAs or the fatty acid biosynthesis inhibitors cerulenin and 5-(tetradecyloxy)-2-furoic acid. We postulate that, by exploiting the stringent response pathway to couple cellular differentiation to its metabolic state, L. pneumophila swiftly acclimates to stresses encountered in its host or the environment, thereby enhancing its overall fitness.

 

 

Rab1 guanine nucleotide exchange factor SidM is a major phosphatidylinositol 4-phosphate-binding effector protein of Legionella pneumophila

Brombacher E, Urwyler S, Ragaz C, Weber SS, Kami K, Overduin M, Hilbi H.

Institute of Microbiology, ETH Zürich, Wolfgang-Pauli-Strasse 10, 8093 Zürich, Switzerland. hilbi@micro.biol.ethz.ch

J Biol Chem. 2009 Feb 20;284(8):4846-56.

ABSTRACT: The causative agent of Legionnaires disease, Legionella pneumophila, forms a replicative vacuole in phagocytes by means of the intracellular multiplication/defective organelle trafficking (Icm/Dot) type IV secretion system and translocated effector proteins, some of which subvert host GTP and phosphoinositide (PI) metabolism. The Icm/Dot substrate SidC anchors to the membrane of Legionella-containing vacuoles (LCVs) by specifically binding to phosphatidylinositol 4-phosphate (PtdIns(4)P). Using a nonbiased screen for novel L. pneumophila PI-binding proteins, we identified the Rab1 guanine nucleotide exchange factor (GEF) SidM/DrrA as the predominant PtdIns(4)P-binding protein. Purified SidM specifically and directly bound to PtdIns(4)P, whereas the SidM-interacting Icm/Dot substrate LidA preferentially bound PtdIns(3)P but also PtdIns(4)P, and the L. pneumophila Arf1 GEF RalF did not bind to any PIs. The PtdIns(4)P-binding domain of SidM was mapped to the 12-kDa C-terminal sequence, termed "P4M" (PtdIns4P binding of SidM/DrrA). The isolated P4M domain is largely helical and displayed higher PtdIns(4)P binding activity in the context of the alpha-helical, monomeric full-length protein. SidM constructs containing P4M were translocated by Icm/Dot-proficient L. pneumophila and localized to the LCV membrane, indicating that SidM anchors to PtdIns(4)P on LCVs via its P4M domain. An L. pneumophila DeltasidM mutant strain displayed significantly higher amounts of SidC on LCVs, suggesting that SidM and SidC compete for limiting amounts of PtdIns(4)P on the vacuole. Finally, RNA interference revealed that PtdIns(4)P on LCVs is specifically formed by host PtdIns 4-kinase IIIbeta. Thus, L. pneumophila exploits PtdIns(4)P produced by PtdIns 4-kinase IIIbeta to anchor the effectors SidC and SidM to LCVs.

 

 

Legionella pneumophila Dot/Icm translocated substrates: a sum of parts

Ensminger AW, Isberg RR.

Department of Molecular Biology and Microbiology, Tufts University School of Medicine, 150 Harrison Avenue, Boston, MA 02111, USA. ralph.isberg@tufts.edu

Curr Opin Microbiol. 2009 Feb;12(1):67-73.

ABSTRACT: Legionella pneumophila is an intracellular pathogen of freshwater amoeba and of alveolar macrophages in human hosts. After phagocytosis, L. pneumophila establishes a unique intracellular vacuolar niche that avoids entry into the lysosomal network. Critical for L. pneumophila intracellular growth is the Dot/Icm type IVB translocation system. Although over 80 substrates of the Dot/Icm apparatus have been identified, individual substrates are often genetically redundant, complicating their analysis. Deletion of critical Dot/Icm translocation system components causes a variety of defects during intracellular growth. Many of these effects on the host cell likely result from the actions of one or more Dot/Icm translocated substrates. Loss of single substrates never generates the profound effects observed in strains lacking translocation system components.

 

 

Modulation of ubiquitin dynamics and suppression of DALIS formation by the Legionella pneumophila Dot/Icm system

Ivanov SS, Roy CR.

Section of Microbial Pathogenesis, Yale University School of Medicine, Boyer Center for Molecular Medicine, New Haven, CT 06536, USA. craig.roy@yale.edu

Cell Microbiol. 2009 Feb;11(2):261-78.

ABSTRACT: Legionella pneumophila is an intracellular pathogen that uses effector proteins translocated by the Dot/Icm type IV secretion system to modulate host cellular processes. Here we investigate the dynamics of subcellular structures containing ubiquitin during L. pneumophila infection of phagocytic host cells. The Dot/Icm system mediated the formation of K48 and K63 poly-ubiquitin conjugates to proteins associated with L. pneumophila-containing vacuoles in macrophages and dendritic cells, suggesting that regulatory events and degradative events involving ubiquitin are regulated by bacterial effectors during infection. Stimulation of TLR2 on the surface of macrophages and dendritic cells by L. pneumophila-derived molecules resulted in the production of ubiquitin-rich dendritic cell aggresome-like structures (DALIS). Cells infected by L. pneumophila with a functional Dot/Icm system, however, failed to produce DALIS. Suppression of DALIS formation did not affect the accumulation of ubiquitinated proteins on vacuoles containing L. pneumophila. Examining other species of Legionella revealed that Legionella jordanis was unable to suppress DALIS formation after creating a ubiquitin-decorated vacuole. Thus, the L. pneumophila Dot/Icm system has the ability to modulate host processes to promote K48 and K63 ubiquitin conjugates on proteins at the vacuole membrane, and independently suppress cellular events required for the formation of DALIS.

  

Large-scale identification of Legionella pneumophila Dot/Icm substrates that modulate host cell vesicle trafficking pathways

Heidtman M, Chen EJ, Moy MY, Isberg RR.

Department of Molecular Biology and Microbiology, Tufts University School of Medicine, 150 Harrison Avenue, Boston, MA 02111, USA. Ralph.Isberg@Tufts.edu

Cell Microbiol. 2009 Feb;11(2):230-48.

ABSTRACT: The bacterial pathogen Legionella pneumophila replicates in a specialized vacuole within host cells. Establishment of the replication vacuole depends on the Dot/Icm translocation system that delivers a large number of protein substrates into the host cell. The functions of most substrates are unknown. Here, we analysed a defined set of 127 confirmed or candidate Dot/Icm substrates for their effect on host cell processes using yeast as a model system. Expression of 79 candidates caused significant yeast growth defects, indicating that these proteins impact essential host cell pathways. Notably, a group of 21 candidates interfered with the trafficking of secretory proteins to the yeast vacuole. Three candidates that caused yeast secretory defects (SetA, Ceg19 and Ceg9) were investigated further. These proteins impinged upon vesicle trafficking at distinct stages and had signals that allowed translocation into host cells by the Dot/Icm system. Ectopically produced SetA, Ceg19 and Ceg9 localized to secretory organelles in mammalian cells, consistent with a role for these proteins in modulating host cell vesicle trafficking. Interestingly, the ability of SetA to cause yeast phenotypes was dependent upon a functional glycosyltransferase domain. We hypothesize that SetA may glycosylate a component of the host cell vesicle trafficking machinery during L. pneumophila infection.

  

Profiling of environmental Legionella pneumophila strains by randomly amplified polymorphic DNA method isolated from geographically nearby buildings

Zeybek Z, Türetgen I, Kimiran Erdem A, Filoğlu G, Cotuk A.

Department of Biology, Faculty of Science, Istanbul University, Istanbul, Turkey, zzeybek@yahoo.com.

Environ Monit Assess. 2009 Feb;149(1-4):323-327.

ABSTRACT: Legionella pneumophila (L. pneumophila) which is also known as etiologic agent Legionnaires Disease lives in natural water and man made water systems. These bacteria belonging to Legionellaceae family are divided 15 serogroups. Phenotypical methods used for the identification of Legionella isolates are not very discriminatory. In this study we investigated genotypic features of eight L. pneumophila serogroup 1 and 18 L. pneumophila serogroup 2-14 strains isolated from different buildings in Istanbul by randomly amplified polymorphic DNA (RAPD) method. Eight L. pneumophila serogroup 1 strains (37.5%) were similar RAPD profile and they were isolated from buildings located in a short distance (about 500 m). Four L. pneumophila serogroup 2-14 strains (22%) were identical genotypically. Three of these strains were isolated from buildings located in a short distance.

  

Cytopathogenicity and molecular subtyping of Legionella pneumophila environmental isolates from 17 hospitals

Garcia-Nuñez M, Pedro-Botet ML, Ragull S, Sopena N, Morera J, Rey-Joly C, Sabria M.

Infectious Diseases Section, Fundació Institut d'Investigació Germans Trias i Pujol, Badalona, Autonomous University of Barcelona. mgarcia.igtp.germanstrias@gencat.net

Epidemiol Infect. 2009 Feb;137(2):188-93.

ABSTRACT: SUMMARYThe cytopathogenicity of 22 Legionella pneumophila isolates from 17 hospitals was determined by assessing the dose of bacteria necessary to produce 50% cytopathic effect (CPED50) in U937 human-derived macrophages. All isolates were able to infect and grow in macrophage-like cells (range log10 CPED50: 2.67-6.73 c.f.u./ml). Five groups were established and related to the serogroup, the number of PFGE patterns coexisting in the same hospital water distribution system, and the possible reporting of hospital-acquired Legionnaires' disease cases. L. pneumophila serogroup 1 isolates had the highest cytopathogenicity (P=0.003). Moreover, a trend to more cytopathogenic groups (groups 1-3) in hospitals with more than one PFGE pattern of L. pneumophila in the water distribution system (60% vs. 17%) and in hospitals reporting cases of hospital-acquired Legionnaires' disease (36.3% vs. 16.6%) was observed. We conclude that the cytopathogenicty of environmental L. pneumophila should be taken into account in evaluating the risk of a contaminated water reservoir in a hospital and hospital acquisition of Legionnaires' disease.

  

SpoT governs Legionella pneumophila differentiation in host macrophages

Dalebroux ZD, Edwards RL, Swanson MS.

Department of Microbiology & Immunology, University of Michigan Medical School, Ann Arbor, MI, USA. mswanson@umich.edu

Mol Microbiol. 2009 Feb 1;71(3):640-658.

ABSTRACT: During its life cycle, Legionella pneumophila alternates between a replicative and a transmissive state. To determine their contributions to L. pneumophila differentiation, the two ppGpp synthetases, RelA and SpoT, were disrupted. Synthesis of ppGpp was required for transmission, as relA spoT mutants were killed during entry to and exit from macrophages. RelA, which senses amino acid starvation induced by serine hydroxamate, is dispensable in macrophages, as relA mutants spread efficiently. SpoT monitors fatty acid biosynthesis (FAB), since following cerulenin treatment, wild-type and relA strains expressed the flaA transmissive gene, but relA spoT mutants did not. As in Escherichia coli, the SpoT response to FAB perturbation likely required an interaction with acyl-carrier protein (ACP), as judged by the failure of the spoT-A413E allele to rescue transmissive trait expression of relA spoT bacteria. Furthermore, SpoT was essential for transmission between macrophages, since secondary infections by relA spoT mutants were restored by induction of spoT, but not relA. To resume replication, ppGpp must be degraded, as mutants lacking spoT hydrolase activity failed to convert from the transmissive to the replicative phase in either bacteriological medium or macrophages. Thus, L. pneumophila requires SpoT to monitor FAB and to alternate between replication and transmission in macrophages.

 

Multiple MyD88-dependent responses contribute to pulmonary clearance of Legionella pneumophila

Archer KA, Alexopoulou L, Flavell RA, Roy CR.

Section of Microbial Pathogenesis, Yale University School of Medicine, Boyer Center for Molecular Medicine, 295 Congress Avenue, New Haven, CT 06536, USA. craig.roy@yale.edu

Cell Microbiol. 2009 Jan;11(1):21-36.

ABSTRACT: MyD88-dependent signalling is important for secretion of early inflammatory cytokines and host protection in response to Legionella pneumophila infection. Although toll-like receptor (TLR)2 contributes to MyD88-dependent clearance of L. pneumophila, TLR-independent functions of MyD88 could also be important. To determine why MyD88 is critical for host protection to L. pneumophila, the contribution of multiple TLRs and IL-18 receptor (IL-18R)-dependent interferon-gamma (IFN-gamma) production in a mouse was examined. Mice deficient for TLR5 or TLR9, or deficient for TLR2 along with either TLR5 or TLR9, were competent for controlling bacterial replication and had no apparent defects in cytokine production compared with control mice. MyD88-dependent production of IFN-gamma in the lung was mediated primarily by natural killer cells and required IL-18R signalling. Reducing IFN-gamma levels did not greatly affect the kinetics of L. pneumophila replication or clearance in infected mice. Additionally, IFN-gamma-deficient mice did not have a susceptibility phenotype as severe as the MyD88-deficient mice and were able to control a pulmonary infection by L. pneumophila. Thus, MyD88-dependent innate immune responses induced by L. pneumophila involve both TLR-dependent responses and IL-18R-dependent production of IFN-gamma by natural killer cells, and these MyD88-dependent pathways can function independently to provide host protection against an intracellular pathogen.

  

NLRC4/IPAF: a CARD carrying member of the NLR family

Sutterwala FS, Flavell RA.

Inflammation Program, Department of Internal Medicine, University of Iowa, Iowa City, IA 52241, USA.

fayyaz-sutterwala@uiowa.edu

Clin Immunol. 2009 Jan;130(1):2-6.

ABSTRACT: The NOD-like receptor (NLR) family of proteins is involved in the regulation of innate immune responses and cell death pathways. Recent findings show that the NLR family member NLRC4 (also known as IPAF) has important roles in innate immune responses to Gram-negative bacteria. Macrophages infected with Legionella pneumophila, Salmonella typhimurium, Shigella flexneri, or Pseudomonas aeruginosa activate caspase-1 in an NLRC4-dependent manner leading to macrophage cell death and the release of proinflammatory cytokines. This review will discuss these findings as well as the role of bacterial type III and type IV secretion systems and flagellin in NLRC4-mediated caspase-1 activation.

 

A hemidominant Naip5 allele in mouse strain MOLF/Ei-derived macrophages restricts Legionella pneumophila intracellular growth

Losick VP, Stephan K, Smirnova II, Isberg RR, Poltorak A.

Department of Molecular Biology and Microbiology, Tufts University School of Medicine, 150 Harrison Ave., Boston, MA 02111, USA. alexander.poltorak@tufts.edu

Infect Immun. 2009 Jan;77(1):196-204.

ABSTRACT: Mouse-derived macrophages have the unique ability to restrict or permit Legionella pneumophila intracellular growth. The common inbred mouse strain C57BL/6J (B6) restricts L. pneumophila growth, whereas macrophages derived from A/J mice allow >10(3)-fold bacterial growth within three days. This phenotypic difference was mapped to the mouse Naip5 allele. The B6 restrictive Naip5 allele is dominant, and six amino acid changes in its product were predicted to control permissiveness. By using the wild-derived mouse strain MOLF/Ei, we found that MOLF/Ei-derived macrophages also restrict L. pneumophila growth, yet the Naip5 protein is identical to the A/J Naip5 at the six-amino-acid signature. The MOLF/Ei restrictive trait, unlike that of B6-derived macrophages, was not dominant over the A/J trait. In spite of this phenotypic difference, the L. pneumophila growth restriction in MOLF/Ei macrophages was mapped to the Naip5 region as well, indicating that the originally predicted change in the A/J Naip5 allele may not be critical for restriction. In the product of the A/J Naip5 permissive allele, there are four unique amino acid changes that map to a NACHT-like domain. Similar misregulating mutations have been identified in the NACHT domains of Nod-like receptor (NLR) proteins. Therefore, one of these mutations may be critical for restriction of L. pneumophila intracellular growth, and this parallels results found with human NLR variants with defects in the innate immune response.

  

The PmrA/PmrB two-component system of Legionella pneumophila is a global regulator required for intracellular replication within macrophages and protozoa

Al-Khodor S, Kalachikov S, Morozova I, Price CT, Abu Kwaik Y.

Department of Microbiology and Immunology, College of Medicine, University of Louisville, Room 412, Louisville, KY 40202, USA. abukwaik@louisville.edu

Infect Immun. 2009 Jan;77(1):374-86.

ABSTRACT: To examine the role of the PmrA/PmrB two-component system (TCS) of Legionella pneumophila in global gene regulation and in intracellular infection, we constructed pmrA and pmrB isogenic mutants by allelic exchange. Genome-wide microarray gene expression analyses of the pmrA and pmrB mutants at both the exponential and the postexponential phases have shown that the PmrA/PmrB TCS has a global effect on the expression of 279 genes classified into nine groups of genes encoding eukaryotic-like proteins, Dot/Icm apparatus and secreted effectors, type II-secreted proteins, regulators of the postexponential phase, stress response genes, flagellar biosynthesis genes, metabolic genes, and genes of unknown function. Forty-one genes were differentially regulated in the pmrA or pmrB mutant, suggesting a possible cross talk with other TCSs. The pmrB mutant is more sensitive to low pH than the pmrA mutant and the wild-type strain, suggesting that acidity may trigger this TCS. The pmrB mutant exhibits a significant defect in intracellular proliferation within human macrophages, Acanthamoeba polyphaga, and the ciliate Tetrahymena pyriformis. In contrast, the pmrA mutant is defective only in the ciliate. Despite the intracellular growth defect within human macrophages, phagosomes harboring the pmrB mutant exclude late endosomal and lysosomal markers and are remodeled by the rough endoplasmic reticulum. Similar to the dot/icm mutants, the intracellular growth defect of the pmrB mutant is totally rescued in cis within communal phagosomes harboring the wild-type strain. We conclude that the PmrA/PmrB TCS has a global effect on gene expression and is required for the intracellular proliferation of L. pneumophila within human macrophages and protozoa. Differences in gene regulation and intracellular growth phenotypes between the pmrA and pmrB mutant suggests a cross talk with other TCSs.

 

The Legionella pneumophila replication vacuole: making a cosy niche inside host cells

Isberg RR, O'Connor TJ, Heidtman M.

Howard Hughes Medical Institute, Tufts University School of Medicine, Boston, Massachusetts 02111, USA. Ralph.Isberg@Tufts.edu

Nat Rev Microbiol. 2009 Jan;7(1):13-24.

ABSTRACT: The pathogenesis of Legionella pneumophila is derived from its growth within lung macrophages after aerosols are inhaled from contaminated water sources. Interest in this bacterium stems from its ability to manipulate host cell vesicular-trafficking pathways and establish a membrane-bound replication vacuole, making it a model for intravacuolar pathogens. Establishment of the replication compartment requires a specialized translocation system that transports a large cadre of protein substrates across the vacuolar membrane. These substrates regulate vesicle traffic and survival pathways in the host cell. This Review focuses on the strategies that L. pneumophila uses to establish intracellular growth and evaluates why this microorganism has accumulated an unprecedented number of translocated substrates that are targeted at host cells.

  

A Legionella pneumophila Effector Protein Encoded in a Region of Genomic Plasticity Binds to Dot/Icm-Modified Vacuoles

Ninio S, Celli J, Roy CR.

Section of Microbial Pathogenesis, Yale University School of Medicine, Boyer Center for Molecular Medicine, New Haven, Connecticut, United States of America. craig.roy@yale.edu

PLoS Pathog. 2009 Jan;5(1):e1000278.

ABSTRACT: Legionella pneumophila is an opportunistic pathogen that can cause a severe pneumonia called Legionnaires' disease. In the environment, L. pneumophila is found in fresh water reservoirs in a large spectrum of environmental conditions, where the bacteria are able to replicate within a variety of protozoan hosts. To survive within eukaryotic cells, L. pneumophila require a type IV secretion system, designated Dot/Icm, that delivers bacterial effector proteins into the host cell cytoplasm. In recent years, a number of Dot/Icm substrate proteins have been identified; however, the function of most of these proteins remains unknown, and it is unclear why the bacterium maintains such a large repertoire of effectors to promote its survival. Here we investigate a region of the L. pneumophila chromosome that displays a high degree of plasticity among four sequenced L. pneumophila strains. Analysis of GC content suggests that several genes encoded in this region were acquired through horizontal gene transfer. Protein translocation studies establish that this region of genomic plasticity encodes for multiple Dot/Icm effectors. Ectopic expression studies in mammalian cells indicate that one of these substrates, a protein called PieA, has unique effector activities. PieA is an effector that can alter lysosome morphology and associates specifically with vacuoles that support L. pneumophila replication. It was determined that the association of PieA with vacuoles containing L. pneumophila requires modifications to the vacuole mediated by other Dot/Icm effectors. Thus, the localization properties of PieA reveal that the Dot/Icm system has the ability to spatially and temporally control the association of an effector with vacuoles containing L. pneumophila through activities mediated by other effector proteins.

  

Proteome analysis of Legionella vacuoles purified by magnetic immunoseparation reveals secretory and endosomal GTPases

Urwyler S, Nyfeler Y, Ragaz C, Lee H, Mueller LN, Aebersold R, Hilbi H.

Institute of Microbiology, Department of Biology, ETH Zurich, Zurich, Switzerland. hilbi@micro.biol.ethz.ch

Traffic. 2009 Jan;10(1):76-87.

ABSTRACT: Legionella pneumophila, the causative agent of Legionnaires' disease, replicates in macrophages and amoebae within 'Legionella-containing vacuoles' (LCVs), which communicate with the early secretory pathway and the endoplasmic reticulum. Formation of LCVs requires the bacterial Icm/Dot type IV secretion system. The Icm/Dot-translocated effector protein SidC selectively anchors to LCVs by binding the host lipid phosphatidylinositol-4-phosphate (PtdIns(4)P). Here, we describe a novel and simple approach to purify intact vacuoles formed by L. pneumophila within Dictyostelium discoideum by using magnetic immunoseparation with an antibody against SidC, followed by density gradient centrifugation. To monitor LCV purification by fluorescence microscopy, we used Dictyostelium producing the LCV marker calnexin-GFP and L. pneumophila labeled with the red fluorescent protein DsRed. A proteome analysis of purified LCVs by liquid chromatography coupled to tandem mass spectrometry revealed 566 host proteins, including known LCV components, such as the small GTPases Arf1, Rab1 and Rab7. Rab8, an endosomal regulator of the late secretory pathway originating from the trans Golgi network, and the endosomal GTPase Rab14 were identified as novel LCV components, which were found to be present on vacuoles harboring wild-type but not Icm/Dot-deficient L. pneumophila. Thus, LCVs also communicate with the late secretory and endosomal pathways. Depletion of Rab8 or Arf1 by RNA interference reduced the amount of SidC on LCVs, indicating that the GTPases promote the recruitment of Legionella effectors by regulating the level of PtdIns(4)P.

 

IFNbeta responses induced by intracellular bacteria or cytosolic DNA in different human cells do not require ZBP1 (DLM-1/DAI)

Lippmann J, Rothenburg S, Deigendesch N, Eitel J, Meixenberger K, van Laak V, Slevogt H, N'guessan PD, Hippenstiel S, Chakraborty T, Flieger A, Suttorp N, Opitz B.

Department of Internal Medicine/Infectious Diseases and Pulmonary Medicine, Charité Universitätsmedizin Berlin, Augustenburger Platz 1, 13353 Berlin, Germany. bastian.opitz@charite.de

Cell Microbiol. 2008 Dec;10(12):2579-88.

ABSTRACT: Intracellular bacteria and cytosolic stimulation with DNA activate type I IFN responses independently of Toll-like receptors, most Nod-like receptors and RIG-like receptors. A recent study suggested that ZBP1 (DLM-1/DAI) represents the long anticipated pattern recognition receptor which mediates IFNalpha/beta responses to cytosolic DNA in mice. Here we show that Legionella pneumophila infection, and intracellular challenge with poly(dA-dT), but not with poly(dG-dC), induced expression of IFNbeta, full-length hZBP1 and a prominent splice variant lacking the first Zalpha domain (hZBP1DeltaZalpha) in human cells. Overexpression of hZBP1 but not hZBP1DeltaZalpha slightly amplified poly(dA-dT)-stimulated IFNbeta reporter activation in HEK293 cells, but had no effect on IFNbeta and IL-8 production induced by bacteria or poly(dA-dT) in A549 cells. We found that mZBP1 siRNA impaired poly(dA-dT)-induced IFNbeta responses in mouse L929 fibroblasts at a later time point, while multiple hZBP1 siRNAs did not suppress IFNbeta or IL-8 expression induced by poly(dA-dT) or bacterial infection in human cells. In contrast, IRF3 siRNA strongly impaired the IFNbeta responses to poly(dA-dT) or bacterial infection. In conclusion, intracellular bacteria and cytosolic poly(dA-dT) activate IFNbeta responses in different human cells without requiring human ZBP1.

 

 The Legionella pneumophila phosphatidylinositol-4 phosphate-binding type IV substrate SidC recruits endoplasmic reticulum vesicles to a replication-permissive vacuole

Ragaz C, Pietsch H, Urwyler S, Tiaden A, Weber SS, Hilbi H.

ETH Zürich, Institute of Microbiology, Wolfgang-Pauli-Strasse 10, 8093 Zürich, Switzerland. hilbi@micro.biol.ethz.ch

Cell Microbiol. 2008 Dec;10(12):2416-33.

ABSTRACT: Legionella pneumophila, the causative agent of Legionnaires' disease, uses the intracellular multiplication/defective organelle trafficking (Icm/Dot) type IV secretion system to establish within amoebae and macrophages an endoplasmic reticulum (ER)-derived replication-permissive compartment, the Legionella-containing vacuole (LCV). The Icm/Dot substrate SidC and its paralogue SdcA anchor to LCVs via phosphatidylinositol-4 phosphate [PtdIns(4)P]. Here we identify the unique 20 kDa PtdIns(4)P-binding domain of SidC, which upon heterologous expression in Dictyostelium binds to LCVs and thus is useful as a PtdIns(4)P-specific probe. LCVs harbouring L. pneumophilaDeltasidC-sdcA mutant bacteria recruit ER and ER-derived vesicles less efficiently and carry endosomal but not lysosomal markers. The phenotypes are complemented by supplying sidC on a plasmid. L. pneumophilaDeltasidC-sdcA grows at wild-type rate in calnexin-negative LCVs, suggesting that communication with the ER is dispensable for establishing a replicative compartment. The amount of SidC and calnexin is directly proportional on isolated LCVs, and in a cell-free system, the recruitment of calnexin-positive vesicles to LCVs harbouring DeltasidC-sdcA mutant bacteria is impaired. Beads coated with purified SidC or its 70 kDa N-terminal fragment recruit ER vesicles in Dictyostelium and macrophage lysates. Our results establish SidC as an L. pneumophila effector protein, which anchors to PtdIns(4)P on LCVs and recruits ER vesicles to a replication-permissive vacuole.

 

Type IV secretion systems: tools of bacterial horizontal gene transfer and virulence

Juhas M, Crook DW, Hood DW.

Clinical Microbiology and Infectious Diseases, Nuffield Department of Clinical Laboratory Sciences, University of Oxford, Oxford OX3 9DU, UK. mario.juhas@ndcls.ox.ac.uk

Cell Microbiol. 2008 Dec;10(12):2377-86.

ABSTRACT: Type IV secretion systems (T4SSs) are multisubunit cell-envelope-spanning structures, ancestrally related to bacterial conjugation machines, which transfer proteins and nucleoprotein complexes across membranes. T4SSs mediate horizontal gene transfer, thus contributing to genome plasticity and the evolution of pathogens through dissemination of antibiotic resistance and virulence genes. Moreover, T4SSs are also used for the delivery of bacterial effector proteins across the bacterial membrane and the plasmatic membrane of eukaryotic host cell, thus contributing directly to pathogenicity. T4SSs are usually encoded by multiple genes organized into a single functional unit. Based on a number of features, the organization of genetic determinants, shared homologies and evolutionary relationships, T4SSs have been divided into several groups. Type F and P (type IVA) T4SSs resembling the archetypal VirB/VirD4 system of Agrobacterium tumefaciens are considered to be the paradigm of type IV secretion, while type I (type IVB) T4SSs are found in intracellular bacterial pathogens, Legionella pneumophila and Coxiella burnetii. Several novel T4SSs have been identified recently and their functions await investigation. The most recently described GI type T4SSs play a key role in the horizontal transfer of a wide variety of genomic islands derived from a broad spectrum of bacterial strains.

 

 Characterization of the posttranscriptional modifications in Legionella pneumophila small-subunit ribosomal RNA

Emmerechts G, Maes L, Herdewijn P, Anné J, Rozenski J.

Laboratory for Medicinal Chemistry, Rega Institute for Medical Research, Katholieke Universiteit Leuven, Minderbroedersstraat 10, Leuven, Belgium. Jef.Rozenski@rega.kuleuven.ac.be

Chem Biodivers. 2008 Dec;5(12):2640-53.

ABSTRACT: It is generally accepted that posttranscriptional modifications in RNA play a role in the fine-tuning of RNA function and the maintenance of RNA structure. This article describes the characterization of the posttranscriptional modifications in Legionella pneumophila 16S rRNA by mass spectrometry and reverse transcriptase assays. Eight modified nucleotides were identified and mapped in the 16S rRNA sequence. Situation of these data in relation to general 16S rRNA modification patterns shows that L. pneumophila is relatively less modified, and that the majority of the L. pneumophila 16S rRNA modifications are conserved among the bacteria characterized so far (Escherichia coli, Clostridium acetobutylicum, Thermus thermophilus, and Thermotoga maritima).

 

Mannose-binding lectin genotypes in susceptibility to community-acquired pneumonia

Endeman H, Herpers BL, de Jong BA, Voorn GP, Grutters JC, van Velzen-Blad H, Biesma DH.

Department of Intensive Care, Diakonessenhuis, Utrect, the Netherlands. henrik.endeman@planet.nl

Chest. 2008 Dec;134(6):1135-40.

ABSTRACT: BACKGROUND: Community-acquired pneumonia (CAP) is most frequently caused by Streptococcus pneumoniae, Haemophilus influenzae, atypical pathogens, and respiratory viruses. Susceptibility to CAP can be increased by single-nucleotide polymorphisms (SNPs) within the mannose-binding lectin (MBL) gene. We questioned whether MBL polymorphisms are associated with the susceptibility to and outcome of CAP and its most common pathogens. METHODS: All adult patients presenting with CAP in a 23-month period were included in this study. Frequencies of SNPs were determined for the promoter X/Y and the three coding SNPs in exon 1 (A/0). Six genotypes were constructed representing patients with sufficient and deficient serum levels of MBL. The results of the patients with CAP were compared with control subjects. RESULTS: In 199 patients and 223 control subjects, MBL genotypes were determined. There were no differences in MBL genotype frequencies between patients with CAP in general, pneumonia caused by S pneumoniae or H influenzae, and control subjects. The frequency of sufficient MBL genotypes was nonsignificantly higher in patients with pneumonia with Legionella sp and Mycoplasma pneumoniae. In Legionella spp, the sufficient YA/YA genotype was significantly more frequent than in control subjects (odds ratio [OR], 5.43; confidence interval [CI], 1.32 to 22.41; p = 0.02). The frequency of the MBL-deficient genotype was significantly higher in patients with viral (co)infections (OR, 2.36; CI, 1.06 to 5.26; p = 0.03) and nonsignificantly higher in patients with pneumococcal pneumonia and viral (co)infections. MBL genotypes had no effect on outcome. CONCLUSIONS: MBL genotypes play a limited role in pneumococcal pneumonia. Sufficient MBL genotypes were more frequently found in a small group of patients with atypical pneumonia, and MBL-deficient genotypes were more frequently found in patients with viral (co)infections.

 

Loss of RNase R induces competence development in Legionella pneumophila

Charpentier X, Faucher SP, Kalachikov S, Shuman HA.

Department of Microbiology, Columbia University Medical Center, New York, NY 10032, USA. has7@columbia.edu

J Bacteriol. 2008 Dec;190(24):8126-36.

ABSTRACT: RNase R is a processive 3'-5' exoribonuclease with a high degree of conservation in prokaryotes. Although some bacteria possess additional hydrolytic 3'-5' exoribonucleases such as RNase II, RNase R was found to be the only predicted one in the facultative intracellular pathogen Legionella pneumophila. This provided a unique opportunity to study the role of RNase R in the absence of an additional RNase with similar enzymatic activity. We investigated the role of RNase R in the biology of Legionella pneumophila under various conditions and performed gene expression profiling using microarrays. At optimal growth temperature, the loss of RNase R had no major consequence on bacterial growth and had a moderate impact on normal gene regulation. However, at a lower temperature, the loss of RNase R had a significant impact on bacterial growth and resulted in the accumulation of structured RNA degradation products. Concurrently, gene regulation was affected and specifically resulted in an increased expression of the competence regulon. Loss of the exoribonuclease activity of RNase R was sufficient to induce competence development, a genetically programmed process normally triggered as a response to environmental stimuli. The temperature-dependent expression of competence genes in the rnr mutant was found to be independent of previously identified competence regulators in Legionella pneumophila. We suggest that a physiological role of RNase R is to eliminate structured RNA molecules that are stabilized by low temperature, which in turn may affect regulatory networks, compromising adaptation to cold and thus resulting in decreased viability.

 

Possible effects of microbial ecto-nucleoside triphosphate diphosphohydrolases on host-pathogen interactions

Sansom FM, Robson SC, Hartland EL.

Department of Microbiology and Immunology, University of Melbourne, Parkville, Victoria 3010, Australia. hartland@unimelb.edu.au

Microbiol Mol Biol Rev. 2008 Dec;72(4):765-81.

ABSTRACT: In humans, purinergic signaling plays an important role in the modulation of immune responses through specific receptors that recognize nucleoside tri- and diphosphates as signaling molecules. Ecto-nucleoside triphosphate diphosphohydrolases (ecto-NTPDases) have important roles in the regulation of purinergic signaling by controlling levels of extracellular nucleotides. This process is key to pathophysiological protective responses such as hemostasis and inflammation. Ecto-NTPDases are found in all higher eukaryotes, and recently it has become apparent that a number of important parasitic pathogens of humans express surface-located NTPDases that have been linked to virulence. For those parasites that are purine auxotrophs, these enzymes may play an important role in purine scavenging, although they may also influence the host response to infection. Although ecto-NTPDases are rare in bacteria, expression of a secreted NTPDase in Legionella pneumophila was recently described. This ecto-enzyme enhances intracellular growth of the bacterium and potentially affects virulence. This discovery represents an important advance in the understanding of the contribution of other microbial NTPDases to host-pathogen interactions. Here we review other progress made to date in the characterization of ecto-NTPDases from microbial pathogens, how they differ from mammalian enzymes, and their association with organism viability and virulence. In addition, we postulate how ecto-NTPDases may contribute to the host-pathogen interaction by reviewing the effect of selected microbial pathogens on purinergic signaling. Finally, we raise the possibility of targeting ecto-NTPDases in the development of novel anti-infective agents based on potential structural and clear enzymatic differences from the mammalian ecto-NTPDases.

 

A novel role for neutrophils as critical activators of NK cells

Spörri R, Joller N, Hilbi H, Oxenius A.

ETH Zurich, Institute for Microbiology, Zürich, Switzerland. roman.spoerri@micro.biol.ethz.ch

J Immunol. 2008 Nov 15;181(10):7121-30.

ABSTRACT: Neutrophils are essential players in innate immune responses to bacterial infection. Despite the striking resistance of Legionella pneumophila (Lpn) to bactericidal neutrophil function, neutrophil granulocytes are important effectors in the resolution of legionellosis. Indeed, mice depleted of neutrophils were unable to clear Lpn due to a lack of the critical cytokine IFN-gamma, which is produced by NK cells. We demonstrate that this can be ascribed to a previously unappreciated role of neutrophils as major NK cell activators. In response to Lpn infection, neutrophils activate caspase-1 and produce mature IL-18, which is indispensable for the activation of NK cells. Furthermore, we show that the IL-12p70 response in Lpn-infected neutropenic mice is also severely reduced and that the Lpn-induced IFN-gamma production by NK cells is strictly dependent on IL-12. However, since dendritic cells, and not neutrophils, are the source of Lpn-induced IL-12, its paucity is a consequence of the absence of IFN-gamma produced by NK cells rather than the absence of neutrophils per se. Therefore, neutrophil-derived IL-18, in combination with dendritic cell-produced IL-12, triggers IFN-gamma synthesis in NK cells in Lpn-infected mice. We propose a novel central role for neutrophils as essential IL-18 producers and hence NK cell "helpers" in bacterial infection.

 

TNF receptor 1 and 2 contribute in different ways to resistance to Legionella pneumophila-induced mortality in mice

Fujita M, Ikegame S, Harada E, Ouchi H, Inoshima I, Watanabe K, Yoshida S, Nakanishi Y.

Research Institute for Diseases of the Chest, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan. mfujita@fukuoka-u.ac.jp

Cytokine. 2008 Nov;44(2):298-303.

ABSTRACT: Legionella pneumophila is one of the most important pathogens which cause community-acquired pneumonia. Although TNF-alpha is considered to play an important role in response to bacteria, the role of the TNF-alpha receptor on L. pneumophila infection remains to be elucidated. To investigate this, we infected TNF receptor deficient mice with L. pneumophila. L. pneumophila was inoculated intranasally into TNF receptor (TNFR)-1-knock-out mice or TNFR2-knock-out mice. The mortality rate, histology of the lung, bacterial growth in the lung, and bronchoalveolar lavage (BAL) fluids were investigated. The bacterial growth of L. pneumophila in the macrophages was also studied. Almost all the mice survived after an intranasal inoculation of 1x10(6)CFU/head of L. pneumophila, but more than 90% mice were killed after inoculation of 1x10(8)CFU/head of L. pneumophila. In the case of TNFR1-knock-out mice and TNFR2-knock-out mice, a high mortality rate was observed after inoculation of 1x10(7)CFU/head of L. pneumophila in comparison to wild-type mice. The lung histology from both the TNFR1-knock-out mice documented severe lung injury at day 3 after inoculation. The clearance of L. pneumophila in the lung of the TNFR1-knock-out mice was slower than those from both the TNFR2-knock-out mice and the wild-type mice. Moreover, L. pneumophila growth in the peritoneal macrophages from the TNFR1-knock-out mice was observed. Interestingly, a lack of neutrophils accumulation in the BAL fluids and a dysregulation of cytokines (IFN-gamma, interleukin-12, and TNF-alpha) were observed in the TNFR1-knock-out mice. On the contrary, large accumulation of neutrophils in BAL fluids was observed in TNFR2-knock-out mice. These data suggested that a TNFR1 deficiency led to a compromise of the innate immunity against L. pneumophila, while a TNFR2 deficiency induced an excessive inflammatory response and resulted in death. The present study confirmed that TNFR1 and TNFR2 play a crucial, but different role in the control of L. pneumophila-induced mortality.

 

Synergistic contribution of the Legionella pneumophila lqs genes to pathogen-host interactions

Tiaden A, Spirig T, Carranza P, Brüggemann H, Riedel K, Eberl L, Buchrieser C, Hilbi H.

Institute of Microbiology, ETH Zürich, Wolfgang-Pauli-Strasse 10, 8093 Zürich, Switzerland. hilbi@micro.biol.ethz.ch

J Bacteriol. 2008 Nov;190(22):7532-47.

ABSTRACT: The causative agent of Legionnaires' disease, Legionella pneumophila, is a natural parasite of environmental protozoa and employs a biphasic life style to switch between a replicative and a transmissive (virulent) phase. L. pneumophila harbors the lqs (Legionella quorum sensing) cluster, which includes genes encoding the autoinducer synthase LqsA, the sensor kinase LqsS, the response regulator LqsR, and a homologue of HdeD, which is involved in acid resistance in Escherichia coli. LqsR promotes host-cell interactions as an element of the stationary-phase virulence regulatory network. Here, we characterize L. pneumophila mutant strains lacking all four genes of the lqs cluster or only the hdeD gene. While an hdeD mutant strain did not have overt physiological or virulence phenotypes, an lqs mutant showed an aberrant morphology in stationary growth phase and was defective for intracellular growth, efficient phagocytosis, and cytotoxicity against host cells. Cytotoxicity was restored upon reintroduction of the lqs genes into the chromosome of an lqs mutant strain. The deletion of the lqs cluster caused more-severe phenotypes than deletion of only lqsR, suggesting a synergistic effect of the other lqs genes. A transcriptome analysis indicated that in the stationary phase more than 380 genes were differentially regulated in the lqs mutant and wild-type L. pneumophila. Genes involved in protein production, metabolism, and bioenergetics were upregulated in the lqs mutant, whereas genes encoding virulence factors, such as effectors secreted by the Icm/Dot type IV secretion system, were downregulated. A proteome analysis revealed that a set of Icm/Dot substrates is not produced in the absence of the lqs gene cluster, which confirms the findings from DNA microarray assays and mirrors the virulence phenotype of the lqs mutant strain.

 

 Paradoxically high resistance of natural killer T (NKT) cell-deficient mice to Legionella pneumophila: another aspect of NKT cells for modulation of host responses

Hayakawa K, Tateda K, Fuse ET, Matsumoto T, Akasaka Y, Ishii T, Nakayama T, Taniguchi M, Kaku M, Standiford TJ, Yamaguchi K.

Department of Microbiology and Infectious Diseases, Toho University, School of Medicine, Tokyo, Japan. kazu@med.toho-u.ac.jp

J Med Microbiol. 2008 Nov;57(Pt 11):1340-8.

ABSTRACT: In the present study, we examined the roles of natural killer T (NKT) cells in host defence against Legionella pneumophila in a mouse model. The survival rate of NKT cell-deficient Jalpha281 knock-out (KO) mice was significantly higher than that of wild-type mice. There was no bacterial overgrowth in the lungs, but Jalpha281 KO mice showed enhanced pulmonary clearance at a later stage of infection, compared with their wild-type counterparts. The severity of lung injury in L. pneumophila-infected Jalpha281 KO mice was less, as indicated by lung permeability measurements, such as lung weight and bronchoalveolar lavage fluid albumin concentration. Recruitment of inflammatory cells in the lungs was approximately twofold greater in Jalpha281 KO mice on day 3. Interestingly, higher values of interleukin (IL)-1beta and IL-18, and increased caspase-1 activity were noted in the lungs of Jalpha281 KO mice from an early time point (6 h). Exogenous alpha-galactosylceramide, a ligand of NKT cells, induced IL-12 and gamma interferon at 6 h, but suppressed IL-1beta at later time points in wild-type, whereas no effects were evident in Jalpha281 KO mice, as expected. Systemic administration of heat-killed L. pneumophila, but not Escherichia coli LPS, reproduced exaggerated production of IL-1beta in the lungs of Jalpha281 KO mice. These results demonstrate that NKT cells play a role in host defence against L. pneumophila, which is characterized by enhanced lung injury and decreased accumulation of inflammatory cells in the lungs. The regulation of IL-1beta, IL-18 and caspase-1 may be associated with the modulating effect of host responses by NKT cells.

 

A Dot/Icm-translocated ankyrin protein of Legionella pneumophila is required for intracellular proliferation within human macrophages and protozoa

Al-Khodor S, Price CT, Habyarimana F, Kalia A, Abu Kwaik Y.

Department of Microbiology and Immunology, Room 413, College of Medicine, University of Louisville, KY 40202, USA. abukwaik@louisville.edu

Mol Microbiol. 2008 Nov;70(4):908-23.

ABSTRACT: The Dot/Icm type IV secretion system of Legionella pneumophila translocates numerous bacterial effectors into the host cell and is essential for bacterial proliferation within macrophages and protozoa. We have recently shown that L. pneumophila strain AA100/130b harbours 11 genes encoding eukaryotic-like ankyrin (Ank) proteins, a family of proteins involved in various essential eukaryotic cellular processes. In contrast to most Dot/Icm-exported substrates, which have little or no detectable role in intracellular proliferation, a mutation in ankB results in a severe growth defect in intracellular replication within human monocyte-derived macrophages (hMDMs), U937 macrophages and Acanthamoeba polyphaga. Single cell analyses of coinfections of hMDMs have shown that the intracellular growth defect of the ankB mutant is totally rescued in cis within communal phagosomes harbouring the wild type strain. Interestingly, distinct from dot/icm structural mutants, the ankB mutant is also rescued in trans within cells harbouring the wild type strain in a different phagosome, indicating that AnkB is a trans-acting secreted effector. Using adenylate cyclase fusions to AnkB, we show that AnkB is translocated into the host cell via the Dot/Icm secretion system in an IcmSW-dependent manner and that the last three C-terminal amino acid residues are essential for translocation. Distinct from the dot/icm structural mutants, the ankB mutant-containing phagosomes exclude late endosomal and lysosomal markers and their phagosomes are remodelled by the rough endoplasmic reticulum. We show that at the postexponential phase of growth, the LetA/S and PmrA/B Two Component Systems confer a positive regulation on expression of the ankB gene, whereas RpoS, LetE and RelA suppress its expression. Our data show that the eukaryotic-like AnkB protein is a Dot/Icm-exported effector that plays a major role in intracellular replication of L. pneumophila within macrophages and protozoa, and its expression is temporally controlled by regulators of the postexponential phase of growth.

 

 

Type IV secretion-dependent activation of host MAP kinases induces an increased proinflammatory cytokine response to Legionella pneumophila

Shin S, Case CL, Archer KA, Nogueira CV, Kobayashi KS, Flavell RA, Roy CR, Zamboni DS.

Section of Microbial Pathogenesis, Yale University School of Medicine, New Haven, Connecticut, USA. craig.roy@yale.edu

PLoS Pathog. 2008 Nov;4(11):e1000220.

ABSTRACT: The immune system must discriminate between pathogenic and nonpathogenic microbes in order to initiate an appropriate response. Toll-like receptors (TLRs) detect microbial components common to both pathogenic and nonpathogenic bacteria, whereas Nod-like receptors (NLRs) sense microbial components introduced into the host cytosol by the specialized secretion systems or pore-forming toxins of bacterial pathogens. The host signaling pathways that respond to bacterial secretion systems remain poorly understood. Infection with the pathogen Legionella pneumophila, which utilizes a type IV secretion system (T4SS), induced an increased proinflammatory cytokine response compared to avirulent bacteria in which the T4SS was inactivated. This enhanced response involved NF-kappaB activation by TLR signaling as well as Nod1 and Nod2 detection of type IV secretion. Furthermore, a TLR- and RIP2-independent pathway leading to p38 and SAPK/JNK MAPK activation was found to play an equally important role in the host response to virulent L. pneumophila. Activation of this MAPK pathway was T4SS-dependent and coordinated with TLR signaling to mount a robust proinflammatory cytokine response to virulent L. pneumophila. These findings define a previously uncharacterized host response to bacterial type IV secretion that activates MAPK signaling and demonstrate that coincident detection of multiple bacterial components enables immune discrimination between virulent and avirulent bacteria.

 

Non-opsonic phagocytosis of Legionella pneumophila by macrophages is mediated by phosphatidylinositol 3-kinase

Tachado SD, Samrakandi MM, Cirillo JD.

Department of Microbial and Molecular Pathogenesis, Texas A&M Health Science Center, College Station, Texas, USA. jdcirillo@medicine.tamhsc.edu

PLoS ONE. 2008 Oct 2;3(10):e3324.

ABSTRACT: BACKGROUND: Legionella pneumophila, is an intracellular pathogen that causes Legionnaires' disease in humans, a potentially lethal pneumonia. L. pneumophila has the ability to enter and replicate in the host and is essential for pathogenesis. METHODOLOGY/PRINCIPAL FINDINGS: Phagocytosis was measured by cell invasion assays. Construction of PI3K mutant by PCR cloning and expression of dominant negative mutant was detected by Western blot. PI3K activity was measured by 32P labeling and detection of phospholipids products by thin layer chromatography. Infection of macrophages with virulent L. pneumophila stimulated the formation of phosphatidylinositol 3-phosphate (PIP3), a phosphorylated lipid product of PI3K whereas two structurally distinct phosphatidylinositol 3 kinase (PI3K) inhibitors, wortmannin and LY294002, reduced L. pneumophila entry into macrophages in a dose-dependent fashion. Furthermore, PI3K activation led to Akt stimulation, a serine/threonine kinase, which was also inhibited by wortmannin and LY294002. In contrast, PI3K and protein kinase B (PKB/Akt) activities were lower in macrophages infected with an avirulent bacterial strain. Only virulent L. pneumophila increased lipid kinase activity present in immunoprecipitates of the p85alpha subunit of class I PI3K and tyrosine phosphorylated proteins. In addition, macrophages expressing a specific dominant negative mutant of PI3K reduced L. pneumophila entry into these cells. CONCLUSION/SIGNIFICANCE: Entry of L. pneumophila is mediated by PI3K/Akt signaling pathway. These results suggest an important role for PI3K and Akt in the L. pneumophila infection process. They point to possible novel strategies for undermining L. pneumophila host uptake and reducing pathogenesis of Legionnaires' disease.

 

Identification of a hypervariable region containing new Legionella pneumophila Icm/Dot translocated substrates by using the conserved icmQ regulatory signature

Zusman T, Degtyar E, Segal G.

Department of Molecular Microbiology and Biotechnology, George S Wise Faculty of Life Sciences, Tel-Aviv University, Ramat-Aviv, Tel-Aviv 69978, Israel. GilS@tauex.tau.ac.il

Infect Immun. 2008 Oct;76(10):4581-91.

ABSTRACT: Legionella pneumophila is an intracellular pathogen that has been shown to utilize the Icm/Dot type IV secretion system for pathogenesis. This system was shown to be composed of Icm/Dot complex components, accessory proteins, and a large number of translocated substrates. In this study, comparison of the icmQ regulatory regions from many Legionella species revealed a conserved regulatory sequence that includes the icmQ -10 promoter element. Mutagenesis of this conserved regulatory element indicated that each of the nucleotides in it affects the level of expression of the icmQ gene but not in a uniform fashion. A genomic analysis discovered that four additional genes in L. pneumophila contain this conserved regulatory sequence, which was found to function similarly in these genes as well. Examination of these four genes indicated that they are dispensable for intracellular growth, but two of them were found to encode new Icm/Dot translocated substrates (IDTS). Comparison of the genomic regions encoding these two IDTS among the four available L. pneumophila genomic sequences indicated that one of these genes is located in a hypervariable genomic region, which was shown before to contain an IDTS-encoding gene. Translocation analysis that was performed for nine proteins encoded from this hypervariable genomic region indicated that six of them are new IDTS which are translocated into host cells in an Icm/Dot-dependent manner. Furthermore, a bioinformatic analysis indicated that additional L. pneumophila genomic regions that contain several neighboring IDTS-encoding genes are hypervariable in gene content.

 

Importance of type II secretion for survival of Legionella pneumophila in tap water and in amoebae at low temperatures

Söderberg MA, Dao J, Starkenburg SR, Cianciotto NP.

Department of Microbiology and Immunology, Northwestern University Medical School, Chicago, IL 60611, USA. n-cianciotto@northwestern.edu

Appl Environ Microbiol. 2008 Sep;74(17):5583-8.

ABSTRACT: Legionella pneumophila type II secretion mutants showed reduced survival in both tap water at 4 to 17 degrees C and aquatic amoebae at 22 to 25 degrees C. Wild-type supernatants stimulated the growth of these mutants, indicating that secreted factors promote low-temperature survival. There was a correlation between low-temperature survival and secretion function when 12 additional Legionella species were examined.

 

Legionella pneumophila EnhC is required for efficient replication in tumour necrosis factor alpha-stimulated macrophages

Liu M, Conover GM, Isberg RR.

Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, MA 02115, USA. ralph.isberg@tufts.edu

Cell Microbiol. 2008 Sep;10(9):1906-23.

ABSTRACT: Legionella pneumophila enhC(-) mutants were originally identified as being defective for uptake into host cells. In this work, we found that the absence of EnhC resulted in defective intracellular growth when dissemination of intracellular bacteria to neighbouring cells was expected to occur. No such defect was observed during growth within the amoeba Dictyostelium discoideum. Culture supernatants containing the secreted products of infected macrophages added to host cells restricted the growth of the DeltaenhC strain, while tumour necrosis factor alpha (TNF-alpha), at concentrations similar to those found in macrophage culture supernatants, could reproduce the growth restriction exerted by culture supernatants on L. pneumophilaDeltaenhC. The absence of EnhC also caused defective trafficking of the Legionella-containing vacuole in TNF-alpha-treated macrophages. EnhC was shown to be an envelope-associated protein largely localized to the periplasm, with its expression induced in post-exponential phase, as is true for many virulence-associated proteins. Furthermore, the absence of EnhC appeared to affect survival under stress conditions, as the DeltaenhC mutant was more susceptible to H(2)O(2) treatment than the wild-type strain. EnhC therefore is a unique virulence factor that is required for growth specifically when macrophages have heightened potential to restrict microbial replication.

 

Iron depletion limits intracellular bacterial growth in macrophages

Paradkar PN, De Domenico I, Durchfort N, Zohn I, Kaplan J, Ward DM.

Department of Pathology, School of Medicine, University of Utah, Salt Lake City, Utah 84132, USA. diane.mcveyward@path.utah.edu

Blood. 2008 Aug 1;112(3):866-74.

ABSTRACT: Many intracellular pathogens infect macrophages and these pathogens require iron for growth. Here we demonstrate in vitro that the intracellular growth of Chlamydia psittaci, trachomatis, and Legionella pneumophila is regulated by the levels of intracellular iron. Macrophages that express cell surface ferroportin, the only known cellular iron exporter, limit the intracellular growth of these bacteria. Hepcidin is an antimicrobial peptide secreted by the liver in response to inflammation. Hepcidin binds to ferroportin mediating its internalization and degradation. Addition of hepcidin to infected macrophages enhanced the intracellular growth of these pathogens. Macrophages from flatiron mice, a strain heterozygous for a loss-of-function ferroportin mutation, showed enhanced intracellular bacterial growth independent of the presence of exogenous hepcidin. Macrophages, from wild-type or flatiron mice, incubated with the oral iron chelator deferriprone or desferasirox showed reduced intracellular bacterial growth suggesting that these chelators might be therapeutic in chronic intracellular bacterial infections.

 

In vivo effect of adhesion inhibitor heparin on Legionella pneumophila pathogenesis in a murine pneumonia model

Ader F, Le Berre R, Fackeure R, Raze D, Menozzi FD, Viget N, Faure K, Kipnis E, Guery B, Jarraud S, Etienne J, Chidiac C.

Université Lyon 1, INSERM U851, Centre National de référence des Légionelles, Faculté de Médecine Laennec, 7 rue Guillaume Paradin, 69372, Lyon cedex 08, France. florence.ader@univ-lyon1.fr.

Intensive Care Med. 2008 Aug;34(8):1511-9.

ABSTRACT: OBJECTIVE: To examine the effect of intratracheal heparin instillation on Legionella pneumophila-related acute lung injury (ALI) and systemic dissemination. DESIGN: Prospective, controlled experimental study. SETTING: University research laboratory. INTERVENTIONS: A/J mice received 5[Symbol: see text]mug of sulfated heparin intratracheally co-instilled with 10(6) or 10(8) colony-forming units (CFU) of a virulent isolate of L. pneumophila. MEASUREMENTS AND RESULTS: ALI was assessed in control groups (PBS and PBS-heparin) and on days 1, 2 and 3 post-infection, in terms of the lung wet-to-dry (W/D) weight ratios and of lung endothelial permeability to radio-labeled albumin (Perm-I(125)). Lung bacterial loads were measured and systemic spread was assessed by blood and target organ culture. The alveolar inflammatory response was evaluated by measuring the cytokine levels (TNF-alpha, IFN-gamma, IL-6 and IL-12p70) in bronchoalveolar lavage fluids (BALF). Co-instilled heparin improved mouse survival after the 10(8) CFU challenge (p[Symbol: see text]<[Symbol: see text]0.01). On day 2, heparin co-instillation significantly reduced the W/D ratio and Perm-I(125) (p[Symbol: see text]<[Symbol: see text]0.01 and p[Symbol: see text]<[Symbol: see text]0.001 respectively), improved lung bacterial clearance (p[Symbol: see text]<[Symbol: see text]0.001), prevented systemic dissemination (blood, liver, spleen, kidneys and brain cultures, all p[Symbol: see text]<[Symbol: see text]0.05) and significantly increased IFN-gamma and IL-12p70 levels in BALF (p[Symbol: see text]<[Symbol: see text]0.05). CONCLUSIONS: Heparin co-instillation during intratracheal L. pneumophila challenge has a protective effect on the alveolar-capillary barrier and prevents bacterial dissemination. These results tend to confirm the competitive inhibition by heparin of L. pneumophila attachment to lung epithelium in vivo, and point to the possible involvement of a heparan-sulfate adhesin in L. pneumophila binding to pneumocytes.

 

Legionella eukaryotic-like type IV substrates interfere with organelle trafficking

de Felipe KS, Glover RT, Charpentier X, Anderson OR, Reyes M, Pericone CD, Shuman HA.

Integrated Program in Cellular, Molecular, and Biophysical Studies, Columbia University Medical Center, New York, New York, United States of America. has7@columbia.edu

PLoS Pathog. 2008 Aug 1;4(8):e1000117.

ABSTRACT: Legionella pneumophila, the causative agent of Legionnaires' disease, evades phago-lysosome fusion in mammalian and protozoan hosts to create a suitable niche for intracellular replication. To modulate vesicle trafficking pathways, L. pneumophila translocates effector proteins into eukaryotic cells through a Type IVB macro-molecular transport system called the Icm-Dot system. In this study, we employed a fluorescence-based translocation assay to show that 33 previously identified Legionella eukaryotic-like genes (leg) encode substrates of the Icm-Dot secretion system. To assess which of these proteins may contribute to the disruption of vesicle trafficking, we expressed each gene in yeast and looked for phenotypes related to vacuolar protein sorting. We found that LegC3-GFP and LegC7/YlfA-GFP caused the mis-secretion of CPY-Invertase, a fusion protein normally restricted to the yeast vacuole. We also found that LegC7/YlfA-GFP and its paralog LegC2/YlfB-GFP formed large structures around the yeast vacuole while LegC3-GFP localized to the plasma membrane and a fragmented vacuole. In mammalian cells, LegC2/YlfB-GFP and LegC7/YlfA-GFP were found within large structures that co-localized with anti-KDEL antibodies but excluded the lysosomal marker LAMP-1, similar to what is observed in Legionella-containing vacuoles. LegC3-GFP, in contrast, was observed as smaller structures which had no obvious co-localization with KDEL or LAMP-1. Finally, LegC3-GFP caused the accumulation of many endosome-like structures containing undigested material when expressed in the protozoan host Dictyostelium discoideum. Our results demonstrate that multiple Leg proteins are Icm/Dot-dependent substrates and that LegC3, LegC7/YlfA, and LegC2/YlfB may contribute to the intracellular trafficking of L. pneumophila by interfering with highly conserved pathways that modulate vesicle maturation.

 

Histone acetylation and flagellin are essential for Legionella pneumophila-induced cytokine expression

Schmeck B, Lorenz J, N'guessan PD, Opitz B, van Laak V, Zahlten J, Slevogt H, Witzenrath M, Flieger A, Suttorp N, Hippenstiel S.

FORSYS Junior Research Group, Systems Biology of Lung Inflammation, Charité-Universitätsmedizin, Berlin, Germany. Bernd.Schmeck@charite.de

J Immunol. 2008 Jul 15;181(2):940-7.

ABSTRACT: Legionella pneumophila causes severe pneumonia. Acetylation of histones is thought to be an important regulator of gene transcription, but its impact on L. pneumophila-induced expression of proinflammatory cytokines is unknown. L. pneumophila strain 130b induced the expression of the important chemoattractant IL-8 and genome-wide histone modifications in human lung epithelial A549 cells. We analyzed the IL-8-promoter and found that histone H4 was acetylated and H3 was phosphorylated at Ser(10) and acetylated at Lys(14), followed by transcription factor NF-kappaB. Recruitment of RNA polymerase II to the IL-8 promoter corresponded with increases in gene transcription. Histone modification and IL-8 release were dependent on p38 kinase and NF-kappaB pathways. Legionella-induced IL-8 expression was decreased by histone acetylase (HAT) inhibitor anacardic acid and enhanced by histone deacetylase (HDAC) inhibitor trichostatin A. After Legionella infection, HATs p300 and CREB-binding protein were time-dependently recruited to the IL-8 promoter, whereas HDAC1 and HDAC5 first decreased and later reappeared at the promoter. Legionella specifically induced expression of HDAC5 but not of other HDACs in lung epithelial cells, but knockdown of HDAC1 or 5 did not alter IL-8 release. Furthermore, Legionella-induced cytokine release, promoter-specific histone modifications, and RNA polymerase II recruitment were reduced in infection with flagellin-deletion mutants. Legionella-induced histone modification as well as HAT-/HDAC-dependent IL-8 release could also be shown in primary lung epithelial cells. In summary, histone acetylation seems to be important for the regulation of proinflammatory gene expression in L. pneumophila infected lung epithelial cells. These pathways may contribute to the host response in Legionnaires' disease.

 

An in vivo gene deletion system for determining temporal requirement of bacterial virulence factors

Liu Y, Gao P, Banga S, Luo ZQ

Department of Biological Sciences, Purdue University, 915 West State Street, West Lafayette, IN 47907, USA. luoz@purdue.edu

Proc Natl Acad Sci U S A. 2008 Jul 8;105(27):9385-90.

ABSTRACT: Analysis of phenotypes associated with specific mutants has been instrumental in determining the roles of a bacterial gene in a biological process. However, this technique does not allow one to address whether a specific gene or gene set is necessary to maintain such a process once it has been established. In the study of microbial pathogenesis, it is important but difficult to determine the temporal requirement of essential pathogenic determinants in the entire infection cycle. Here we report a Cre/loxP-based genetic system that allowed inducible deletion of specific bacterial genes after the pathogen had been phagocytosed by host cells. Using this system, we have examined the temporal requirement of the Dot/Icm type IV protein transporter of Legionella pneumophila during infection. We found that deletion of single essential dot/icm genes did not prevent the internalized bacteria from completing one cycle of intracellular replication. Further analyses indicate that the observed phenotypes were due to the high stability of the examined Dot/Icm protein. However, postinfection deletion within 8 h of the gene coding for the Dot/Icm substrate, SdhA, abolishes intracellular bacterial growth. This result indicates that the Dot/Icm transporter is important for intracellular bacterial growth after the initial biogenesis of the vacuole. Our study has provided a technical concept for analyzing the temporal requirement of specific bacterial proteins or protein complexes in infection or development.

 

Significant role for ladC in initiation of Legionella pneumophila infection

Newton HJ, Sansom FM, Dao J, Cazalet C, Bruggemann H, Albert-Weissenberger C, Buchrieser C, Cianciotto NP, Hartland EL. Hartland@unimelb.edu.au

Australian Bacterial Pathogenesis Program, Department of Microbiology, Monash University, Victoria 3800, Australia.

Infect Immun. 2008 Jul;76(7):3075-85.

ABSTRACT: Previously, we identified ladC in a cohort of genes that were present in Legionella pneumophila but absent in other Legionella species. Here we constructed a ladC mutant of L. pneumophila and assessed its ability to replicate in mammalian cell lines and Acanthamoeba castellanii. The ladC mutant was recovered in significantly lower numbers than wild-type L. pneumophila at early time points, which was reversed upon transcomplementation with ladC but not ladC(N430A/R434A), encoding a putative catalytically inactive derivative of the protein. In fact, complementation of ladC::Km with ladC(N430A/R434A) resulted in a severe replication defect within human and amoeba cell models of infection, which did not follow a typical dominant negative phenotype. Using differential immunofluorescence staining to distinguish adherent from intracellular bacteria, we found that the ladC mutant exhibited a 10-fold reduction in adherence to THP-1 macrophages but no difference in uptake by THP-1 cells. When tested in vivo in A/J mice, the competitive index of the ladC mutant dropped fivefold over 72 h, indicating a significant attenuation compared to wild-type L. pneumophila. Although localization of LadC to the bacterial inner membrane suggested that the protein may be involved in signaling pathways that regulate virulence gene expression, microarray analysis indicated that ladC does not influence the transcriptional profile of L. pneumophila in vitro or during A. castellanii infection. Although the mechanism by which LadC modulates the initial interaction between the bacterium and host cell remains unclear, we have established that LadC plays an important role in L. pneumophila infection.

 

Toll-like receptor 9 regulates the lung macrophage phenotype and host immunity in murine pneumonia caused by Legionella pneumophila

Bhan U, Trujillo G, Lyn-Kew K, Newstead MW, Zeng X, Hogaboam CM, Krieg AM, Standiford TJ.

University of Michigan Medical Center, Division of Pulmonary and Critical Care Medicine, Ann Arbor, MI 48109-2200, USA. tstandif@umich.edu

Infect Immun. 2008 Jul;76(7):2895-904.

ABSTRACT: Experiments were performed to determine the contribution of TLR9 to the generation of protective immunity against the intracellular respiratory bacterial pathogen Legionella pneumophila. In initial studies, we found that the intratracheal (i.t.) administration of L. pneumophila to mice deficient in TLR9 (TLR9(-/-)) resulted in significantly increased mortality, which was associated with an approximately 10-fold increase in the number of lung CFU compared to that of wild-type BALB/c mice. Intrapulmonary bacterial challenge in TLR9(-/-) mice resulted in the reduced accumulation of myeloid dendritic cells (DC) and activated CD4(+) T cells. Lung macrophages isolated from Legionella-infected TLR9(-/-) mice displayed the impaired internalization of bacteria and evidence of alternative rather than classical activation, as manifested by the markedly reduced expression of nitric oxide and type 1 cytokines, whereas the expression of Fizz-1 and arginase-1 was enhanced. The adoptive transfer of bone marrow-derived DC from syngeneic wild-type, but not TLR9(-/-), mice administered i.t. reconstituted anti-legionella immunity and restored the macrophage phenotype in TLR9(-/-) mice. Finally, the i.t., but not intraperitoneal, administration of the TLR9 agonist molecule CpG oligodeoxynucleotide stimulated protective immunity in Legionella-infected mice. In total, our findings indicate that TLR9 is required for effective innate immune responses against the intracellular bacterial pathogen L. pneumophila, and approaches to maximize TLR9-mediated responses may serve as a means to augment antibacterial immunity in pneumonia.

 

Identification and characterization of a new conjugation/type IVA secretion system (trb/tra) of Legionella pneumophila Corby localized on two mobile genomic islands

Glöckner G, Albert-Weissenberger C, Weinmann E, Jacobi S, Schunder E, Steinert M, Hacker J, Heuner K.

Leibniz Institute for Age Research - Fritz Lipmann Institute, D-07745 Jena, Germany. klaus.heuner@mail.uni-wuerzburg.de

Int J Med Microbiol. 2008 Jul;298(5-6):411-28.

ABSTRACT: Horizontal gene transfer probably contributes to evolution of Legionella pneumophila and its adaptation to different environments. Although horizontal gene transfer was observed in Legionella, the mechanism is still not specified. In this study we identified and analysed a new type of conjugation/type IVA secretion system (trb/tra) of L. pneumophila Corby, a virulent human isolate. Two similar versions of this conjugation system were identified, localized on two different genomic islands (Trb-1, 42,710 bp and Trb-2, 34,434 bp). Trb-1 and Trb-2 are integrated within the tRNA(Pro) gene (lpc2778) and the tmRNA gene (lpc0164), respectively. Both islands exhibit an oriT region and both can be excised from the chromosome forming episomal circles. Trb-1 was analysed in more detail. It is active and can be horizontally transferred to other Legionella strains by conjugation and then integrated into the genome in a site-specific manner within the tRNA(Pro) gene. We characterized the sequence of the excision and integration sites of Trb-1 in three different L. pneumophila strains. Here we demonstrate that L. pneumophila exhibits a functional oriT region and that genomic islands in Legionella can be mobilized and conjugated to other species of Legionella. Thus, we describe for the first time a mechanism that may explain the observed horizontal transfer of chromosomal DNA in Legionella.

 

Differential 2-D protein gel electrophoresis analysis of Legionella pneumophila wild type and Tat secretion mutants

De Buck E, Höper D, Lammertyn E, Hecker M, Anné J.

Laboratory of Bacteriology, Rega Institute for Medical Research, Katholieke Universiteit Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium. jozef.anne@rega.kuleuven.be

Int J Med Microbiol. 2008 Jul;298(5-6):449-61.

ABSTRACT: The twin-arginine translocation (Tat) pathway is a secretory pathway for translocation of folded proteins with two arginines in their signal peptide across the cytoplasmic membrane. Recently, we showed the presence of the Tat secretion pathway in Legionella pneumophila Philadelphia-1 and its role in intracellular replication and biofilm formation. To analyse the importance of the Tat pathway in protein export and its role in L. pneumophila virulence, a comparative 2-D protein gel electrophoresis analysis was performed on supernatants of the wild type and two Tat secretion mutants in order to identify possible Tat substrates. Twenty proteins were identified as differential proteins, eight of which were present in a lower quantity in the supernatant of the tat mutants. Among these, one protein with a typical twin-arginine motif in its signal peptide was identified as the 3',5'-cyclic nucleotide phosphodiesterase. Two other proteins that resulted as differential proteins from this study were flagellin and LvrE, which were studied in more detail and their Tat-dependence was further confirmed with specific antibodies. LvrE was shown to play a role in intracellular growth in differentiated U937 cells.

 

The Legionella autoinducer synthase LqsA produces an alpha-hydroxyketone signaling molecole

Spirig T, Tiaden A, Kiefer P, Buchrieser C, Vorholt JA, Hilbi H.

Institute of Microbiology, ETH Zürich, Zürich, Switzerland. hilbi@micro.biol.ethz.ch

J Biol Chem. 2008 Jun 27;283(26):18113-23.

ABSTRACT: The opportunistic pathogen Legionella pneumophila replicates in human lung macrophages and in free-living amoebae. To accommodate the transfer between host cells, L. pneumophila switches from a replicative to a transmissive phase. L. pneumophila harbors a gene cluster homologous to the Vibrio cholerae cqsAS quorum sensing system, encoding a putative autoinducer synthase (lqsA) and a sensor kinase (lqsS), which flank a response regulator (lqsR). LqsR is an element of the L. pneumophila virulence regulatory network, which promotes pathogen-host cell interactions and inhibits entry into the replicative growth phase. Here, we show that lqsA functionally complements a V. cholerae cqsA autoinducer synthase deletion mutant and, upon expression in L. pneumophila or Escherichia coli, produces the diffusible signaling molecule LAI-1 (Legionella autoinducer-1). LAI-1 is distinct from CAI-1 (Cholerae autoinducer-1) and was identified as 3-hydroxypentadecan-4-one using liquid chromatography coupled to high resolution tandem mass spectrometry. The activity of both LqsA and CqsA was abolished upon mutation of a conserved lysine, and covalent binding of the cofactor pyridoxal 5'-phosphate to this lysine was confirmed by mass spectrometry. Thus, LqsA and CqsA belong to a family of pyridoxal 5'-phosphate-dependent autoinducer synthases, which produce the alpha-hydroxyketone signaling molecules LAI-1 and CAI-1.

 

 Ankyrin repeat proteins comprise a diverse family of bacterial type IV effectors

Pan X, Lührmann A, Satoh A, Laskowski-Arce MA, Roy CR.

Section of Microbial Pathogenesis, Yale University School of Medicine, 295 Congress Avenue, New Haven, CT 06536, USA. craig.roy@yale.edu

Science. 2008 Jun 20;320(5883):1651-4.

ABSTRACT: Specialized secretion systems are used by many bacteria to deliver effector proteins into host cells that can either mimic or disrupt the function of eukaryotic factors. We found that the intracellular pathogens Legionella pneumophila and Coxiella burnetii use a type IV secretion system to deliver into eukaryotic cells a large number of different bacterial proteins containing ankyrin repeat homology domains called Anks. The L. pneumophila AnkX protein prevented microtubule-dependent vesicular transport to interfere with fusion of the L. pneumophila-containing vacuole with late endosomes after infection of macrophages, which demonstrates that Ank proteins have effector functions important for bacterial infection of eukaryotic host cells.

 

Host cell processes that influence the intracellular survival of Legionella pneumophila

Shin S, Roy CR.

Section of Microbial Pathogenesis, Yale University School of Medicine, 295 Congress Avenue, Room 345, New Haven, CT 06536, USA. sunny.shin@yale.edu

Cell Microbiol. 2008 Jun;10(6):1209-20.

ABSTRACT: Key to the pathogenesis of intracellular pathogens is their ability to manipulate host cell processes, permitting the establishment of an intracellular replicative niche. In turn, the host cell deploys defence mechanisms that limit intracellular infection. The bacterial pathogen Legionella pneumophila, the aetiological agent of Legionnaire's Disease, has evolved virulence mechanisms that allow it to replicate within protozoa, its natural host. Many of these tactics also enable L. pneumophila's survival and replication inside macrophages within a membrane-bound compartment known as the Legionella-containing vacuole. One of the virulence factors indispensable for L. pneumophila's intracellular survival is a type IV secretion system, which translocates a large repertoire of bacterial effectors into the host cell. These effectors modulate multiple host cell processes and in particular, redirect trafficking of the L. pneumophila phagosome and mediate its conversion into an ER-derived organelle competent for intracellular bacterial replication. In this review, we discuss how L. pneumophila manipulates host cells, as well as host cell processes that either facilitate or impede its intracellular survival.

 

Role for the Ankyrin eukaryotic-like genes of Legionella pneumophila in parasitism of protozoan hosts and human macrophages

Habyarimana F, Al-Khodor S, Kalia A, Graham JE, Price CT, Garcia MT, Kwaik YA.

Department of Microbiology and Immunology, Room MS-410, College of Medicine, University of Louisville, Louisville, KY 40292, USA. abukwaik@louisville.edu

Environ Microbiol. 2008 Jun;10(6):1460-74.

ABSTRACT: Legionella pneumophila is a ubiquitous organism in the aquatic environment where it is capable of invasion and intracellular proliferation within various protozoan species and is also capable of causing pneumonia in humans. In silico analysis showed that the three sequenced L. pneumophila genomes each contained a common multigene family of 11 ankyrin (ank) genes encoding proteins with approximately 30-35 amino acid tandem Ankyrin repeats that are involved in protein-protein interactions in eukaryotic cells. To examine whether the ank genes are involved in tropism of protozoan hosts, we have constructed isogenic mutants of L. pneumophila in ten of the ank genes. Among the mutants, the DeltaankH and DeltaankJ mutants exhibit significant defects in robust intracellular replication within A. polyphaga, Hartmanella vermiformis and Tetrahymena pyriformis. A similar defect is also exhibited in human macrophages. Most of the ank genes are upregulated by L. pneumophila upon growth transition into the post-exponential phase in vitro and within Acanthamoeba polyphaga, and this upregulation is mediated, at least in part, by RpoS. Single-cell analyses have shown that upon co-infection of the wild-type strain with the ankH or ankJ mutant, the replication defect of the mutant is rescued within communal phagosomes harbouring the wild-type strain, similar to dot/icm mutants. Therefore, at least two of the L. pneumophila eukaryotic-like Ank proteins play a role in intracellular replication of L. pneumophila within amoeba, ciliated protozoa and human macrophages. The Ank proteins may not be involved in host tropism in the aquatic environment. Many of the L. pneumophila eukaryotic-like ank genes are triggered upon growth transition into post-exponential phase in vitro as well as within A. polyphaga. Our data suggest a role for AnkH and AnkJ in modulation of phagosome biogenesis by L. pneumophila independent of evasion of lysosomal fusion and recruitment of the rough endoplasmic reticulum.

 

Characterization of anti-Legionella activity of warnericin RK and delta-lysin I from Staphylococcus warneri

Verdon J, Berjeaud JM, Lacombe C, Héchard Y.

Université de Poitiers, Laboratoire de Chimie et Microbiologie de l'Eau, CNRS UMR 6008, 40 avenue du recteur Pineau, 86022 Poitiers, France.

yann.hechard@univ-poitiers.fr

Peptides. 2008 Jun;29(6):978-84.

ABSTRACT: Legionella pneumophila, the causative agent of Legionnaires' disease, is a waterborne bacteria. It can multiply in man-made water systems and infect people who inhale contaminated droplets. We have previously reported a Staphylococcus warneri strain that display an anti-Legionella activity. In this work, we characterized three anti-Legionella peptides that are produced by S. warneri. One peptide, warnericin RK, is original, while the two others are delta-lysin I and delta-lysin II, whose genes were previously described. Due to high sequence similarity of the two delta-lysins, further characterization was performed only on delta-lysin I. Warnericin RK and delta-lysin I displayed the same antibacterial spectrum, which is almost restricted to the Legionella genus. Also, both peptides have a hemolytic activity. These results led to the hypothesis that warnericin RK and delta-lysin I share a similar mode of action, and that Legionella should have a specific feature that may explain the high specificity of these antibacterial peptides.

 

NAIP and Ipaf control Legionella pneumophila replication in human cells

Vinzing M, Eitel J, Lippmann J, Hocke AC, Zahlten J, Slevogt H, N'guessan PD, Günther S, Schmeck B, Hippenstiel S, Flieger A, Suttorp N, Opitz B.

Department of Internal Medicine/Infectious Diseases and Pulmonary Medicine, Charité Universitätsmedizin Berlin, Berlin, Germany. bastian.opitz@charite.de

J Immunol. 2008 May 15;180(10):6808-15.

ABSTRACT: In mice, different alleles of the mNAIP5 (murine neuronal apoptosis inhibitory protein-5)/mBirc1e gene determine whether macrophages restrict or support intracellular replication of Legionella pneumophila, and whether a mouse is resistant or (moderately) susceptible to Legionella infection. In the resistant mice strains, the nucleotide-binding oligomerization domain (Nod)-like receptor (NLR) family member mNAIP5/mBirc1e, as well as the NLR protein mIpaf (murine ICE protease-activating factor), are involved in recognition of Legionella flagellin and in restriction of bacterial replication. Human macrophages and lung epithelial cells support L. pneumophila growth, and humans can develop severe pneumonia (Legionnaires disease) after Legionella infection. The role of human orthologs to mNAIP5/mBirc1e and mIpaf in this bacterial infection has not been elucidated. Herein we demonstrate that flagellin-deficient L. pneumophila replicate more efficiently in human THP-1 macrophages, primary monocyte-derived macrophages, and alveolar macrophages, and in A549 lung epithelial cells compared with wild-type bacteria. Additionally, we note expression of the mNAIP5 ortholog hNAIP in all cell types examined, and expression of hIpaf in human macrophages. Gene silencing of hNAIP or hIpaf in macrophages or of hNAIP in lung epithelial cells leads to an enhanced bacterial growth, and overexpression of both molecules strongly reduces Legionella replication. In contrast to experiments with wild-type L. pneumophila, hNAIP or hIpaf knock-down affects the (enhanced) replication of flagellin-deficient Legionella only marginally. In conclusion, hNAIP and hIpaf mediate innate intracellular defense against flagellated Legionella in human cells.

 

 

Enzymatic properties of an ecto-nucleoside triphosphate diphosphohydrolase from Legionella pneumophila: substrate specificity and requirement for virulence

Sansom FM, Riedmaier P, Newton HJ, Dunstone MA, Müller CE, Stephan H, Byres E, Beddoe T, Rossjohn J, Cowan PJ, d'Apice AJ, Robson SC, Hartland EL.

Department of Microbiology and Immunology and Medicine, University of Melbourne, Parkville, Victoria 3010, Australia. hartland@unimelb.edu.au

J Biol Chem. 2008 May 9;283(19):12909-18.

ABSTRACT: Legionella pneumophila is the predominant cause of Legionnaires disease, a severe and potentially fatal form of pneumonia. Recently, we identified an ecto-nucleoside triphosphate diphosphohydrolase (NTPDase) from L. pneumophila, termed Lpg1905, which enhances intracellular replication of L. pneumophila in eukaryotic cells. Lpg1905 is the first prokaryotic member of the CD39/NTPDase1 family of enzymes, which are characterized by the presence of five apyrase conserved regions and the ability to hydrolyze nucleoside tri- and diphosphates. Here we examined the substrate specificity of Lpg1905 and showed that apart from ATP and ADP, the enzyme catalyzed the hydrolysis of GTP and GDP but had limited activity against CTP, CDP, UTP, and UDP. Based on amino acid residues conserved in the apyrase conserved regions of eukaryotic NTPDases, we generated five site-directed mutants, Lpg1905E159A, R122A, N168A, Q193A, and W384A. Although the mutations E159A, R122A, Q193A, and W384A abrogated activity completely, N168A resulted in decreased activity caused by reduced affinity for nucleotides. When introduced into the lpg1905 mutant strain of L. pneumophila, only N168A partially restored the ability of L. pneumophila to replicate in THP-1 macrophages. Following intratracheal inoculation of A/J mice, none of the Lpg1905 mutants was able to restore virulence to an lpg1905 mutant during lung infection, thereby demonstrating the importance of NTPDase activity to L. pneumophila infection. Overall, the kinetic studies undertaken here demonstrated important differences to mammalian NTPDases and different sensitivities to NTPDase inhibitors that may reflect underlying structural variations.

 

Exclusion of actin-binding protein p57/coronin-1 from bacteria-containing phagosomes in macrophages infected with Legionella

Hayashi T, Miyake M, Fukui T, Sugaya N, Daimon T, Itoh S, Oku T, Tsuji T, Toyoshima S, Imai Y.

Laboratory of Microbiology and Immunology and the Global COE Program, University of Shizuoka School of Pharmaceutical Sciences, 52-1 Yada, Suruga-ku, Shizuoka, Shizuoka 422-8526, Japan.

Biol Pharm Bull. 2008 May;31(5):861-5.

ABSTRACT: Legionella pneumophila, the causative agent of Legionnaires' disease, is a human pathogen that multiplies within alveolar macrophages. L. pneumophila establishes specialized phagosomes in which it evades the host defense through largely unknown mechanisms. Here we analyzed the role of an actin-binding protein, p57/coronin-1, a member of the coronin protein family, during Legionella infection. On fluorescence microscopy, p57/coronin-1 and F-actin were found to be co-localized at the sites on the plasma membrane where L. pneumophila adhered to U937 human macrophage-like cells. The localization of p57/coronin-1 at the sites of bacterial adherence was inhibited by treatment with cytochalasin D (an inhibitor of actin polymerization), suggesting that p57/coronin-1 is involved in the actin-dependent uptake of L. pneumophila into U937 cells. In addition, we showed that p57/coronin-1 was excluded from phagosomes containing live L. pneumophila throughout the infection, whereas transient accumulation of p57/coronin-1 was observed on phagosomes containing Texas-Red-labeled opsonized zymosan (TROpZ) or heat-killed L. pneumophila at an early stage of phagocytosis. The exclusion of p57/coronin-1 from phagosomes containing live another Legionella species Legionella gratiana at an early stage of infection was also observed. Taken together, these results suggest that the endocytic pathways of live Legionella species are distinct from general phagocytic pathways, which lead to lysosomal degradation.

 

Legionella pneumophila infection induces programmed cell death, caspase activation, and release of high-mobility group box 1 protein in A549 alveolar epithelial cells: inhibition by methyl prednisolone

Furugen M, Higa F, Hibiya K, Teruya H, Akamine M, Haranaga S, Yara S, Koide M, Tateyama M, Mori N, Fujita J.

Department of Medicine and Therapeutics, Control and Prevention of Infectious Diseases, Graduate School of Medicine, University of the Ryukyus, 207 Uehara, Nishihara-Town, Okinawa 903-0215, Japan. k068737@eve.u-ryukyu.ac.jp

Respir Res. 2008 May 1;9:39.

ABSTRACT: BACKGROUND: Legionella pneumophila pneumonia often exacerbates acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). Apoptosis of alveolar epithelial cells is considered to play an important role in the pathogenesis of ALI and ARDS. In this study, we investigated the precise mechanism by which A549 alveolar epithelial cells induced by L. pneumophila undergo apoptosis. We also studied the effect of methyl prednisolone on apoptosis in these cells. METHODS: Nuclear deoxyribonucleic acid (DNA) fragmentation and caspase activation in L. pneumophila-infected A549 alveolar epithelial cells were assessed using the terminal deoxyribonucleotidyl transferase-mediated triphosphate (dUTP)-biotin nick end labeling method (TUNEL method) and colorimetric caspase activity assays. The virulent L. pneumophila strain AA100jm and the avirulent dotO mutant were used and compared in this study. In addition, we investigated whether methyl prednisolone has any influence on nuclear DNA fragmentation and caspase activation in A549 alveolar epithelial cells infected with L. pneumophila. RESULTS: The virulent strain of L. pneumophila grew within A549 alveolar epithelial cells and induced subsequent cell death in a dose-dependent manner. The avirulent strain dotO mutant showed no such effect. The virulent strains of L. pneumophila induced DNA fragmentation (shown by TUNEL staining) and activation of caspases 3, 8, 9, and 1 in A549 cells, while the avirulent strain did not. High-mobility group box 1 (HMGB1) protein was released from A549 cells infected with virulent Legionella. Methyl prednisolone (53.4 muM) did not influence the intracellular growth of L. pneumophila within alveolar epithelial cells, but affected DNA fragmentation and caspase activation of infected A549 cells. CONCLUSION: Infection of A549 alveolar epithelial cells with L. pneumophila caused programmed cell death, activation of various caspases, and release of HMGB1. The dot/icm system, a major virulence factor of L. pneumophila, is involved in the effects we measured in alveolar epithelial cells. Methyl prednisolone may modulate the interaction of Legionella and these cells.

 

Exclusion of Actin-Binding Protein p57/Coronin-1 from Bacteria-Containing Phagosomes in Macrophages Infected with Legionella

Hayashi T, Miyake M, Fukui T, Sugaya N, Daimon T, Itoh S, Oku T, Tsuji T, Toyoshima S, Imai Y.

Laboratory of Microbiology and Immunology and the Global COE Program, University of Shizuoka School of Pharmaceutical Sciences.

miyakem@ys7.u-shizuoka-ken.ac.jp

Biol Pharm Bull. 2008 May;31(5):861-5.

ABSTRACT: Legionella pneumophila, the causative agent of Legionnaires' disease, is a human pathogen that multiplies within alveolar macrophages. L. pneumophila establishes specialized phagosomes in which it evades the host defense through largely unknown mechanisms. Here we analyzed the role of an actin-binding protein, p57/coronin-1, a member of the coronin protein family, during Legionella infection. On fluorescence microscopy, p57/coronin-1 and F-actin were found to be co-localized at the sites on the plasma membrane where L. pneumophila adhered to U937 human macrophage-like cells. The localization of p57/coronin-1 at the sites of bacterial adherence was inhibited by treatment with cytochalasin D (an inhibitor of actin polymerization), suggesting that p57/coronin-1 is involved in the actin-dependent uptake of L. pneumophila into U937 cells. In addition, we showed that p57/coronin-1 was excluded from phagosomes containing live L. pneumophila throughout the infection, whereas transient accumulation of p57/coronin-1 was observed on phagosomes containing Texas-Red-labeled opsonized zymosan (TROpZ) or heat-killed L. pneumophila at an early stage of phagocytosis. The exclusion of p57/coronin-1 from phagosomes containing live another Legionella species Legionella gratiana at an early stage of infection was also observed. Taken together, these results suggest that the endocytic pathways of live Legionella species are distinct from general phagocytic pathways, which lead to lysosomal degradation.

 

Genotypic comparison of clinical Legionella isolates and patient-related environmental isolates in The Netherlands, 2002-2006

Den Boer JW, Bruin JP, Verhoef LP, Van der Zwaluw K, Jansen R, Yzerman EP.

Municipal Health Service Kennemerland, and Regional Public Health Laboratory Kennemerland, Haarlem, The Netherlands. jwdenboer@hdk.nl

Clin Microbiol Infect. 2008 May;14(5):459-66.

ABSTRACT: This study investigated the hypothesis that the genotype distribution of Legionella isolates from sporadic patients with Legionnaires' disease differs from that of Legionella strains in the environment. An amplified fragment-length polymorphism (AFLP) assay was used to genotype patient-derived and environmental Legionella isolates. The three Legionella pneumophila genotypes isolated most frequently from human respiratory secretions were AFLP types 004 Lyon, 010 London and 006 Copenhagen. These genotypes were cultured significantly less frequently from environmental samples (50% vs. 4%; p <0.001). The most frequently observed L. pneumophila serogroup 1 genotype among patient-derived isolates was 004 Lyon (32%). This genotype was cultured from only one of 6458 environmental samples. The positive sample contained 1.26 x 10(6) CFU/mL and originated from a whirlpool spa that had not been disinfected and had been maintained at 36 degrees C for several months. Overall, the distribution of genotypes differed significantly among patient and environmental isolates. A possible explanation is that virulent strains may exist in potential environmental sources at undetectable concentrations.

 

Enzymatic Properties of an Ecto-nucleoside Triphosphate Diphosphohydrolase from Legionella pneumophila: substrate specificity and requirement for virulence

Sansom FM, Riedmaier P, Newton HJ, Dunstone MA, Müller CE, Stephan H, Byres E, Beddoe T, Rossjohn J, Cowan PJ, d'Apice AJ, Robson SC, Hartland EL.

Departments of Microbiology and Immunology and Medicine, University of Melbourne, Parkville, Victoria 3010, Australia, the Departments of Microbiology and Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3800, Australia. hartland@unimelb.edu.au

J Biol Chem. 2008 May 9;283(19):12909-18.

ABSTRACT: Legionella pneumophila is the predominant cause of Legionnaires disease, a severe and potentially fatal form of pneumonia. Recently, we identified an ecto-nucleoside triphosphate diphosphohydrolase (NTPDase) from L. pneumophila, termed Lpg1905, which enhances intracellular replication of L. pneumophila in eukaryotic cells. Lpg1905 is the first prokaryotic member of the CD39/NTPDase1 family of enzymes, which are characterized by the presence of five apyrase conserved regions and the ability to hydrolyze nucleoside tri- and diphosphates. Here we examined the substrate specificity of Lpg1905 and showed that apart from ATP and ADP, the enzyme catalyzed the hydrolysis of GTP and GDP but had limited activity against CTP, CDP, UTP, and UDP. Based on amino acid residues conserved in the apyrase conserved regions of eukaryotic NTPDases, we generated five site-directed mutants, Lpg1905E159A, R122A, N168A, Q193A, and W384A. Although the mutations E159A, R122A, Q193A, and W384A abrogated activity completely, N168A resulted in decreased activity caused by reduced affinity for nucleotides. When introduced into the lpg1905 mutant strain of L. pneumophila, only N168A partially restored the ability of L. pneumophila to replicate in THP-1 macrophages. Following intratracheal inoculation of A/J mice, none of the Lpg1905 mutants was able to restore virulence to an lpg1905 mutant during lung infection, thereby demonstrating the importance of NTPDase activity to L. pneumophila infection. Overall, the kinetic studies undertaken here demonstrated important differences to mammalian NTPDases and different sensitivities to NTPDase inhibitors that may reflect underlying structural variations.

 

Proteomic characterization of the whole secretome of Legionella pneumophila and functional analysis of outer membrane vesicles

Galka F, Wai SN, Kusch H, Engelmann S, Hecker M, Schmeck B, Hippenstiel S, Uhlin BE, Steinert M.

Institut für Mikrobiologie, Technische Universität Braunschweig, Spielmannstr. 7, D-38106 Braunschweig, Germany. m.steinert@tu-bs.de

Infect Immun. 2008 May;76(5):1825-36.

ABSTRACT: Secretion of effector molecules is one of the major mechanisms by which the intracellular human pathogen Legionella pneumophila interacts with host cells during infection. Specific secretion machineries which are responsible for the subfraction of secreted proteins (soluble supernatant proteins [SSPs]) and the production of bacterial outer membrane vesicles (OMVs) both contribute to the protein composition of the extracellular milieu of this lung pathogen. Here we present comprehensive proteome reference maps for both SSPs and OMVs. Protein identification and assignment analyses revealed a total of 181 supernatant proteins, 107 of which were specific to the SSP fraction and 33 of which were specific to OMVs. A functional classification showed that a large proportion of the identified OMV proteins are involved in the pathogenesis of Legionnaires' disease. Zymography and enzyme assays demonstrated that the SSP and OMV fractions possess proteolytic and lipolytic enzyme activities which may contribute to the destruction of the alveolar lining during infection. Furthermore, it was shown that OMVs do not kill host cells but specifically modulate their cytokine response. Binding of immunofluorescently stained OMVs to alveolar epithelial cells, as visualized by confocal laser scanning microscopy, suggested that there is delivery of a large and complex group of proteins and lipids in the infected tissue in association with OMVs. On the basis of these new findings, we discuss the relevance of protein sorting and compartmentalization of virulence factors, as well as environmental aspects of the vesicle-mediated secretion.

  

An ortholog of OxyR in Legionella pneumophila is expressed postexponentially and negatively regulates the alkyl hydroperoxide reductase (ahpC2D) operon

LeBlanc JJ, Brassinga AK, Ewann F, Davidson RJ, Hoffman PS.

Department of Pathology and Laboratory Medicine, Queen Elizabeth II Health Sciences Center, Halifax, Nova Scotia, Canada. psh2n@virginia.edu

J Bacteriol. 2008 May;190(10):3444-55.

ABSTRACT: Legionella pneumophila expresses two peroxide-scavenging alkyl hydroperoxide reductase systems (AhpC1 and AhpC2D) that are expressed differentially during the bacterial growth cycle. Functional loss of the postexponentially expressed AhpC1 system is compensated for by increased expression of the exponentially expressed AhpC2D system. In this study, we used an acrylamide capture of DNA-bound complexes (ACDC) technique and mass spectrometry to identify proteins that bind to the promoter region of the ahpC2D operon. The major protein captured was an ortholog of OxyR (OxyR(Lp)). Genetic studies indicated that oxyR(Lp) was an essential gene expressed postexponentially and only partially complemented an Escherichia coli oxyR mutant (GS077). Gel shift assays confirmed specific binding of OxyR(Lp) to ahpC2D promoter sequences, but not to promoters of ahpC1 or oxyR(Lp); however, OxyR(Lp) weakly bound to E. coli OxyR-regulated promoters (katG, oxyR, and ahpCF). DNase I protection studies showed that the OxyR(Lp) binding motif spanned the promoter and transcriptional start sequences of ahpC2 and that the protected region was unchanged by treatments with reducing agents or hydrogen peroxide (H(2)O(2)). Moreover, the OxyR(Lp) (pBADLpoxyR)-mediated repression of an ahpC2-gfp reporter construct in E. coli GS077 (the oxyR mutant) was not reversed by H(2)O(2) challenge. Alignments with other OxyR proteins revealed several amino acid substitutions predicted to ablate thiol oxidation or conformational changes required for activation. We suggest these mutations have locked OxyR(Lp) in an active DNA-binding conformation, which has permitted a divergence of function from a regulator of oxidative stress to a cell cycle regulator, perhaps controlling gene expression during postexponential differentiation.

  

Multiple-locus variable-number tandem repeat analysis of Legionella pneumophila using multi-colored capillary electrophoresis

Nederbragt AJ, Balasingham A, Sirevåg R, Utkilen H, Jakobsen KS, Anderson-Glenna MJ.

University of Oslo, Department of Biology, Centre for Ecological and Evolutionary Synthesis, P.O. Box 1066 Blindern, N-0316 Oslo, Norway. k.s.jakobsen@bio.uio.no

J Microbiol Methods. 2008 May;73(2):111-7.

ABSTRACT: Several methods for typing of Legionella pneumophila exist, one of which is an 8-locus variable-number of tandem repeats analysis (MLVA). This method is based on separating and sizing amplified VNTR PCR products by agarose gel electrophoresis. In the present work, the existing L. pneumophila MLVA-8 assay is adapted to capillary electrophoresis. The assay was multiplexed by using multiple fluorescent labeling dyes and tested on a panel of L. pneumophila strains with known genotypes. The results from the capillary electrophoresis-based assay are shown to be equivalent to, and in a few cases more sensitive than, the gel-based genotyping assay. The assay presented here allows for a swift, automated and precise typing of L. pneumophila from patient or environmental samples and represents an improvement over the current gel-based method.

 

Lorraine strain of Legionella pneumophila serogroup 1, France

Ginevra C, Forey F, Campèse C, Reyrolle M, Che D, Etienne J, Jarraud S.

Université de Lyon, Faculté Laennec, 7 rue Guillaume Paradin, F-69 372 Lyon CEDEX 08, France. christophe.ginevra@univ-lyon1.fr

Emerg Infect Dis. 2008 Apr;14(4):673-5.

Letter.

 

Outer-membrane proteomic maps and surface-exposed proteins of Legionella pneumophila using cellular fractionation and fluorescent labelling

Khemiri A, Galland A, Vaudry D, Chan Tchi Song P, Vaudry H, Jouenne T, Cosette P.

UMR 6522 CNRS, Faculty of Sciences, University of Rouen, 76821, Mont Saint Aignan Cedex, France. pascal.cosette@univ-rouen.fr

Anal Bioanal Chem. 2008 Apr;390(7):1861-71.

ABSTRACT: Bacterial surface-associated proteins play crucial roles in host-pathogen interactions and pathogenesis. The identification of these proteins represents an important goal of bacterial proteomics for vaccine development, but also for environmental concerns such as microbial biosensing. Here, we developed such an approach for Legionella pneumophila, a bacterium that causes severe pneumonia. We propose a complementary strategy consisting of (1) a fluorescent labelling of surface-exposed proteins in parallel with (2) a fractionation of the outer-membrane protein extract. These two distinct protein populations were subsequently separated using two-dimensional gel electrophoresis and characterised by mass spectrometry. Within these populations, we found proteins which were expected for the compartments studied, but also a great number of proteins never experimentally described, and also a non-negligible fraction of proteins never described in these fractions. These data provided new routes of inspection for transport and host recognition for Legionella pneumophila. In addition, these results on the membranome and surfaceome show that Legionella in the stationary phase of growth possesses the major determinants to infect host cells.

  

Packaging of live Legionella pneumophila into pellets expelled by Tetrahymena spp. does not require bacterial replication and depends on a Dot/Icm-mediated survival mechanism

Berk SG, Faulkner G, Garduño E, Joy MC, Ortiz-Jimenez MA, Garduño RA.

Department of Microbiology and Immunology, Dalhousie University, Sir Charles Tupper Building, 7th floor, 5850 College Street, Halifax, Nova Scotia B3H-1X5, Canada. rafael.garduno@dal.ca

Appl Environ Microbiol. 2008 Apr;74(7):2187-99.

ABSTRACT: The freshwater ciliate Tetrahymena sp. efficiently ingested, but poorly digested, virulent strains of the gram-negative intracellular pathogen Legionella pneumophila. Ciliates expelled live legionellae packaged in free spherical pellets. The ingested legionellae showed no ultrastructural indicators of cell division either within intracellular food vacuoles or in the expelled pellets, while the number of CFU consistently decreased as a function of time postinoculation, suggesting a lack of L. pneumophila replication inside Tetrahymena. Pulse-chase feeding experiments with fluorescent L. pneumophila and Escherichia coli indicated that actively feeding ciliates maintain a rapid and steady turnover of food vacuoles, so that the intravacuolar residence of the ingested bacteria was as short as 1 to 2 h. L. pneumophila mutants with a defective Dot/Icm virulence system were efficiently digested by Tetrahymena sp. In contrast to pellets of virulent L. pneumophila, the pellets produced by ciliates feeding on dot mutants contained very few bacterial cells but abundant membrane whorls. The whorls became labeled with a specific antibody against L. pneumophila OmpS, indicating that they were outer membrane remnants of digested legionellae. Ciliates that fed on genetically complemented dot mutants produced numerous pellets containing live legionellae, establishing the importance of the Dot/Icm system to resist digestion. We thus concluded that production of pellets containing live virulent L. pneumophila depends on bacterial survival (mediated by the Dot/Icm system) and occurs in the absence of bacterial replication. Pellets of virulent L. pneumophila may contribute to the transmission of Legionnaires' disease, an issue currently under investigation.

 

The manifold phospholipases A of Legionella pneumophila - identification, export, regulation, and their link to bacterial virulence

Banerji S, Aurass P, Flieger A.

Research Group Pathogenesis of Legionella Infections, Robert Koch-Institut, Nordufer 20, D-13353 Berlin, Germany. fliegera@rki.de

Int J Med Microbiol. 2008 Apr;298(3-4):169-81.

ABSTRACT: The intracellular lung pathogen Legionella pneumophila expresses secreted and cell-associated phospholipase A (PLA) and lysophospholipase A (LPLA) activities belonging to at least three enzyme families. The first family consists of three secreted PLA and LPLA activities displaying the amino acid signature motif 'GDSL'; PlaA, PlaC and PlaD. The second group contains the cell-associated and very potent PLA/LPLA, PlaB. The third group, the patatin-like proteins, comprises 11 members. One patatin-like protein, PatA/VipD, shows LPLA and PLA activities and interferes with vesicular trafficking when expressed in yeast and therefore is possibly involved in the intracellular infection process. Likewise, members of the first two phospholipase families have roles in bacterial virulence because phospholipases are important virulence factors that have been shown to promote bacterial survival, spread and host cell modification/damage. The GDSL enzyme PlaA detoxifies cytolytic lysophospholipids, and PlaB shows contact-dependent haemolytic activity. PlaC acylates cholesterol, a lipid present in eukaryotic hosts but not in the bacterium. Many of the L. pneumophila PLAs are exported by the type II Lsp or the type IVB Dot/Icm secretion systems involved in virulence factor export. Moreover, the regulation of lipolytic activities depends on the transcriptional regulators LetA/S and RpoS, inducing the expression of virulence traits, and on posttranscriptional activators like the zinc metalloprotease ProA.

 

Lgt: a family of cytotoxic glucosyltransferases produced by Legionella pneumophila

Belyi Y, Tabakova I, Stahl M, Aktories K.

Gamaleya Research Institute, Moscow 123098, Russia. Klaus.Aktories@pharmakol.uni-freiburg.de

J Bacteriol. 2008 Apr;190(8):3026-35.

ABSTRACT: Legionella pneumophila is a facultative intracellular pathogen responsible for severe lung disease in humans, known as legionellosis or Legionnaires' disease. Previously, we reported on the approximately 60-kDa glucosyltransferase (Lgt1) from Legionella pneumophila, which modified eukaryotic elongation factor 1A. In the present study, using L. pneumophila Philadelphia-1, Lens, Paris, and Corby genome databases, we identified several genes coding for proteins with considerable sequence homology to Lgt1. These new enzymes form three subfamilies, termed Lgt1 to -3, glucosylate mammalian elongation factor eEF1A at serine-53, inhibit its activity, and subsequently kill target eukaryotic cells. Expression studies on L. pneumophila grown in broth medium or in Acanthamoeba castellanii revealed that production of Lgt1 was maximal at stationary phase of broth culture or during the late phase of Legionella-host cell interaction, respectively. In contrast, synthesis of Lgt3 peaked during the lag phase of liquid culture and at early steps of bacterium-amoeba interaction. Thus, the data indicate that members of the L. pneumophila glucosyltransferase family are differentially regulated, affect protein synthesis of host cells, and represent potential virulence factors of Legionella.

  

Application of unstable Gfp variants to the kinetic study of Legionella pneumophila icm gene expression during infection

Barysheva OV, Fujii J, Takaesu G, Yoshida S.

Department of Bacteriology, Faculty of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan. ksyusha@bact.med.kyushu-u.ac.jp

Microbiology. 2008 Apr;154(Pt 4):1015-25.

ABSTRACT: An unstable type of green fluorescent protein (Gfp) tagged with a C-terminal extension, which is a target for tail-specific protease, was used as a reporter gene in Legionella pneumophila. To analyse Gfp expression in legionellae, transcriptional fusions of unstable gfp with the Legionella-specific icm (intracellular multiplication) promoters (P(icmS), P(icmT) and P(icmQ)) were constructed. Infection studies using J774.1 macrophages as the host, and L. pneumophila strains carrying P(icmS)-gfp, P(icmT)-gfp and P(icmQ)-gfp fusions, indicated that the icmS, icmT and icmQ genes could be expressed intracellularly. Expression of icmS, icmT and icmQ genes in infected cells was examined by flow cytometry. Furthermore, fluorescent intracellular legionellae were detected directly by confocal microscopy. Real-time quantitative RT-PCR revealed the differences in the gene expression of icmS, and that of icmT and icmQ, during infection. Expression of icmS was high in the late stage of infection, while that of icmT and icmQ was high in the early phase only. We show that unstable gfp is a useful reporter gene whose expression in legionellae can be followed in real-time, and that it allows analysis of promoter activities in legionellae and monitoring of the infection process.

 

Solution structure of the Legionella pneumophila Mip-rapamycin complex

Ceymann A, Horstmann M, Ehses P, Schweimer K, Paschke AK, Steinert M, Faber C.

Department of Experimental Physics 5, University of Würzburg, Würzburg, Germany. asceyman@physik.uni-wuerzburg.de

BMC Struct Biol. 2008 Mar 17;8:17.

ABSTRACT: BACKGROUND: Legionella pneumphila is the causative agent of Legionnaires' disease. A major virulence factor of the pathogen is the homodimeric surface protein Mip. It shows peptidyl-prolyl cis/trans isomerase activty and is a receptor of FK506 and rapamycin, which both inhibit its enzymatic function. Insight into the binding process may be used for the design of novel Mip inhibitors as potential drugs against Legionnaires' disease. RESULTS: We have solved the solution structure of free Mip77-213 and the Mip77-213-rapamycin complex by NMR spectroscopy. Mip77-213 showed the typical FKBP-fold and only minor rearrangements upon binding of rapamycin. Apart from the configuration of a flexible hairpin loop, which is partly stabilized upon binding, the solution structure confirms the crystal structure. Comparisons to the structures of free FKBP12 and the FKBP12-rapamycin complex suggested an identical binding mode for both proteins. CONCLUSION: The structural similarity of the Mip-rapamycin and FKBP12-rapamycin complexes suggests that FKBP12 ligands may be promising starting points for the design of novel Mip inhibitors. The search for a novel drug against Legionnaires' disease may therefore benefit from the large variety of known FKBP12 inhibitors.

 

Biosynthesis of CMP-N,N'-diacetyllegionaminic acid from UDP-N,N'-diacetylbacillosamine in Legionella pneumophila

Glaze PA, Watson DC, Young NM, Tanner ME.

Department of Chemistry, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z1. mtanner@chem.ubc.ca

Biochemistry. 2008 Mar 11;47(10):3272-82.

ABSTRACT: Legionaminic acid is a nine-carbon alpha-keto acid that is similar in structure to other members of the sialic acid family that includes neuraminic acid and pseudaminic acid. It is found as a component of the lipopolysaccharide in several bacterial species and is perhaps best known for its presence in the O-antigen of the causative agent of Legionnaires' disease, Legionella pneumophila. In this work, the enzymes responsible for the biosynthesis and activation of N, N'-diacetyllegionaminic acid are identified for the first time. A cluster of three L. pneumophila genes bearing homology to known sialic acid biosynthetic genes ( neuA,B,C) were cloned and overexpressed in Escherichia coli. The NeuC homologue was found to be a hydrolyzing UDP- N, N'-diacetylbacillosamine 2-epimerase that converts UDP- N, N'-diacetylbacillosamine into 2,4-diacetamido-2,4,6-trideoxymannose and UDP. Stereochemical and isotopic labeling studies showed that the enzyme utilizes a mechanism involving an initial anti elimination of UDP to form a glycal intermediate and a subsequent syn addition of water to generate product. This is similar to the hydrolyzing UDP- N-acetylglucosamine 2-epimerase (NeuC) of sialic acid biosynthesis, but the L. pneumophila enzyme would not accept UDP-GlcNAc as an alternate substrate. The NeuB homologue was found to be a N, N'-diacetyllegionaminic acid synthase that condenses 2,4-diacetamido-2,4,6-trideoxymannose with phosphoenolpyruvate (PEP), although the in vitro activity of the recombinant enzyme (isolated as a MalE fusion protein) was very low. The synthase activity was dependent on the presence of a divalent metal ion, and the reaction proceeded via a C-O bond cleavage process, similar to the reactions catalyzed by the sialic acid and pseudaminic acid synthases. Finally, the NeuA homologue was shown to possess the CMP- N, N'-diacetyllegionaminic acid synthetase activity that generates the activated form of legionaminic acid used in lipopolysaccharide biosynthesis. Together, the three enzymes constitute a pathway that converts a UDP-linked bacillosamine derivative into a CMP-linked legionaminic acid derivative.

 

A Legionella pneumophila peptidyl-prolyl cis-trans isomerase present in culture supernatants is necessary for optimal growth at low temperatures

Söderberg MA, Cianciotto NP.

Department of Microbiology and Immunology, Northwestern University Medical School, Chicago, IL 60611, USA. n-cianciotto@northwestern.edu

Appl Environ Microbiol. 2008 Mar;74(5):1634-8.

ABSTRACT: Several Legionella pneumophila proteins were highly expressed in low-temperature supernatants. One of these proteins was the peptidyl-prolyl isomerase PpiB. Mutants lacking ppiB exhibited reduced growth at 17 degrees C. Since PpiB lacked a signal sequence and was present in 17 degrees C supernatants of type II and type IV secretion mutants, this protein may be secreted by a novel mechanism.

 

Clonal population structure of Legionella pneumophila inferred from allelic profiling

Edwards MT, Fry NK, Harrison TG.

Respiratory and Systemic Infection Laboratory, Health Protection Agency Centre for Infections, London, UK. Norman.Fry@HPA.org.uk

Microbiology. 2008 Mar;154(Pt 3):852-64.

ABSTRACT: The population structure of Legionella pneumophila was investigated by analysing nucleotide sequences from six loci (flaA, pilE, asd, mip, mompS and proA) of 335 globally distributed isolates from clinical and environmental sources over a 29-year period (1977-2006). Data were obtained from unrelated isolates from Europe (n=270), Japan (n=31), Canada (n=7), the USA (n=24) and Australia (n=1). The country of origin of two strains was unknown. Analysis of these isolates indicated significant linkage disequilibrium between the six loci. Application of six sequence-based recombination detection tests did not reveal evidence of recombination, but estimates of rates of recombination and mutation made by a seventh test suggested that recombination could have occurred at a rate similar to, but probably lower than, that of mutation. Genealogies inferred under models with and without recombination were congruent with each other, providing no definitive evidence regarding recombination, and were in agreement with sequence clusters identified by graph methods. Further evidence supporting the distinct nature of two of the three subspecies of L. pneumophila, subsp. fraseri and subsp. pascullei, was also found. The ratios of non-synonymous to synonymous nucleotide polymorphisms for each of the allele sets were examined and revealed that the putative virulence loci mompS and pilE are under diversifying pressure, while the allelic regions of three other loci linked to virulence (flaA, proA and mip) do not appear to be.

 

A Legionella pneumophila peptidyl-prolyl cis-trans isomerase present in culture supernatants is necessary for optimal growth at low temperatures

Söderberg MA, Cianciotto NP.

Department of Microbiology and Immunology, Northwestern University Medical School, Chicago, IL 60611, USA. n-cianciotto@northwestern.edu

Appl Environ Microbiol. 2008 Mar;74(5):1634-8.

ABSTRACT: Several Legionella pneumophila proteins were highly expressed in low-temperature supernatants. One of these proteins was the peptidyl-prolyl isomerase PpiB. Mutants lacking ppiB exhibited reduced growth at 17 degrees C. Since PpiB lacked a signal sequence and was present in 17 degrees C supernatants of type II and type IV secretion mutants, this protein may be secreted by a novel mechanism.

 

The response regulator CpxR directly regulates expression of several Legionella pneumophila icm/dot components as well as new translocated substrates

Altman E, Segal G.

Department of Molecular Microbiology & Biotechnology, George S. Wise Faculty of Life Sciences, Tel-Aviv University, Tel-Aviv 69978, Israel. GilS@tauex.tau.ac.il

J Bacteriol. 2008 Mar;190(6):1985-96.

ABSTRACT: Legionella pneumophila has been shown to utilize the icm/dot type IV secretion system for pathogenesis. This system was shown to be composed of icm/dot complex components and accessory proteins, as well as a large number of translocated substrates. Bioinformatic analysis of the regulatory regions of all the genes revealed that several icm/dot genes, as well as two genes encoding icm/dot translocated substrates, contain the conserved CpxR regulatory element, a regulator that has been shown previously to control the expression of the icmR gene. An experimental analysis, which included a comparison of gene expression in a L. pneumophila wild-type strain and gene expression in a cpxR deletion mutant, construction of mutants with mutations in the CpxR conserved regulatory elements, controlled expression studies, and mobility shift assays, demonstrated the direct relationship between the CpxR regulator and the expression of the genes. Furthermore, genomic analysis identified nine additional genes that contain a putative CpxR regulatory element; five of these genes (two legA genes and three ceg genes) were suggested previously to be putative icm/dot translocated substrates. The three ceg genes identified, which were shown previously to contain a putative PmrA regulatory element, were found here to be regulated by both CpxR and PmrA. The other six genes (two legA genes and four new genes products were found to be regulated by CpxR. Moreover, using the CyaA translocation assay, these nine gene products were found to be translocated into host cells in an Icm/Dot-dependent manner. Our results establish that the CpxR regulator is a fundamental regulator of the icm/dot type IV secretion system in L. pneumophila.

 

Legionella translocates an E3 ubiquitin ligase that has multiple U-boxes with distinct functions

Kubori T, Hyakutake A, Nagai H.

The 21st Century COE Program, Research Institute for Microbial Diseases, Osaka University, Yamadaoka 3-1, Suita, Osaka 565-0871, Japan. hnagai@biken.osaka-u.ac.jp

Mol Microbiol. 2008 Mar;67(6):1307-19.

ABSTRACT: Legionella pneumophila has a Dot/Icm type IV secretion system used to translocate a number of 'effector proteins' which subvert host cell functions. In this study, we identified 19 novel Dot/Icm substrate proteins using a systematic screening technique. A blast analysis revealed that one of the substrates, which we named LubX (LegionellaU-box protein), contains two domains that have a remarkable similarity to the U-box, a domain found in eukaryotic E3 ubiquitin ligases. The expression of LubX is induced upon infection, and most of the LubX produced was translocated into the host cells. LubX has ubiquitin ligase activity in conjunction with UbcH5a or UbcH5c E2 enzymes and mediates polyubiquitination of host Clk1 (Cdc2-like kinase 1). We demonstrate that one of the U-boxes (U-box 1) is critical to the ubiquitin ligation, and the other U-box (U-box 2) mediates interaction with Clk1. Thus, the two U-boxes of LubX have distinct functions, and U-box 2 plays a non-canonical role in substrate binding. Although we demonstrate that inhibition of Clk kinase results in a marked reduction of Legionella growth within mouse macrophages, the consequence of Clk1 ubiquitination is still being elucidated. Together, these data suggest that Clk1 is the target host molecule which Legionella modulates during infection.

 

The type II secretion system of Legionella pneumophila elaborates two aminopeptidases, as well as a metalloprotease that contributes to differential infection among protozoan hosts

Rossier O, Dao J, Cianciotto NP.

Department of Microbiology-Immunology, Northwestern University Medical School, 320 East Superior Ave., Chicago, IL 60611-3010, USA.

n-cianciotto@northwestern.edu

Appl Environ Microbiol. 2008 Feb;74(3):753-61.

ABSTRACT: Legionella pneumophila, the agent of Legionnaires' disease, is an intracellular parasite of aquatic amoebae and human macrophages. A key factor for L. pneumophila in intracellular infection is its type II protein secretion system (Lsp). In order to more completely define Lsp output, we recently performed a proteomic analysis of culture supernatants. Based upon the predictions of that analysis, we found that L. pneumophila secretes two distinct aminopeptidase activities encoded by the genes lapA and lapB. Whereas lapA conferred activity against leucine, phenylalanine, and tyrosine aminopeptides, lapB was linked to the cleavage of lysine- and arginine-containing substrates. To assess the role of secreted aminopeptidases in intracellular infection, we examined the relative abilities of lapA and lapB mutants to infect human U937 cell macrophages as well as Hartmannella vermiformis and Acanthamoeba castellanii amoebae. Although these experiments identified a dispensable role for LapA and LapB, they uncovered a previously unrecognized role for the type II-dependent ProA (MspA) metalloprotease. Whereas proA mutants were not defective for macrophage or A. castellanii infection, they (but not their complemented derivatives) were impaired for growth upon coculture with H. vermiformis. Thus, ProA represents the first type II effector implicated in an intracellular infection event. Furthermore, proA represents an L. pneumophila gene that shows differential importance among protozoan infection models, suggesting that the legionellae might have evolved some of its factors to especially target certain of their protozoan hosts.

 

Phosphatidylcholine synthesis is required for optimal function of Legionella pneumophila virulence determinants

Conover GM, Martinez-Morales F, Heidtman MI, Luo ZQ, Tang M, Chen C, Geiger O, Isberg RR.

Department of Molecular Biology and Microbiology, Tufts University School of Medicine, 150 Harrison Avenue, Boston, MA 02111, USA. ralph.isberg@tufts.edu

Cell Microbiol. 2008 Feb;10(2):514-28.

ABSTRACT: The function of phosphatidylcholine (PC) in the bacterial cell envelope remains cryptic. We show here that productive interaction of the respiratory pathogen Legionella pneumophila with host cells requires bacterial PC. Synthesis of the lipid in L. pneumophila was shown to occur via either phospholipid N-methyltransferase (PmtA) or phosphatidylcholine synthase (PcsA), but the latter pathway was demonstrated to be of predominant importance. Loss of PC from the cell envelope caused lowered yields of L. pneumophila within macrophages as well as loss of high multiplicity cytotoxicity, while mutants defective in PC synthesis could be complemented either by reintroduction of PcsA or by overproduction of PmtA. The lowered yields and reduced cytotoxicity in mutants with defective PC biosynthesis were due to three related defects. First, there was a poorly functioning Dot/Icm apparatus, which delivers substrates required for intracellular growth into the cytosol of infected cells. Second, there was reduced bacterial binding to macrophages, possibly due to loss of PC or a PC derivative on the bacterium that is recognized by the host cell. Finally, strains lacking PC had low steady-state levels of flagellin protein, a deficit that had been previously associated with the phenotypes of lowered cytotoxicity and poor cellular adhesion.

 

 

Molecular mechanisms of host-pathogen interactions and their potential for the discovery of new drug targets

Briken V.

Department of Cell Biology and Molecular Genetics and Maryland Pathogen Research Institute, University of Maryland, Microbiology Bldg. 231, Room 2201, College Park, MD, 20742, USA. vbriken@umd.edu

Curr Drug Targets. 2008 Feb;9(2):150-7.

ABSTRACT: Vaccines and chemotherapy have undeniably been the discoveries in the field of biomedical research that have exerted the biggest impact on the improvement of public health. Nevertheless, the development of bacterial resistance to antibiotics has co-evolved over time with the discovery of new drugs. This entails the necessity for continuous research on new anti-infectious agents. The current review highlights recent discoveries in the molecular mechanisms of specific host pathogen interactions and their potential for drug discovery. The focus is on facultative and obligate intracellular pathogens (Mycobacterium, Chlamydia and Legionella) and their manipulation of host cells in regard to inhibition of phagosome, maturation and cell death. Furthermore, the composition and role of the SecA2 and the ESX-1 secretion pathways in bacterial virulence and manipulation of infected host cells is discussed. The central hypothesis proposed in this review is that the characterization of bacterial proteins and lipids involved in host cell manipulation (modulins) will provide an abundance of new drug targets. One advantage of targeting such bacterial modulins for drug development is that these anti-modulin drugs will not disrupt the beneficial host microflora and therefore have fewer side effects.

 

Occurrence and distribution of sequence types among Legionella pneumophila strains isolated from patients in Germany: common features and differences to other regions of the world

Borchardt J, Helbig JH, Lück PC.

Institute of Medical Microbiology and Hygiene, TU Dresden, Fiedlerstrasse 42, Dresden, 01307, Germany. Christian.Lueck@tu-dresden.de

Eur J Clin Microbiol Infect Dis. 2008 Jan;27(1):29-36.

ABSTRACT: A total of 105 unrelated clinical isolates of Legionella pneumophila were randomly selected from the German National Legionella strain collection and typed by monoclonal antibody (MAb) subgrouping and a seven-gene locus sequence-based typing (SBT) scheme. According to the case definitions of the European Working Group for Legionella Infections, 19 of the isolates tested were travel-associated, 38 were community-acquired and 48 were of nosocomial origin. Eighty-four of these strains belonged to serogroup 1, 20 belonged to other serogroups, and one isolate could not be serogrouped. The majority of strains among the travel-associated and community-acquired cases were MAb3-1-positive. The most common sequence type (1, 4, 3, 1, 1, 1, 1) was found in 20 isolates in 11 cities; other allelic profiles also found in Europe (2, 3, 9, 10, 2, 1, 6), (1, 3, 9, 10, 2, 1, 6), (2, 6, 17, 14, 13, 11, 11) and (3, 4, 1, 1, 1, 9, 1) were detected among the German isolates but at a low frequency. In contrast, some SBT are unique to Germany, including (3, 4, 1, 3, 35, 9, 11), which was found among five isolates from patients in Berlin. In concordance with European data, a significant portion of the L. pneumophila strains isolated from patients in Germany belong to clones that occur throughout the world and which are responsible for the majority of clinical cases.

 

Presence in Legionella pneumophila of a mammalian-like mitochondrial permeability transition pore?

Khemiri A, Jouenne T, Cosette P.

IBBR Group, UMR 6522 CNRS, University of Rouen, Proteomic Platform of the Institute for Peptide Research (IFRMP23), Mont-Saint-Aignan, France. pascal.cosette@univ-rouen.fr

FEMS Microbiol Lett. 2008 Jan;278(2):171-6.

ABSTRACT: The genome of Legionella pneumophila reveals the presence of a large number of genes coding for eukaryotic-like proteins. By using database searches and homology investigations, we identified three proteins in L. pneumophila whose sequences share similarities with that of eukaryotic polypeptides (lpg0211, lpg1974 and lpg1982). In eukaryotes, the corresponding proteins (PBR, peripheral benzodiazepine receptor; VDAC, voltage-dependant anion channel; and CypD, cyclophilin D) participate in the formation of the mammalian mitochondrial permeability transition pore (MPTP), a complex involved in cell apoptosis. Intriguingly, the presence of these proteins has never been reported in the same bacterium and constitutes, up to now, a unique feature of L. pneumophila. In Legionella, we hypothesize that these proteins are recruited in a multiprotein complex close to the MPTP that may regulate intracellular survival and/or proliferation. 

 

The Legionella pneumophila response regulator LqsR promotes host cell interactions as an element of the virulence regulatory network controlled by RpoS and LetA

Tiaden A, Spirig T, Weber SS, Brüggemann H, Bosshard R, Buchrieser C, Hilbi H.

Institute of Microbiology, ETH Zürich, Wolfgang-Pauli-Strasse 10, 8093 Zürich, Switzerland. hilbi@micro.biol.ethz.ch

Cell Microbiol. 2007;9(12):2903-20.

ABSTRACT:Legionella pneumophila is an opportunistic human pathogen that replicates within environmental amoebae including Acanthamoeba castellanii and Dictyostelium discoideum. The Icm/Dot type IV secretion system promotes phagocytosis and intracellular replication of L. pneumophila in an endoplasmic reticulum-derived 'Legionella-containing vacuole' (LCV). L. pneumophila adopts a biphasic life cycle consisting of a replicative growth phase and a transmissive (stationary) phase, the latter of which is characterized by the preferential expression of genes required for motility and virulence. A bioinformatic analysis of the L. pneumophila genome revealed a gene cluster homologous to the Vibrio cholerae cqsAS genes, encoding a putative quorum sensing autoinducer synthase (lqsA) and a sensor kinase (lqsS), which flank a novel response regulator (lqsR). We report here that an L. pneumophila lqsR deletion mutant grew in broth with the same rate as wild-type bacteria, but entered the replicative growth phase earlier. Overexpression of lqsR led to an elongated morphology of the bacteria. The lqsR mutant strain was found to be more salt-resistant and impaired for intracellular growth in A. castellanii, D. discoideum and macrophages, formation of the ER-derived LCV and toxicity. Moreover, L. pneumophila lacking LqsR, as well as strains lacking the stationary sigma factor RpoS or the two-component response regulator LetA, were phagocytosed less efficiently by A. castellanii, D. discoideum or macrophages. The expression of lqsR was dependent on RpoS and, to a lesser extent, also on LetA. DNA microarray experiments revealed that lqsR regulates the expression of genes involved in virulence, motility and cell division, consistent with a role for LqsR in the transition from the replicative to the transmissive (virulent) phase. Our findings indicate that LqsR is a novel pleiotropic regulator involved in RpoS- and LetA-controlled interactions of L. pneumophila with phagocytes.

 

Receptor-mediated uptake of Legionella pneumophila by Acanthamoeba castellanii and Naegleria lovaniensis

Declerck P, Behets J, De Keersmaecker B, Ollevier F.

Laboratory of Aquatic Ecology, Zoological Institute, Katholieke Universiteit Leuven, Leuven, Belgium. Priscilla.declerck@bio.kuleuven.be

J Appl Microbiol. 2007 Dec;103(6):2697-703.

ABSTRACT: AIMS: Investigation of the attachment and uptake of Legionella pneumophila by Acanthamoeba castellanii and Naegleria lovaniensis, as these are two critical steps in the subsequent bacterial survival in both amoeba hosts. METHODS AND RESULTS: Initially, the mode of Legionella uptake was examined using inhibitors of microfilament-dependent and receptor-mediated uptake phagocytosis. Secondly, the minimum saccharide structure to interfere with L. pneumophila uptake was determined by means of selected saccharides. Bacterial attachment and uptake by each of the amoeba species occurred through a receptor-mediated endocytosis, which required de novo synthesis of host proteins. Legionella pneumophila showed a high affinity to the alpha1-3D-mannobiose domain of the mannose-binding receptor located on A. castellanii. In contrast, L. pneumophila bacteria had a high affinity for the GalNAcbeta1-4Gal domain of the N-acetyl-D-galactosamine receptor of N. lovaniensis. CONCLUSIONS: Our data pointed to a remarkable adaptation of L. pneumophila to invade different amoeba hosts, as the uptake by both amoeba species is mediated by two different receptor families. SIGNIFICANCE AND IMPACT OF THE STUDY: The fact that L. pneumophila is taken up by two different amoeba species using different receptor families adds further complexity to the host-parasite interaction process, as 14 amoeba species are known to be appropriate Legionella hosts.

 

Sel1 repeat protein LpnE is a Legionella pneumophila virulence determinant that influences vacuolar trafficking

Newton HJ, Sansom FM, Dao J, McAlister AD, Sloan J, Cianciotto NP, Hartland EL.

Australian Bacterial Pathogenesis Program, Department of Microbiology, Monash University, Victoria 3800, Australia. Liz.Hartland@med.monash.edu.au

Infect Immun. 2007 Dec;75(12):5575-85.

ABSTRACT: The environmental pathogen Legionella pneumophila possesses five proteins with Sel1 repeats (SLRs) from the tetratricopeptide repeat protein family. Three of these proteins, LpnE, EnhC, and LidL, have been implicated in the ability of L. pneumophila to efficiently establish infection and/or manipulate host cell trafficking events. Previously, we showed that LpnE is important for L. pneumophila entry into macrophages and epithelial cells. In further virulence studies here, we show that LpnE is also required for efficient infection of Acanthamoeba castellanii by L. pneumophila and for replication of L. pneumophila in the lungs of A/J mice. In addition, we found that the role of LpnE in host cell invasion is dependent on the eight SLR regions of the protein. A truncated form of LpnE lacking the two C-terminal SLR domains was unable to complement the invasion defect of an lpnE mutant of L. pneumophila 130b in both the A549 and THP-1 cell lines. The lpnE mutant displayed impaired avoidance of LAMP-1 association, suggesting that LpnE influenced trafficking of the L. pneumophila vacuole, similar to the case for EnhC and LidL. We also found that LpnE was present in L. pneumophila culture supernatants and that its export was independent of both the Lsp type II secretion system and the Dot/Icm type IV secretion system. The fact that LpnE was exported suggested that the protein may interact with a eukaryotic protein. Using LpnE as bait, we screened a HeLa cell cDNA library for interacting partners, using the yeast two-hybrid system. Examination of the protein-protein interaction between LpnE and a eukaryotic protein, obscurin-like protein 1, suggested that LpnE can interact with eukaryotic proteins containing immunoglobulin-like folds via the SLR regions. This investigation has further characterized the contribution of LpnE to L. pneumophila virulence and, more specifically, the importance of the SLR regions to LpnE function.

 

Altered Inflammatory Responses in TLR5-Deficient Mice Infected with Legionella pneumophila

Hawn TR, Berrington WR, Smith IA, Uematsu S, Akira S, Aderem A, Smith KD, Skerrett SJ.

University of Washington School of Medicine, Seattle, WA 98195. thawn@u.washington.edu

J Immunol. 2007 Nov 15;179(10):6981-7.

ABSTRACT: Legionella pneumophila (Lp), an important cause of morbidity and mortality from pneumonia, infects alveolar macrophages (AMs) and is recognized by several TLRs as well as Birc1e (NAIP5) and IL-1 converting enzyme-protease activating factor. We examined the role of TLR5 during the murine response to aerosolized Lp infection. At 4 h after infection, Tlr5(-/-) mice had lower numbers of polymorphonuclear neutrophils (PMNs) in their broncho-alveolar lavage fluid in comparison to wild-type (WT) mice. At 24 and 72 h, the PMN recruitment was similar. WT mice infected with a flagellin-deficient strain (LpFlaA-) also showed an impaired early PMN response at 4 h compared with those infected with the WT strain. There was no consistent difference in bacterial counts at any of the time points when comparing the Tlr5(-/-) and WT mice. However, at 6 days after infection, the Tlr5(-/-) mice had increased leukocytic infiltrates in the alveolar and peribronchial interstitial spaces that were consistent with organizing pneumonia. We also examined the role of TLR5 during macrophage infection. In contrast to bone marrow-derived macrophages, AMs secreted TNF-alpha after stimulation with purified flagellin. In addition, WT, but not Tlr5(-/-), AMs produced TNF-alpha after stimulation with Lp. Live LpFlaA- did not induce TNF-alpha secretion in AM. These results suggested that AMs recognize Lp flagellin and that a majority of the Lp-induced TNF-alpha response is TLR5-mediated. Thus, TLR5 mediates recognition of Lp in AMs and performs a distinct role during the in vivo pulmonary immune response through regulation of early PMN recruitment and subsequent later development of pneumonia.

 

Legionella pneumophila proteins that regulate Rab1 membrane cycling

Ingmundson A, Delprato A, Lambright DG, Roy CR.

Section of Microbial Pathogenesis, Yale University School of Medicine, Boyer Center for Molecular Medicine, 295 Congress Avenue, New Haven, Connecticut 06536, USA. craig.roy@yale.edu

Nature. 2007 Nov 15;450(7168):365-9.

ABSTRACT: Rab1 is a GTPase that regulates the transport of endoplasmic-reticulum-derived vesicles in eukaryotic cells. The intracellular pathogen Legionella pneumophila subverts Rab1 function to create a vacuole that supports bacterial replication by a mechanism that is not well understood. Here we describe L. pneumophila proteins that control Rab1 activity directly. We show that a region in the DrrA (defect in Rab1 recruitment A) protein required for recruitment of Rab1 to membranes functions as a guanine nucleotide dissociation inhibitor displacement factor. A second region of the DrrA protein stimulated Rab1 activation by functioning as a guanine nucleotide exchange factor. The LepB protein was found to inactivate Rab1 by stimulating GTP hydrolysis, indicating that LepB has GTPase-activating protein activity that regulates removal of Rab proteins from membranes. Thus, L. pneumophila encodes proteins that regulate three distinct biochemical reactions critical for Rab GTPase membrane cycling to redirect Rab1 to the pathogen-occupied vacuole and to control Rab1 function.

 

A bifunctional bacterial protein links GDI displacement to Rab1 activation

Machner MP, Isberg RR.

Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, MA 02111, USA. ralph.isberg@tufts.edu

Science. 2007 Nov 9;318(5852):974-7.

ABSTRACT: Rab guanosine triphosphatases (GTPases) regulate vesicle trafficking in eukaryotic cells by reversibly associating with lipid membranes. Inactive Rab GTPases are maintained in the cytosol by binding to GDP-dissociation inhibitor (GDI). It is believed that specialized proteins are required to displace GDI from Rab GTPases before Rab activation by guanosine diphosphate-guanosine 5'-triphosphate (GDP-GTP) exchange factors (GEFs). Here, we found that SidM from Legionella pneumophila could act as both GEF and GDI-displacement factor (GDF) for Rab1. Rab1 released from GDI was inserted into liposomal membranes and was used as a substrate for SidM-mediated nucleotide exchange. During host cell infection, recruitment of Rab1 to Legionella-containing vacuoles depended on the GDF activity of SidM. Thus, GDF and GEF activity can be promoted by a single protein, and GDF activity can coordinate Rab1 recruitment from the GDI-bound pool.

 

Legionella pathogenicity: Genome structure, regulatory networks and the host cell response

Steinert M, Heuner K, Buchrieser C, Albert-Weissenberger C, Glöckner G.

Institut für Mikrobiologie, Technische Universität Braunschweig, Spielmannstr. 7, D-38106 Braunschweig, Germany; Institut für Molekulare Infektionsbiologie, Universität Würzburg, Röntgenring 11, D-97070 Würzburg, Germany. m.steinert@tu-bs.de

Int J Med Microbiol. 2007 Nov;297(7-8):577-87.

ABSTRACT: Legionella spp. the causative agent of Legionnaires' disease is naturally found in fresh water where the bacteria parasitize intracellularly within protozoa. Upon aerosol formation via man-made water systems, Legionella can enter the human lung and cause a severe form of pneumonia. Here we review results from systematic comparative genome analysis of Legionella species with different pathogenic potentials. The complete genomes reveal that horizontal gene transfer has played an important role during the evolution of Legionella and indicate the importance of secretion machineries for the intracellular lifestyle of this pathogen. Moreover, we highlight recent findings on the in vivo transcriptional program of L. pneumophila and the regulatory networks involved in the biphasic life cycle. In order to understand how Legionella effectively subvert host cell functions for its own benefit the transcriptional host cell response upon infection of the model amoeba Dictyostelium discoideum was studied. The use of this model organism made it possible to develop a roadmap of host cell factors which significantly contribute to the uptake of L. pneumophila and the establishment of an ER-associated replicative vacuole.

 

Random mutagenesis of Legionella pneumophila reveals genes associated with lipopolysaccharide synthesis and recognition by typing monoclonal antibodies

Wagner C, Krönert C, Lück PC, Jacobs E, Cianciotto NP, Helbig JH.

Medizinische Fakultät TU Dresden, Institut Medizinische Mikrobiologie und Hygiene, Dresden, Germany. j.helbig@mailbox.tu-dresden.de

J Appl Microbiol. 2007 Nov;103(5):1975-82.   

ABSTRACT: AIMS: To use random mutagenesis for the characterization of Legionella pneumophila lipopolysaccharide (LPS) components and serotypes. METHODS AND RESULTS: Five strains belonging to different serogroups and/or monoclonal subgroups were mutagenized using a mini-Tn10 transposon. Exactly 11 819 mutants were checked for alterations in LPS using at least 11 monoclonal antibodies (mAbs) that define L. pneumophila serotypes. Among the mutants, five different mini-Tn10 insertions were identified. Four mutants originating from serogroup-1 did not lose their serogroup-specific epitope, but did sustain subtler changes that resulted in switches to different mAb subgroups. In contrast, a mutant from serogroup-6 lost its serogroup-specific epitope, while retaining a serogroup-cross-reacting epitope. CONCLUSIONS: Random mutagenesis is a valuable tool for LPS epitope mapping. While some characteristics of L. pneumophila LPS can be altered, others appear resistant to mutagenesis. This underscores both the flexibility and rigidity of LPS architecture in L. pneumophila. SIGNIFICANCE AND IMPACT OF THE STUDY: Losses of L. pneumophila LPS epitopes can result in new serotypes, changes that might escape detection by current DNA-based typing schemes. But, as the frequency of these changes is rare, based upon our observations, serotyping should remain an important tool for identifying L. pneumophila in water systems that are implicated in human infection.

 

Expression of Legionella pneumophila paralogous lipid A biosynthesis genes under different growth conditions

Albers U, Tiaden A, Spirig T, Al Alam D, Goyert SM, Gangloff SC, Hilbi H.

ETH Zürich, Institute of Microbiology, Wolfgang-Pauli Strasse 10, HCI G405, 8093 Zürich, Switzerland. hilbi@micro.biol.ethz.ch

Microbiology. 2007 Nov;153(Pt 11):3817-29. 

ABSTRACT: Legionella pneumophila is an opportunistic pathogen that in the environment colonizes biofilms and replicates within amoebae. The bacteria employ the intracellular multiplication/defective organelle trafficking (Icm/Dot) type IV secretion system to grow intracellularly in a specific vacuole. Using Acanthamoeba castellanii as a host cell, we have previously identified lcsC (Legionella cytotoxic suppressor), a paralogue of the lipid A disaccharide synthase lpxB, as a cytotoxic factor of L. pneumophila. A bioinformatic analysis of the genome revealed that L. pneumophila is unique in harbouring two paralogues of lpxB and two and three paralogues of the lipid A biosynthesis acyltransferases lpxA and lpxD, respectively. LcsC (lpxB1) forms a transcriptional unit with gnnA, encoding a putative UDP-GlcNAc oxidase in the biosynthetic pathway leading to 3-aminoglucosamine analogues of lipid A. LpxB2 clusters with lpxD2, lpxA2 and lpxL paralogues, encoding secondary acyltransferases. LcsC/lpxB1 and lpxB2 were found to partially complement the growth defect of an Escherichia coli lpxB conditional mutant strain, indicating that both corresponding enzymes possess lipid A disaccharide synthase activity. The two L. pneumophila lpxB paralogues are not functionally equivalent, since expression of lcsC/lpxB1 but not lpxB2 in an L. pneumophila icmG mutant is cytotoxic for A. castellanii, and LPS purified from the two strains triggers CD14-dependent tumour necrosis factor (TNF)alpha production by macrophages with a different potency. The lpxB and lpxA paralogues are expressed under various growth conditions, including broth, biofilms and in A. castellanii. While the flagellar gene flaA is mainly expressed in late stationary phase, the lpxB and lpxA paralogues are preferentially expressed in the exponential and early stationary phases. Upon exposure to hypotonic stress and nutrient deprivation, lpxA1, and to a lesser extent lcsC/lpxB1, is upregulated. The differential regulation of lpxB or lpxA paralogues in response to changing environmental conditions might allow L. pneumophila to adapt its lipid A structure.

 

Legionella pneumophila exhibits plasminogen activator activity

Vranckx L, De Buck E, Anné J, Lammertyn E.

Laboratory for Bacteriology, Rega Institute for Medical Research, Katholieke Universiteit Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium. Jozef.Anne@rega.kuleuven.be

Microbiology. 2007 Nov;153(Pt 11):3757-65

ABSTRACT: Based on their localization at the boundary of the bacterial cell and its environment, outer-membrane proteins (Omps) are important determinants for interaction of bacteria with their host cell. Therefore, they can be considered as important determinants for virulence. Looking for Legionella pneumophila Omps potentially involved in virulence, we identified a gene encoding a homologue of the plasminogen activator (Pla) of Yersinia pestis. Pla belongs to the class of omptins, a family of surface proteases/adhesins that exhibit different virulence-associated functions. In this report we describe the cloning and identification of the plasminogen activator homologue Lpa of L. pneumophila and demonstrate its outer-membrane localization. Transcriptional analysis of the Lpa region revealed expression of the gene in both exponential and stationary growth phase and showed that transcription of the lpa gene is directed by its own promoter. We also show, to our knowledge for the first time, that L. pneumophila has the capacity to convert plasminogen into plasmin by the action of the outer-membrane Lpa protein.

 

Complete nucleotide sequence of pLD-TEX-KL, a 66-kb plasmid of Legionella dumoffii TEX-KL strain

Qin T, Hirakawa H, Iida K, Oshima K, Hattori M, Tashiro K, Kuhara S, Yoshida S.

Department of Bacteriology, Faculty of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan. qtian@bact.med.kyushu-u.ac.jp

Plasmid. 2007 Nov;58(3):261-8.

ABSTRACT: The complete nucleotide sequence of a large (66kb) plasmid pLD-TEX-KL of Legionella dumoffii TEX-KL strain was determined. Of the 57 predicted open reading frames (ORFs), 39 (68%) encoded proteins similar to previously known proteins, five (9%) were assigned with putative functions, three (5%) encoded conserved hypothetical proteins, and 10 (18%) had no homology to any genes present in the current open databases. The ORFs with similar functions were organized in a modular structure; thus, transfer region was identified, as well as a putative heavy-metal ion transporter system (hel). The transfer region encoded homologs of the Salmonella entrica serovar Typhi conjugative system components involved in conjugation. In addition, we also found a potential protein that was analogous to the DNA polymerase III epsilon subunit. It is rarely found that plasmid encode the DNA polymerase.

 

A bacterial ecto-triphosphate diphosphohydrolase similar to human CD39 is essential for intracellular multiplication of Legionella pneumophila

Sansom FM, Newton HJ, Crikis S, Cianciotto NP, Cowan PJ, d'Apice AJ, Hartland EL.

Australian Bacterial Pathogenesis Program, Department of Microbiology, Monash University, Victoria 3800, Australia.  liz.hartland@med.monash.edu.au

Cell Microbiol. 2007 Aug;9(8):1922-35.

ABSTRACT: As part of its pathogenesis, Legionella pneumophila persists within human alveolar macrophages in non-acidified organelles that do not mature into phagolysosomes. Two L. pneumophila genes, lpg0971 and lpg1905, are predicted to encode ecto-nucleoside triphosphate diphosphohydrolases (ecto-NTPDases) that share sequence similarity with human CD39/NTPDase1. The predicted products possess five apyrase conserved domains that are typical of eukaryotic ecto-NTPDases. In this study, we found that an lpg1905 mutant was recovered in lower numbers from macrophages, alveolar epithelial cells and the amoeba, Hartmannella vermiformis compared with wild-type L. pneumophila and an lpg0971 mutant. Similar to human CD39, recombinant purified Lpg1905 exhibited ATPase and ADPase activity and possessed the ability to inhibit platelet aggregation. Mutation of a conserved Glu159 residue that is essential for CD39 activity inhibited ATPase and ADPase activity of Lpg1905. In addition, enzyme activity was inhibited in the presence of the specific ecto-NTPDase inhibitor, ARL67156. The entry and replication defect of the lpg1905 mutant was reversed upon transcomplementation with lpg1905 but not lpg1905E159A encoding an enzymatically inactive form of the protein. Although several protozoan parasites exhibit ecto-NTPDase activity, including Toxoplasma gondii, Trichomonas vaginalis and Trypanosoma cruzi, this is the first time a bacterial ecto-NTPDase has been implicated in virulence.

 

Effector proteins translocated by Legionella pneumophila: strength in numbers

Ninio S, Roy CR.

Section of Microbial Pathogenesis, Yale University School of Medicine, Boyer Center for Molecular Medicine, 295 Congress Avenue, New Haven, CT 06536, USA. shira.ninio@yale.edu

Trends Microbiol. 2007 Aug;15(8):372-80.

ABSTRACT: The Gram-negative bacterium Legionella pneumophila is a parasite of eukaryotic cells. It has evolved to survive and replicate in a wide range of protozoan hosts and can also infect human alveolar macrophages as an opportunistic pathogen. Crucially for the infection process, L. pneumophila uses a type IV secretion system called Dot/Icm to translocate bacterial proteins into host cells. In recent years a large number of Dot/Icm-translocated proteins have been identified. The study of these proteins, referred to as effectors, is providing valuable insight into the mechanism by which an intracellular pathogen can manipulate eukaryotic cellular processes to traffic and replicate in host cells.

 

Host cell-dependent secretion and translocation of the LepA and LepB effectors of Legionella pneumophila

Chen J, Reyes M, Clarke M, Shuman HA.

Department of Microbiology, Columbia University Medical Center, New York, NY 10032, USA. has7@columbia.edu

Cell Microbiol. 2007 Jul;9(7):1660-71.

ABSTRACT: Legionella pneumophila is the Gram-negative bacterial agent of Legionnaires' disease, an acute, often fatal pneumonia. L. pneumophila infects alveolar macrophages, evading the antimicrobial defences of the phagocyte by preventing fusion of the phagosome with lysosomes and avoiding phagosome acidification. The bacteria then modulate the composition of the vacuole so that it takes on the characteristics of the endoplasmic reticulum. Similar events occur when the bacteria infect unicellular protozoa. It is thought that replication in fresh water protozoa provides an environmental reservoir for the organism. Several effector proteins are delivered to the host by the Icm/Dot type IV secretion system (TFSS). Some of these have been shown to participate in the trafficking of the Legionella phagosome. Here we describe the ability of the Icm/Dot TFSS to translocate two effectors, LepA and LepB, that play a role in the non-lytic release of Legionella from protozoa. We report that translocation of the Lep proteins is inhibited by agents that depolymerize actin filaments and that effectors may be secreted into the extracellular medium upon cell contact. Depletion of the Lep proteins by deletion of their genes results in increased ability to lyse red blood cells. In contrast, overexpression of Lep-containing hybrid proteins appears to specifically inhibit the activity of the Icm/Dot TFSS and may prevent the delivery of other effectors that are critical for intracellular multiplication.

 

Rapid escape of the dot/icm mutants of Legionella pneumophila into the cytosol of mammalian and protozoan cells

Molmeret M, Santic' M, Asare R, Carabeo RA, Abu Kwaik Y.

Department of Microbiology and Immunology, University of Louisville College of Medicine, Louisville, KY 40292, USA. abukwaik@louisville.edu

Infect Immun. 2007 Jul;75(7):3290-304.

ABSTRACT: The Legionella pneumophila-containing phagosome evades endocytic fusion and intercepts endoplasmic reticulum (ER)-to-Golgi vesicle traffic, which is believed to be mediated by the Dot/Icm type IV secretion system. Although phagosomes harboring dot/icm mutants are thought to mature through the endosomal-lysosomal pathway, colocalization studies with lysosomal markers have reported contradictory results. In addition, phagosomes harboring the dot/icm mutants do not interact with endocytosed materials, which is inconsistent with maturation of the phagosomes in the endosomal-lysosomal pathway. Using multiple strategies, we show that the dot/icm mutants defective in the Dot/Icm structural apparatus are unable to maintain the integrity of their phagosomes and escape into the cytoplasm within minutes of entry into various mammalian and protozoan cells in a process independent of the type II secretion system. In contrast, mutants defective in cytoplasmic chaperones of Dot/Icm effectors and rpoS, letA/S, and letE regulatory mutants are all localized within intact phagosomes. Importantly, non-dot/icm L. pneumophila mutants whose phagosomes acquire late endosomal-lysosomal markers are all located within intact phagosomes. Using high-resolution electron microscopy, we show that phagosomes harboring the dot/icm transporter mutants do not fuse to lysosomes but are free in the cytoplasm. Inhibition of ER-to-Golgi vesicle traffic by brefeldin A does not affect the integrity of the phagosomes harboring the parental strain of L. pneumophila. We conclude that the Dot/Icm transporter is involved in maintaining the integrity of the L. pneumophila phagosome, independent of interception of ER-to-Golgi vesicle traffic, which is a novel function of type IV secretion systems.

 

Legionella pneumophila adaptation to intracellular life and the host response: clues from genomics and transcriptomics

Jules M, Buchrieser C.

Unité de Génomique des Microorganismes Pathogènes and CNRS URA 2171, Institut Pasteur, 25-28 Rue du Dr. Roux, 75724 Paris Cedex 15, France. cbuch@pasteur.fr

FEBS Lett. 2007 Jun 19;581(15):2829-38.

ABSTRACT : Legionella pneumophila is the causative agent of the pneumonia-like Legionnaires' disease. The bacterium's survival and spread depend on the ability to replicate inside eukaryotic phagocytic cells. A particular feature of Legionella is its dual host system allowing the intracellular growth in protozoa like Acanthamoeba castellanii, and during infection in human alveolar macrophages. Genome analysis and comparisons as well as expression profiling of the pathogen and the host helped to identify regulatory circuits mediating adaptation of the L. pneumophila transcriptome to the intracellular environment and gave clues for the metabolic needs of intracellular Legionella. This review will summarize what is currently known about intracellular gene expression of L. pneumophila, the transcriptional host response of the model host Dictyostelium discoideum and will present hypotheses drawn from these data with respect to subversion of host cell functions and virulence of L. pneumophila.

 

The Nod-like receptor family member Naip5/Birc1e restricts Legionella pneumophila growth independently of caspase-1 activation

Lamkanfi M, Amer A, Kanneganti TD, Muñoz-Planillo R, Chen G, Vandenabeele P, Fortier A, Gros P, Núñez G.

Department of Pathology and Comprehensive Cancer Center, University of Michigan Medical School, 1500 East Medical Center Drive, Ann Arbor, MI 48109, USA. bclx@umich.edu

J Immunol. 2007 Jun 15;178(12):8022-7.

ABSTRACT: Similar to Ipaf and caspase-1, the Nod-like receptor protein Naip5 restricts intracellular proliferation of Legionella pneumophila, the causative agent of a severe form of pneumonia known as Legionnaires' disease. Thus, Naip5 has been suggested to regulate Legionella replication inside macrophages through the activation of caspase-1. In this study, we show that cytosolic delivery of recombinant flagellin activated caspase-1 in A/J macrophages carrying a mutant Naip5 allele, and in C57BL/6 (B6) macrophages congenic for the mutant Naip5 allele (B6-Naip5(A/J)), but not in Ipaf(-/-) cells. In line with these results, A/J and B6-Naip5(A/J) macrophages induced high levels of caspase-1 activation and IL-1beta secretion when infected with Legionella. In addition, transgenic expression of a functional Naip5 allele in A/J macrophages did not alter Legionella-induced caspase-1 activation and IL-1beta secretion. Notably, defective Naip5 signaling renders B6-Naip5(A/J) macrophages permissive for Legionella proliferation despite normal caspase-1 activation. These results indicate that the restriction of intracellular Legionella replication is more complex than previously appreciated and requires both Ipaf-dependent caspase-1 activation as well as functional Naip5 signaling.

 

Iron acquisition by Legionella pneumophila

Cianciotto NP.

Department of Microbiology-Immunology, Northwestern University Medical School, 320 East Superior Street, Chicago, IL 60611-3010, USA.

n-cianciotto@northwestern.edu

Biometals. 2007 Jun;20(3-4):323-31.

ABSTRACT: For nearly 20 years, it was believed that Legionella pneumophila does not produce siderophores. Yet, we have now determined that L. pneumophila secretes a siderophore (legiobactin) that is detectable by the CAS assay. We have optimized conditions for legiobactin expression, shown its biological activity, and found genes (lbtAB) involved in its production and secretion. LbtA is homologous with siderophore synthetases from E. coli (aerobactin), Sinorhizobium (rhizobactin), and Bordetella (alcaligin), while LbtB is a member of the major facilitator superfamily of multidrug efflux pumps. Mutants lacking lbtAB produce 40-70% less CAS reactivity. The lbtA mutant is also defective for growth in deferrated media containing citrate, indicating that legiobactin is required in conditions of severe iron limitation. lbtAB mutants grow normally in macrophages and amoebae host cells as well as within the lungs of mice. L. pneumophila does express lbtA in macrophages, suggesting that legiobactin has a dispensable role in infection. Legiobactin is iron repressed and does not react in the Csáky and Arnow assays. Anion-exchange HPLC has been used to purify legiobactin, and thus far, structural analysis suggests that the molecule is similar but not identical to rhizobactin, rhizoferrin, and alcaligin. The residual CAS reactivity present in supernatants of the lbtAB mutants suggests that L. pneumophila might produce a second siderophore. Besides siderophores, we have determined that ferrous iron transport, encoded by feoB, is critical for L. pneumophila growth in low-iron conditions, in host cells, and in the mammalian lung. Some of our other studies have discovered a critical, yet undefined, role for the L. pneumophila cytochrome c maturation locus in low-iron growth, intracellular infection, and virulence.

 

Early trafficking and intracellular replication of Legionella longbeachaea within an ER-derived late endosome-like phagosome

Asare R, Abu Kwaik Y.

Department of Microbiology, Immunology, University of Louisville College of Medicine, Louisville, KY 40292, USA. abukwaik@louisville.edu

Cell Microbiol. 2007 Jun;9(6):1571-87.

ABSTRACT: Legionella pneumophila is the predominant cause of Legionnaires' disease in the USA and Europe in contrast to Legionella longbeachaea, which is the leading cause of the disease in Western Australia. The ability of L. pneumophila to replicate intracellularly is triggered at the post-exponential phase along with expression of other virulence traits, such as motility. We show that while motility of L. longbeachaea is triggered upon growth transition into post-exponential phase, its ability to proliferate intracellularly is totally independent of the bacterial growth phase. Within macrophages, L. pneumophila replicates in a phagosome that excludes early and late endocytic markers and is surrounded by the rough endoplasmic reticulum (RER). In contrast, the L. longbeachaea phagosome colocalizes with the early endosomal marker early endosomal antigen 1 (EEA1) and the late endosomal markers lysosomal associated membrane glycoprotein 2 (LAMP-2) and mannose 6-phosphate receptor (M6PR), and is surrounded by the RER. The L. longbeachaea phagosome does not colocalize with the vacuolar ATPase (vATPase) proton pump, and the lysosomal luminal protease Cathepsin D, or the lysosomal tracer Texas red Ovalbumin (TROV). Intracellular proliferation of L. longbeachaea occurs in LAMP-2-positive phagosomes that are remodelled by the RER. Despite their distinct trafficking, both L. longbeachaea and L. pneumophila can replicate in communal phagosomes whose biogenesis is predominantly modulated by L. longbeachaea into LAMP-2-positive phagosomes. In addition, the L. pneumophila dotA mutant is rescued for intracellular replication if it co-inhabits the phagosome with L. longbeachaea. During late stages of infection, L. longbeachaea escape into the cytoplasm, prior to lysis of the macrophage, similar to L. pneumophila. We conclude that the L. longbeachaea phagosome matures to a non-acidified late endosome-like stage that is remodelled by the RER, indicating an idiosyncratic trafficking of L. longbeachaea compared with other intracellular pathogens, and a divergence in its intracellular lifestyle from L. pneumophila. In addition, re-routing biogenesis of the L. pneumophila phagosome into a late endosome controlled by L. longbeachaea has no effect on intracellular replication.

 

Host-dependent trigger of caspases and apoptosis by Legionella pneumophila

Santic M, Asare R, Doric M, Abu Kwaik Y.

Department of Microbiology and Immunology, University of Louisville College of Medicine, 319 Abraham Flexner Way 55A, Louisville, KY 40202, USA. abukwaik@louisville.edu

Infect Immun. 2007 Jun;75(6):2903-13.

ABSTRACT: The Dot/Icm system of Legionella pneumophila triggers activation of caspase-3 during early stages of infection of human macrophages, but apoptosis is delayed until late stages of infection. During early stages of infection of mouse macrophages, the organism triggers rapid caspase-1-mediated cytotoxicity, which is mediated by bacterial flagellin. However, it is not known whether caspase-1 is triggered by L. pneumophila in human macrophages or whether caspase-3 is activated in permissive or nonpermissive mouse macrophages. Using single-cell analyses, we show that the wild-type strain of L. pneumophila does not trigger caspase-1 activation throughout the intracellular infection of human monocyte-derived macrophages (hMDMs), even when the flagellated bacteria escape into the cytoplasm during late stages. Using single-cell analyses, we show that the Dot/Icm system of L. pneumophila triggers caspase-3 but not caspase-1 within permissive A/J mouse bone marrow-derived primary macrophages by 2 to 8 h, but apoptosis is delayed until late stages of infection. While L. pneumophila triggers a Dot/Icm-dependent activation of caspase-1 in nonpermissive BALB/c mouse-derived macrophages, caspase-3 is not activated at any stage of infection. We show that robust intrapulmonary replication of the wild-type strain of L. pneumophila in susceptible A/J mice is associated with late-stage Dot/Icm-dependent pulmonary apoptosis and alveolar inflammation. In the lungs of nonpermissive BALB/c mice, L. pneumophila does not replicate and does not trigger pulmonary apoptosis or alveolar inflammation. Thus, similar to hMDMs, L. pneumophila does not trigger caspase-1 but triggers caspase-3 activation during early and exponential replication in permissive A/J mouse-derived macrophages, and apoptosis is delayed until late stages of infection. The Dot/Icm type IV secretion system is essential for pulmonary apoptosis in the genetically susceptible A/J mice.

 

Legionella pneumophila infection up-regulates dendritic cell Toll-like receptor 2 (TLR2)/TLR4 expression and key maturation markers

Rogers J, Hakki A, Perkins I, Newton C, Widen R, Burdash N, Klein T, Friedman H.

Department of Molecular Medicine, University of South Florida, 12901 Bruce B. Downs Boulevard, Tampa, FL 33612-4799, USA. hfriedma@hsc.usf.edu

Infect Immun. 2007 Jun;75(6):3205-8.

ABSTRACT: Dendritic cells (DCs) have a critical role in linking innate to adaptive immunity, and this transition is regulated by the up-regulation of costimulatory and major histocompatibility complex (MHC) molecules as well as Toll-like receptors. These changes in DCs have been observed to occur following microbial infection, and in the present study, we examined the effect of Legionella pneumophila infection on the expression of these DC markers. We showed that bone marrow-derived DC cultures from BALB/c mice infected with live L. pneumophila resulted in the up-regulation of Toll-like receptors 2 and 4 and the activation of CD40, CD86, and MHC class I/II molecules.

 

Addition of neuA, the gene encoding N-acylneuraminate cytidylyl transferase, increases the discriminatory ability of the consensus sequence-based scheme for typing Legionella pneumophila serogroup 1 strains

Ratzow S, Gaia V, Helbig JH, Fry NK, Lück PC.

Institut für Medizinische Mikrobiologie und Hygiene, TU Dresden, Fiedlerstrasse 42, D-01307 Dresden, Germany. Christian.Lueck@mailbox.tu-dresden.de

J Clin Microbiol. 2007 Jun;45(6):1965-8.

ABSTRACT: The standard sequence-based method for the typing of Legionella pneumophila serogroup 1 strains was extended by using the gspA and neuA alleles. The use of neuA as a seventh allele for typing significantly increased the index of discrimination calculated for a panel of unrelated strains (from 0.932 to 0.963) and subdivided some known large common complexes (e.g., 1,4,3,1,1,1). This modification to the standard method is proposed as the method of choice in the epidemiological investigation of L. pneumophila infections.

 

A pair of highly conserved two-component systems participates in the regulation of the hypervariable FIR proteins in different Legionella species

Feldman M, Segal G.

Department of Molecular Microbiology & Biotechnology, George S. Wise Faculty of Life Sciences, Tel-Aviv University, Ramat-Aviv, Tel-Aviv 69978, Israel. gils@tauex.tau.ac.il

J Bacteriol. 2007 May;189(9):3382-91.

ABSTRACT: Legionella pneumophila and other pathogenic Legionella species multiply inside protozoa and human macrophages by using the Icm/Dot type IV secretion system. The IcmQ protein, which possesses pore-forming activity, and IcmR, which functions as its chaperone, are two essential components of this system. It was previously shown that in 29 Legionella species, a large hypervariable-gene family (fir genes) is located upstream from a conserved icmQ gene, but although nonhomologous, the FIR proteins were found to function similarly together with their corresponding IcmQ proteins. Alignment of the regulatory regions of 29 fir genes revealed that they can be divided into three regulatory groups; the first group contains a binding site for the CpxR response regulator, which was previously shown to regulate the L. pneumophila fir gene (icmR); the second group, which includes most of the fir genes, contains the CpxR binding site and an additional regulatory element that was identified here as a PmrA binding site; and the third group contains only the PmrA binding site. Analysis of the regulatory region of two fir genes, which included substitutions in the CpxR and PmrA consensus sequences, a controlled expression system, as well as examination of direct binding with mobility shift assays, revealed that both CpxR and PmrA positively regulate the expression of the fir genes that contain both regulatory elements. The change in the regulation of the fir genes that occurred during the course of evolution might be required for the adaptation of the different Legionella species to their specific environmental hosts.

 

Birc1e/Naip5 rapidly antagonizes modulation of phagosome maturation by Legionella pneumophila

Fortier A, de Chastellier C, Balor S, Gros P.

Department of Biochemistry, Centre for the Study of Host Resistance, McGill University, Montreal, Quebec, Canada. philippe.gros@mcgill.ca

Cell Microbiol. 2007 Apr;9(4):910-23.

ABSTRACT: Legionella survives intracellularly by preventing fusion with lysosomes, due to phagosome escape from the endocytic pathway at an early stage of phagosome maturation, and by creating a replicative organelle that acquires endoplasmic reticulum (ER) characteristics through sustained interactions and fusion with the ER. Intracellular replication of Legionella pneumophila in mouse macrophages is controlled by the Lgn1 locus. Functional complementation in vivo has identified the Birc1e/Naip5 gene as being responsible for the Lgn1 effect. To understand the function and temporal site of action of Birc1e/Naip5 in susceptibility to L. pneumophila, we examined the biogenesis of Legionella-containing vacuoles (LCVs) formed in permissive A/J macrophages and in their Birc1e/Naip5 transgenic non-permissive counterpart. Birc1e/Naip5 effects on acquisition of lysosomal and ER markers were evident within 1-2 h following infection. A significantly higher proportion of LCVs formed in Birc1e/Naip5 transgenic macrophages had acquired the lysosomal markers cathepsin D and Lamp1 by 2 h post infection, whereas a significantly higher proportion of LCVs formed in permissive macrophages were positively stained for the ER markers BAP31 and calnexin, 6 h post infection. Likewise, studies by electron microscopy showed acquisition of lysosomal contents (horseradish peroxidase), within the first hour following phagocytic uptake, by LCVs formed in Birc1e/Naip5 transgenic macrophages and delivery of the ER marker glucose 6-phosphatase (G6Pase) only to the lumen of LCVs formed in A/J macrophages. Finally, a larger proportion of LCVs formed in A/J macrophages were studded with ribosomes 24 h post infection, compared with LCVs formed in Birc1e/Naip5 transgenic macrophages. These results suggest that sensing of L. pneumophila products by Birc1e/Naip5 in macrophages occurs rapidly following phagocytosis, a process that antagonizes the ability of L. pneumophila to remodel its phagosome into a specialized vacuole with ER characteristics.

 

Genetic susceptibility and caspase activation in mouse and human macrophages are distinct for Legionella longbeachae and L. pneumophila

Asare R, Santic M, Gobin I, Doric M, Suttles J, Graham JE, Price CD, Abu Kwaik Y.

Department of Microbiology and Immunology, University of Louisville College of Medicine, 319 Abraham Flexner Way, Louisville, KY 40292, USA. abukwaik@louisville.edu

Infect Immun. 2007 Apr;75(4):1933-45.

ABSTRACT: Legionella pneumophila is the predominant cause of Legionnaires' disease in the United States and Europe, while Legionella longbeachae is the common cause of the disease in Western Australia. Although clinical manifestations by both intracellular pathogens are very similar, recent studies have shown that phagosome biogeneses of both species within human macrophages are distinct (R. Asare and Y. Abu Kwaik, Cell. Microbiol., in press). Most inbred mouse strains are resistant to infection by L. pneumophila, with the exception of the A/J mouse strain, and this genetic susceptibility is associated with polymorphism in the naip5 allele and flagellin-mediated early activation of caspase 1 and pyropoptosis in nonpermissive mouse macrophages. Here, we show that genetic susceptibility of mice to infection by L. longbeachae is independent of allelic polymorphism of naip5. L. longbeachae replicates within bone marrow-derived macrophages and in the lungs of A/J, C57BL/6, and BALB/c mice, while L. pneumophila replicates in macrophages in vitro and in the lungs of the A/J mouse strain only. Quantitative real-time PCR studies on infected A/J and C57BL/6 mouse bone marrow-derived macrophages show that both L. longbeachae and L. pneumophila trigger similar levels of naip5 expression, but the levels are higher in infected C57BL/6 mouse macrophages. In contrast to L. pneumophila, L. longbeachae has no detectable pore-forming activity and does not activate caspase 1 in A/J and C57BL/6 mouse or human macrophages, despite flagellation. Unlike L. pneumophila, L. longbeachae triggers only a modest activation of caspase 3 and low levels of apoptosis in human and murine macrophages in vitro and in the lungs of infected mice at late stages of infection. We conclude that despite flagellation, infection by L. longbeachae is independent of polymorphism in the naip5 allele and L. longbeachae does not trigger the activation of caspase 1, caspase 3, or late-stage apoptosis in mouse and human macrophages. Neither species triggers caspase 1 activation in human macrophages.

 

Identification of variable-number tandem-repeat (VNTR) sequences in Legionella pneumophila and development of an optimized multiple-locus VNTR analysis typing scheme

Pourcel C, Visca P, Afshar B, D'Arezzo S, Vergnaud G, Fry NK.

Institut de Génétique et Microbiologie, Bât 400, Université Paris-Sud, 91405 Orsay cedex, France.

christine.pourcel@igmors.u-psud.fr

J Clin Microbiol. 2007 Apr;45(4):1190-9.

ABSTRACT: The utility of a genotypic typing assay for Legionella pneumophila was investigated. A multiple-locus variable number of tandem repeats (VNTR) analysis (MLVA) scheme using PCR and agarose gel electrophoresis is proposed based on eight minisatellite markers. Panels of well-characterized strains were examined in a multicenter analysis to validate the assay and to compare its performance to that of other genotyping assays. Excellent typeability, reproducibility, stability, and epidemiological concordance were observed. The MLVA type or profile is composed of a string of allele numbers, corresponding to the number of repeats at each VNTR locus, separated by commas, in a predetermined order. A database containing information from 99 L. pneumophila serogroup 1 strains and four strains of other serogroups and their MLVA profiles, which can be queried online, is available from http://bacterial-genotyping.igmors.u-psud.fr/.

 

Legionella pneumophila inhibits macrophage apoptosis by targeting pro-death members of the Bcl2 protein family

Banga S, Gao P, Shen X, Fiscus V, Zong WX, Chen L, Luo ZQ.

Department of Biological Sciences, Purdue University, 915 West State Street, West Lafayette, IN 47907, USA. luoz@purdue.edu

Proc Natl Acad Sci U S A. 2007 Mar 20;104(12):5121-6.

ABSTRACT: To establish a vacuole that supports bacterial replication, Legionella pneumophila translocates a large number of bacterial proteins into host cells via the Dot/Icm type IV secretion system. Functions of most of these translocated proteins are unknown, but recent investigations suggest their roles in modulating diverse host processes such as vesicle trafficking, autophagy, ubiquitination, and apoptosis. Cells infected by L. pneumophila exhibited resistance to apoptotic stimuli, but the bacterial protein directly involved in this process remained elusive. We show here that SidF, one substrate of the Dot/Icm transporter, is involved in the inhibition of infected cells from undergoing apoptosis to allow maximal bacterial multiplication. Permissive macrophages harboring a replicating sidF mutant are more apoptotic and more sensitive to staurosporine-induced cell death. Furthermore, cells expressing SidF are resistant to apoptosis stimuli. SidF contributes to apoptosis resistance in L. pneumophila-infected cells by specifically interacting with and neutralizing the effects of BNIP3 and Bcl-rambo, two proapoptotic members of Bcl2 protein family. Thus, inhibiting the functions of host pro-death proteins by translocated effectors constitutes a mechanism for L. pneumophila to protect host cells from apoptosis.

 

Population structure and recombination in environmental isolates of Legionella pneumophila

Coscollá M, González-Candelas F.

Institut Cavanilles de Biodiversitat i Biologia Evolutiva and Departament de Genètica, Universitat de València, 46071 València, Spain. fernando.gonzalez@uv.es

Environ Microbiol. 2007 Mar;9(3):643-56. 

ABSTRACT: Legionella pneumophila is a water-borne bacteria responsible for most cases of legionellosis, an emerging disease with an increasing incidence in industrialized countries. Although early analysis based on multilocus enzyme electrophoresis (MLEE) described the population structure of this species as clonal, more recent reports have suggested that recombination also contributes to shaping variation across its genome. We report here the results of analysing the nucleotide sequences of 19 loci in 31 environmental samples of L. pneumophila from a small Spanish region (near Alcoi, province of Alicante) where legionellosis has become almost endemic. We analysed the six loci currently incorporated to the sequence-based typing scheme developed by European Working Group for Legionella Infections (EWGLI) for L. pneumophila and 13 intergenic regions, for which we developed primers anchored in flanking, conserved genes. Our results show that recombination among natural isolates of this species is a common phenomenon, as 20 of the 31 isolates contained at least one locus in which recombination was revealed by at least three different methods. The mapping of the recombination events on the maximum likelihood tree of the concatenate sequence of the 19 loci indicated that at least nine independent recombination events might explain the observed distribution of recombinant loci among isolates. In consequence, we have shown that recombination in L. pneumophila is much more frequent than previously considered and that it does not seem to be restricted to already described pathogenicity islands or other genome constituents which provide it with a high plasticity.

 

Role of Toll-like receptor 2 in recognition of Legionella pneumophila in a murine pneumonia model

Fuse ET, Tateda K, Kikuchi Y, Matsumoto T, Gondaira F, Azuma A, Kudoh S, Standiford TJ, Yamaguchi K.

Department of Microbiology and Infectious Diseases, Toho University School of Medicine, Tokyo 143-8540, Japan. kazu@med.toho-u.ac.jp

J Med Microbiol. 2007 Mar;56(Pt 3):305-12.

ABSTRACT: Legionella pneumophila is an intracellular organism and the major aetiological agent of Legionnaires' disease. Although recent progress has identified Toll-like receptors (TLRs) as receptors for recognition of pathogen-associated molecular patterns in a variety of micro-organisms, understanding the contribution of TLRs to the host response in L. pneumophila infection is still limited. This study examined the roles of TLR2 and TLR4 in murine L. pneumophila pneumonia and an in vitro infection model using bone-marrow-derived macrophages. TLR2-deficient mice, but not TLR4-deficient mice, demonstrated higher lethal sensitivity to pulmonary challenge with L. pneumophila than wild-type mice (P<0.05). Although no differences in pulmonary bacterial burden were observed among the mouse strains examined, lower values of macrophage inflammatory protein-2 (MIP-2), keratinocyte-derived cytokine and interleukin (IL)-6 and higher IL-12 levels were noted in lung homogenates of TLR2-deficient mice compared with the wild-type control and TLR4-deficient mice. Recruitment of inflammatory cells, particularly neutrophils, was severely disturbed in the lungs of TLR2-deficient mice. Reduced MIP-2 production was demonstrated in bone-marrow-derived macrophages from TLR2-deficient mice in response to live L. pneumophila and purified LPS of this strain, but not Escherichia coli LPS. These data highlight the involvement and importance of TLR2 in the pathogenesis of L. pneumophila pneumonia in mice. The results showed that TLR2-mediated recognition of Legionella LPS and subsequent chemokine-dependent cellular recruitment may be a crucial host innate response in L. pneumophila pneumonia.

 

The response regulator PmrA is a major regulator of the icm/dot type IV secretion system in Legionella pneumophila and Coxiella burnetii

Zusman T, Aloni G, Halperin E, Kotzer H, Degtyar E, Feldman M, Segal G.

Department of Molecular Microbiology and Biotechnology, George S. Wise Faculty of Life Sciences, Tel-Aviv University, Ramat-Aviv, Tel-Aviv 69978, Israel. GilS@tauex.tau.ac.il

Mol Microbiol. 2007 Mar;63(5):1508-23.

ABSTRACT: Legionella pneumophila and Coxiella burnetii have been shown to utilize the icm/dot type IV secretion system for pathogenesis and recently a large number of icm/dot-translocated substrates were identified in L. pneumophila. Bioinformatic analysis has revealed that 13 of the genes encoding for L. pneumophila-translocated substrates and five of the C. burnetii icm/dot genes, contain a conserved regulatory element that resembles the target sequence of the PmrA response regulator. Experimental analysis which included the construction of a L. pneumophila pmrA deletion mutant, intracellular growth analysis, comparison of gene expression between L. pneumophila wild type and the pmrA mutant, construction of mutations in the PmrA conserved regulatory element, controlled expression studies as well as mobility shift assays, demonstrated the direct relation between the PmrA regulator and the expression of L. pneumophila icm/dot-translocated substrates and several C. burnetii icm/dot genes. Furthermore, genomic analysis identified 35 L. pneumophila and 68 C. burnetii unique genes that contain the PmrA regulatory element and few of these genes from L. pneumophila were found to be new icm/dot-translocated substrates. Our results establish the PmrA regulator as a fundamental regulator of the icm/dot type IV secretion system in these two bacteria.

 

Collagen binding protein Mip enables Legionella pneumophila to transmigrate through a barrier of NCI-H292 lung epithelial cells and extracellular matrix

Wagner C, Khan AS, Kamphausen T, Schmausser B, Unal C, Lorenz U, Fischer G, Hacker J, Steinert M.

Institut für Molekulare Infektionsbiologie, Universität Würzburg, Röntgenring 11, D-97070 Würzburg, Germany. michael.steinert@mail.uni-wuerzburg.de

Cell Microbiol. 2007 Feb;9(2):450-62.

ABSTRACT: Guinea pigs are highly susceptible to Legionella pneumophila infection and therefore have been the preferred animal model for studies of legionellosis. In this study guinea pig infections revealed that the Legionella virulence factor Mip (macrophage infectivity potentiator) contributes to the bacterial dissemination within the lung tissue and the spread of Legionella to the spleen. Histopathology of infected animals, binding assays with components of the extracellular matrix (ECM), bacterial transmigration experiments across an artificial lung epithelium barrier, inhibitor studies and ECM degradation assays were used to elucidate the underlying mechanism of the in vivo observation. The Mip protein, which belongs to the enzyme family of FK506-binding proteins (FKBP), was shown to bind to the ECM protein collagen (type I, II, III, IV, V, VI). Transwell assays with L. pneumophila and recombinant Escherichia coli HB101 strains revealed that Mip enables these bacteria to transmigrate across a barrier of NCI-H292 lung epithelial cells and ECM (NCI-H292/ECM barrier). Mip-specific monoclonal antibodies and the immunosuppressants rapamycin and FK506, which inhibit the peptidyl prolyl cis/trans isomerase (PPIase) activity of Mip, were able to inhibit this transmigration. By using protease inhibitors we found that the penetration of the NCI-H292/ECM barrier additionally requires a serine protease activity. Degradation assays with (35)S-labelled ECM proteins supported the finding of a concerted action of Mip and a serine protease. The described synergism between the activity of the collagen binding Mip protein and the serine protease activity represents an entirely new mechanism for bacterial penetration of the lung epithelial barrier and has implications for other prokaryotic and eukaryotic pathogens.

 

Legionella pneumophila - a human pathogen that co-evolved with fresh water protozoa

Albert-Weissenberger C, Cazalet C, Buchrieser C.

Unité de Génomique des Microorganismes Pathogènes and CNRS URA 2171, Institut Pasteur, 25 Rue du Dr Roux, 75724, Paris Cedex 15, France.

cbuch@pasteur.fr

Cell Mol Life Sci. 2007 Feb;64(4):432-48.

ABSTRACT: The bacterial pathogen Legionella pneumophila is found ubiquitously in fresh water environments where it replicates within protozoan hosts. When inhaled by humans it can replicate within alveolar macrophages and cause a severe pneumonia, Legionnaires disease. Yet much needs to be learned regarding the mechanisms that allow Legionella to modulate host functions to its advantage and the regulatory network governing its intracellular life cycle. The establishment and publication of the complete genome sequences of three clinical L. pneumophila isolates paved the way for major breakthroughs in understanding the biology of L. pneumophila. Based on sequence analysis many new putative virulence factors have been identified foremost among them eukaryotic-like proteins that may be implicated in many different steps of the Legionella life cycle. This review summarizes what is currently known about regulation of the Legionella life cycle and gives insight in the Legionella-specific features as deduced from genome analysis.

 

Environmental mimics and the Lvh type IVA secretion system contribute to virulence-related phenotypes of Legionella pneumophila

Bandyopadhyay P, Liu S, Gabbai CB, Venitelli Z, Steinman HM.

Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, USA. steinman@aecom.yu.edu

Infect Immun. 2007 Feb;75(2):723-35.

ABSTRACT: Legionella pneumophila, the causative organism of Legionnaires' disease, is a fresh-water bacterium and intracellular parasite of amoebae. This study examined the effects of incubation in water and amoeba encystment on L. pneumophila strain JR32 and null mutants in dot/icm genes encoding a type IVB secretion system required for entry, delayed acidification of L. pneumophila-containing phagosomes, and intracellular multiplication when stationary-phase bacteria infect amoebae and macrophages. Following incubation of stationary-phase cultures in water, mutants in dotA and dotB, essential for function of the type IVB secretion system, exhibited entry and delay of phagosome acidification comparable to that of strain JR32. Following encystment in Acanthamoeba castellanii and reversion of cysts to amoeba trophozoites, dotA and dotB mutants exhibited intracellular multiplication in amoebae. The L. pneumophila Lvh locus, encoding a type IVA secretion system homologous to that in Agrobacterium tumefaciens, was required for restoration of entry and intracellular multiplication in dot/icm mutants following incubation in water and amoeba encystment and was required for delay of phagosome acidification in strain JR32. These data support a model in which the Dot/Icm type IVB secretion system is conditionally rather than absolutely required for L. pneumophila virulence-related phenotypes. The data suggest that the Lvh type IVA secretion system, previously thought to be dispensable, is involved in virulence-related phenotypes under conditions mimicking the spread of Legionnaires' disease from environmental niches. Since environmental amoebae are implicated as reservoirs for an increasing number of environmental pathogens and for drug-resistant bacteria, the environmental mimics developed here may be useful in virulence studies of other pathogens.

 

The Legionella pneumophila effector SidJ is required for efficient recruitment of endoplasmic reticulum proteins to the bacterial phagosome

Liu Y, Luo ZQ.

Department of Biological Sciences, Purdue University, 915 West State Street, West Lafayette, IN 47907, USA. luoz@purdue.edu

Infect Immun. 2007 Feb;75(2):592-603.

ABSTRACT: The virulence of Legionella pneumophila is dependent on the Dot/Icm type IV protein secretion system, which translocates effectors into infected cells. A large number of such translocated proteins have been identified, but few of these proteins are necessary for intracellular replication of the pathogen, making it difficult to correlate these genes with specific cell-biological events associated with L. pneumophila infection. We report here the identification and characterization of a family of two substrates, SidJ and SdjA, with distinctive phenotypes. In contrast to many Dot/Icm substrates, whose expression levels are elevated when bacteria are grown to postexponential phase, SidJ is produced at a constant rate during the entire bacterial growth cycle. Mutation in sidJ causes a significant growth defect in both macrophage and amoeba hosts, but an sdjA mutant is detectably defective only in protozoan hosts. However, in the amoeba host a mutant lacking both sidJ and sdjA does not display a more severe growth defect than the sidJ mutant. Despite its significant intracellular growth defect, the sidJ mutant is still able to effectively evade fusion with lysosomes. Importantly, recruitment of endoplasmic reticulum (ER) proteins by vacuoles containing the sidJ mutant was considerably delayed in both mammalian and amoeba cells. Our results suggest that SidJ modulates host cellular pathways, contributing to the trafficking or retention of ER-derived vesicles to L. pneumophila vacuoles.

 

The twin-arginine translocation pathway is necessary for correct membrane insertion of the Rieske Fe/S protein in Legionella pneumophila

De Buck E, Vranckx L, Meyen E, Maes L, Vandersmissen L, Anné J, Lammertyn E.

Laboratory of Bacteriology, Rega Institute for Medical Research, Katholieke Universiteit Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium. Elke.Lammertyn@rega.kuleuven.be

FEBS Lett. 2007 Jan 23;581(2):259-64.

ABSTRACT: The twin-arginine translocation (Tat) pathway translocates folded proteins across the cytoplasmic membrane. Proteins transported through this secretion system typically carry two arginine residues in their signal peptide that is cleaved off during translocation. Recently, we demonstrated the presence of the Tat pathway in Legionella pneumophila Philadelphia-1 and the Rieske Fe/S protein PetA was one of the predicted Tat substrates. Because we observed that the signal peptide of PetA is not processed and that this protein is still membrane associated in the tat mutants, correct membrane insertion was assayed using a trypsin sensitivity assay. We conclude that the Tat pathway is necessary for correct membrane insertion of L. pneumophila PetA.

 

Legionella pneumophila-induced PKCalpha-, MAPK-, and NF-kappaB-dependent COX-2 expression in human lung epithelium

N'Guessan PD, Etouem MO, Schmeck B, Hocke AC, Scharf S, Vardarova K, Opitz B, Flieger A, Suttorp N, Hippenstiel S.

Department of Internal Medicine/Infectious Diseases and Pulmonary Medicine, Charité-Universitätsmedizin Berlin, Augustenburger Platz 1, 13353 Berlin, Germany.

stefan.hippenstiel@charite.de

Am J Physiol Lung Cell Mol Physiol. 2007 Jan;292(1):L267-77.

ABSTRACT: Legionella pneumophila causes community- and hospital-acquired pneumonia. Lung airway and alveolar epithelial cells comprise an important barrier against airborne pathogens. Cyclooxygenase (COX) and microsomal PGE(2) synthase-1 (mPGES-1)-derived prostaglandins like prostaglandin E(2) (PGE(2)) are considered as important regulators of lung function. Herein we tested the hypothesis that L. pneumophila induced COX-2 and mPGES-1-dependent PGE(2) production in pulmonary epithelial cells. Legionella induced the release of PGE(2) in primary human small airway epithelial cells and A549 cells. This was accompanied by an increased expression of COX-2 and mPGES-1 as well as an increased PLA(2) activity in infected cells. Deletion of the type IV secretion system Dot/Icm did not impair Legionella-related COX-2 expression or PGE(2) release in A549 cells. L. pneumophila induced the degradation of IkappaBalpha and activated NF-kappaB. Inhibition of IKK blocked L. pneumophila-induced PGE(2) release and COX-2 expression. We noted activation of p38 and p42/44 MAP kinase in Legionella-infected A549 cells. Moreover, membrane translocation and activation of PKCalpha was observed in infected cells. PKCalpha and p38 and p42/44 MAP kinase inhibitors reduced PGE(2) release and COX-2 expression. In summary, PKCalpha and p38 and p42/44 MAP kinase controlled COX-2 expression and subsequent PGE(2) release by Legionella-infected lung epithelial cells. These pathways may significantly contribute to the host response in Legionnaires' disease.

 

Anti-apoptotic signalling by the Dot/Icm secretion system of L. pneumophila

Abu-Zant A, Jones S, Asare R, Suttles J, Price C, Graham J, Kwaik YA.

Department of Microbiology, University of Louisville Collage of Medicine, Louisville, KY 40202, USA. abukwaik@louisville.edu

Cell Microbiol. 2007 Jan;9(1):246-64.

ABSTRACT: The Dot/Icm type IV secretion system of Legionella pneumophila triggers robust activation of caspase-3 during early and exponential stages of proliferation within human macrophages, but apoptosis is delayed till late stages of infection, which is novel. As caspase-3 is the executioner of the cell, we tested the hypothesis that L. pneumophila triggers anti-apoptotic signalling within the infected human macrophages to halt caspase-3 from dismantling the cells. Here we show that during early and exponential replication, L. pneumophila-infected human monocyte-derived macrophages (hMDMs) exhibit a remarkable resistance to induction of apoptosis, in a Dot/Icm-dependent manner. Microarray analyses and real-time PCR reveal that during exponential intracellular replication, L. pneumophila triggers upregulation of 12 anti-apoptotic genes that are linked to activation of the nuclear transcription factor kappa-B (NF-kappaB). Our data show that L. pneumophila induces a Dot/Icm-dependent sustained nuclear translocation of the p50 and p65 subunits of NF-kappaB during exponential intracellular replication. Bacterial entry is essential both for the anti-apoptotic phenotype of infected hMDMs and for nuclear translocation of the p65. Using p65-/- and IKKalpha-/- beta-/- double knockout mouse embryonic fibroblast cell lines, we show that nuclear translocation of NF-kappaB is required for the resistance of L. pneumophila-infected cells to apoptosis-inducing agents. In addition, the L. pneumophila-induced nuclear translocation of NF-kappaB requires the activity of IKKalpha and/or IKKbeta. We conclude that although the Dot/Icm secretion system of L. pneumophila elicits an early robust activation of caspase-3 in human macrophages, it triggers a strong anti-apoptotic signalling cascade mediated, at least in part by NF-kappaB, which renders the cells refractory to external potent apoptotic stimuli.

 

Comparison of clinical and environmental isolates of Legionella pneumophila obtained in the UK over 19 years

Harrison TG, Doshi N, Fry NK, Joseph CA.

Respiratory and Systemic infection Laboratory, Agency Centre for Infections, London, UK.

tim.harrison@hpa.org.uk

Clin Microbiol Infect. 2007 Jan;13(1):78-85.

ABSTRACT: Between January 1980 and December 1998, 3458 cases of Legionnaires' disease were reported to the national surveillance scheme in England and Wales. Of these, 463 (13.4%) were reported as proven by culture and isolation of Legionella spp., with 96.3% being Legionella pneumophila. Serogroup (Sgp), monoclonal antibody (mAb) subgrouping and restriction fragment length polymorphism (RFLP) analysis data were obtained for 321 (69.3%) of these, of which 284 were classified as being unrelated to any other isolate in the study. Typing data were also available for 117 unrelated environmental isolates of L. pneumophila obtained from England and Wales, giving a total of 401 unrelated isolates in the study. Of the clinical isolates, 88.0% were Sgp1, compared with only 42.7% of environmental isolates (p <0.001); 79.6% of clinical isolates were subgrouped as mAb2+, compared with only 12.8% of environmental isolates (p <0.001). RFLP typing identified 67 types among the 401 isolates, with clinical isolates showing significantly less diversity than environmental isolates (index of diversity (IOD) 0.944 vs. 0.958; p <0.05), with three RFLP types (1, 5 and 14) accounting for 40.0% of all clinical isolates. Combining the phenotypic and genotypic data resulted in 173 distinct phenons, with clinical isolates showing significantly less diversity than environmental isolates (IOD 0.964 vs. 0.996; p <0.01). Three phenons accounted for 30% of all clinical isolates. These data strongly suggest that some strains of L. pneumophila are more likely to cause human infection than would be expected from their distribution in the environment.

 

Legionella pneumophila-induced NF-kappaB- and MAPK-dependent cytokine release by lung epithelial cells

Schmeck B, N'Guessan PD, Ollomang M, Lorenz J, Zahlten J, Opitz B, Flieger A, Suttorp N, Hippenstiel S.

Dept of Internal Medicine/Infectious Diseases and Respiratory Medicine, Charité - Universitätsmedizin Berlin, Augustenburger Platz 1, 13353 Berlin, Germany.

stefan.hippenstiel@charite.de

Eur Respir J. 2007 Jan;29(1):25-33.

ABSTRACT: Legionella pneumophila causes community-acquired pneumonia with high mortality, but little is known about its interaction with the alveolar epithelium. The aim of this study was to investigate whether L. pneumophila infection of lung epithelial cells (A549) resulted in pro-inflammatory activation. L. pneumophila infection induced liberation of interleukin (IL)-2, -4, -6, -8 and -17, monocyte chemoattractant protein-1, tumour necrosis factor-alpha, IL-1beta, interferon-gamma and granulocyte colony-stimulating factor, but not of IL-5, -7, -10, -12 (p70) or -13 or granulocyte-macrophage colony-stimulating factor. The present study focused on IL-8 and found induction by L. pneumophila strains 130b, Philadelphia 1, Corby and, to a lesser extent, JR32. Knockout of dotA, a central gene involved in type IVB secretion, did not alter IL-8 induction, whereas lack of flagellin significantly reduced IL-8 release by Legionella. Moreover, p38 mitogen-activated protein kinase (MAPK) was activated and kinase inhibition reduced secretion of induced cytokines, with the exception of IL-2 and granulocyte colony-stimulating factor. In contrast, inhibition of the MAPK kinase 1/extracellular signal-regulated kinase pathway only reduced the expression of a few cytokines. L. pneumophila also induced binding of nuclear factor-kappaB subunit RelA/p65 and RNA polymerase II to the il8 promoter, and a specific inhibitor of the inhibitor of nuclear factor-kappaB complex dose-dependently lowered IL-8 expression. Taken together, Legionella pneumophila activated p38 mitogen-activated protein kinase- and nuclear factor-kappaB/RelA pathway-dependent expression of a complex pattern of cytokines by human alveolar epithelial cells, presumably contributing to the immune response in legionellosis.

 

Role of Toll-like receptor 9 in Legionella pneumophila-induced interleukin-12 p40 production in bone marrow-derived dendritic cells and macrophages from permissive and nonpermissive mice

Newton CA, Perkins I, Widen RH, Friedman H, Klein TW.

Department of Molecular Medicine, MDC Box 10, University of South Florida College of Medicine, 12901 Bruce B. Downs Boulevard, Tampa, FL 33612, USA.

tklein@health.usf.edu

Infect Immun. 2007 Jan;75(1):146-51.

ABSTRACT: The progression of Legionella pneumophila infection in macrophages is controlled by the Lgn1 gene locus, which expresses the nonpermissive phenotype in cells from BALB/c mice but the permissive phenotype in cells from A/J mice. Activation of dendritic cells and macrophages by L. pneumophila is mediated by the pathogen recognition receptor Toll-like receptor 2 (TLR2); furthermore, Legionella induces innate and adaptive immune cytokines by the MyD88-dependent pathway. TLR9 is coupled to MyD88 and mediates the production of interleukin-12 (IL-12) in dendritic cells infected with other facultatively intracellular pathogens. In the current study, L. pneumophila growth in dendritic cells from BALB/c and A/J mice was examined along with the role of TLR9 in the induction of IL-12 in these cells. Dendritic cells from both strains were nonpermissive for L. pneumophila intracellular growth, suggesting that the products of the Lgn1 gene locus that control intracellular growth in macrophages do not control the growth of Legionella in dendritic cells. In addition, chloroquine treatment suppressed IL-12 p40 production in response to Legionella treatment in dendritic cells and macrophages from BALB/c and A/J mice. Furthermore, the TLR9 inhibitor ODN2088 suppressed the Legionella-induced IL-12 production in dendritic cells from both mouse strains. These results suggest that L. pneumophila is similar to other intracellular bacteria in that it stimulates the production of immune-transitioning cytokines, such as IL-12, through activation of TLR9 and that this receptor provides a common mechanism for sensing these types of microbes and inducing innate and adaptive immunity.

 

Enhancement of Legionella pneumophila culture isolation from microenvironments by macrophage infectivity potentiator (mip) gene-specific nested polymerase chain reaction

Nintasen R, Utrarachkij F, Siripanichgon K, Bhumiratana A, Suzuki Y, Suthienkul O.

Department of Microbiology, Faculty of Public Health, Mahidol University, 420/1 Rajvithi Road, Rajthewee, Bangkok 10400, Thailand. phost@mahidol.ac.th

Microbiol Immunol. 2007;51(8):777-85.

ABSTRACT: The combination of a Legionella pneumophila culture isolation technique and macrophage infectivity potentiator (mip) gene-specific nested polymerase chain reaction (PCR) is pivotal for effective routine use in an environmental water system laboratory. Detection of Legionella organisms in 169 environmental samples was performed by using modified buffered charcoal yeast extract (MBCYE) agar for conventional culture. Nested PCR specific for L. pneumophila was performed using boiled genomic DNA extracts from filtered and Chelex 100-treated water samples, or by using silica-gel membrane spin column-eluted DNA from concentrated pond, canal and river samples. Overall, the nested PCR was twelvefold more sensitive than the culture method. The target amplicons (471 basepairs) of all 4 biochemically characterized L. pneumophila isolates were sequenced. They had homology at the DNA and protein levels to 3' proximity of the mip-coding gene of L. pneumophila deposited in genome databases. EcoRI- or KpnI-digested PCR fragments with expected sizes were also confirmed in all 52 PCR-positive samples that were isolated from cooling towers and condenser drains. Viable but nonculturable L. pneumophila might have been present in 48 PCR-positive samples. This study demonstrates that detection of the genetically stable mip gene by nested PCR with a modified process of water sample preparation can be rapidly and effectively used to enhance isolation of the L. pneumophila taxon from microenvironments.

 

Interferon-gamma reverses the evasion of Birc1e/Naip5 gene mediated murine macrophage immunity by Legionella pneumophila mutant lacking flagellin

Akamine M, Higa F, Haranaga S, Tateyama M, Mori N, Heuner K, Fujita J.

Department of Medicine and Therapeutics, Control and Prevention of Infectious Diseases, Graduate School of Medicine, University of the Ryukyus, Okinawa, Japan. h066567@eve.u-ryukyu.ac.jp

Microbiol Immunol. 2007;51(3):279-87.

ABSTRACT: Legionella pneumophila is the etiologic agent of Legionnaires' disease. This bacterium contains a single monopolar flagellum, of which the FlaA subunit is a major protein constituent. The murine macrophage resistance against this bacterium is controlled by the Birc1e/Naip5 gene, which belongs to the NOD family. We evaluated the intracellular growth of the flaA mutant bacteria as well as another aflagellated fliA mutant, within bone marrow-derived macrophages from mice with an intact (C57BL/6, BALB/c) or mutated (A/J) Birc1e/Naip5 gene. The flaA mutant L. pneumophila multiplied within C57BL/6 and BALB/c macrophages while the wild-type strain did not. Cell viability was not impaired until 3 days after infection when the flaA mutant bacteria replicated 10(2-3)-fold in macrophages, implying that L. pneumophila inhibited host cell death during the early phase of intracellular replication. The addition of recombinant interferon-gamma (IFN-gamma) to the infected macrophages restricted replication of the flaA mutant within macrophages; these treated cells also showed enhanced nitric oxide production, although inhibition of nitric oxide production did not affect the IFN-gamma induced inhibition of Legionella replication. These findings suggested that IFN-gamma activated macrophages to restrict the intracellular growth of the L. pneumophila flaA mutant by a NO independent pathway.

 

Specific detection of PCR product from Legionella pneumophila strain Philadelphia1 using zinc finger protein Sp2

Ikebukuro K, Kumagai T, Motoki H, Osawa Y, Matuo T, Horiuchi M, Sode K.

Department of Biotechnology, Graduate School of Engineering, Tokyo University of Agriculture & Technology, 2-24-16 Naka-machi, Koganei, Tokyo 184-8588, Japan. ikebu@cc.tuat.ac.jp

Nucleic Acids Symp Ser (Oxf). 2007;(51):285-6.

ABSTRACT: We have developed a novel method to detect PCR products from pathogen genome DNA by Zinc finger protein that can bind to double strand DNA (dsDNA) in sequence specific manner. In this study, we tried to detect Legionella pneumophila strain Philadelphia 1 using Zinc finger protein. We found the specific target DNA sequence for zinc finger protein Sp2 in L. pneumophila strain Philadelphia 1 genome DNA. Specific PCR product was successfully amplified from L. pneumophila strain Philadelphia 1 genome DNA and we used Zinc finger protein Sp2 to detect it. We succeeded in detecting the PCR products from L. pneumophila strain Philadelphia 1 genome DNA with Sp2.

 

Legionella pneumophila type II secretome reveals unique exoproteins and a chitinase that promotes bacterial persistence in the lung

DebRoy S, Dao J, Söderberg M, Rossier O, Cianciotto NP.

Department of Microbiology-Immunology, Northwestern University Medical School, Chicago, IL 60611, USA. n-cianciotto@northwestern.edu

Proc Natl Acad Sci U S A. 2006 Dec 12;103(50):19146-51.

ABSTRACT: Type II protein secretion is critical for Legionella pneumophila infection of amoebae, macrophages, and mice. Previously, we found several enzymes to be secreted by this (Lsp) secretory pathway. To better define the L. pneumophila type II secretome, a 2D electrophoresis proteomic approach was used to compare proteins in wild-type and type II mutant supernatants. We identified 20 proteins that are type II-dependent, including aminopeptidases, an RNase, and chitinase, as well as proteins with no homology to known proteins. Because a chitinase had not been previously reported in Legionella, we determined that wild type secretes activity against both p-nitrophenyl triacetyl chitotriose and glycol chitin. An lsp mutant had a 70-75% reduction in activity, confirming the type II dependency of the secreted chitinase. Newly constructed chitinase (chiA) mutants also had approximately 75% less activity, and reintroduction of chiA restored the mutants to normal levels of activity. Although chiA mutants were not impaired for in vitro intracellular infection, they were defective upon intratracheal inoculation into the lungs of A/J mice, and antibodies against ChiA were detectable in infected animals. In contrast, mutants lacking a secreted phosphatase, protease, or one of several lipolytic enzymes were not defective in vivo. In sum, this study shows that the output of type II secretion is greater in magnitude than previously appreciated and includes previously undescribed proteins. Our data also indicate that an enzyme with chitinase activity can promote infection of a mammalian host.

 

A Legionella pneumophila-translocated substrate that is required for growth within macrophages and protection from host cell death

Laguna RK, Creasey EA, Li Z, Valtz N, Isberg RR.

Howard Hughes Medical Institute and Department of Molecular Biology and Microbiology, Tufts University School of Medicine, 150 Harrison Avenue, Boston, MA 02111, USA. ralph.isberg@tufts.edu

Proc Natl Acad Sci U S A. 2006 Dec 5;103(49):18745-50.

ABSTRACT: Legionella pneumophila requires the Dot/Icm protein translocation system to replicate within host cells as a critical component of Legionnaire's pneumonia. None of the known individual substrates of the translocator have been shown to be essential for intracellular replication. We demonstrate here that mutants lacking the Dot/Icm substrate SdhA were severely impaired for intracellular growth within mouse bone marrow macrophages, with the defect absolute in triple mutants lacking sdhA and its two paralogs. The defect caused by the absence of the sdhA family was less severe during growth within Dictyostelium discoideum amoebae, indicating that the requirement for SdhA shows cell-type specificity. Macrophages harboring the L. pneumophila sdhA mutant showed increased nuclear degradation, mitochondrial disruption, membrane permeability, and caspase activation, indicating a role for SdhA in preventing host cell death. Defective intracellular growth of the sdhA(-) mutant could be partially suppressed by the action of caspase inhibitors, but caspase-independent cell death pathways eventually aborted replication of the mutant.

 

Identification of non-dot/icm suppressors of the Legionella pneumophila DeltadotL lethality phenotype

Vincent CD, Buscher BA, Friedman JR, Williams LA, Bardill P, Vogel JP.

Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri 63110, USA. jvogel@borcim.wustl.edu

J Bacteriol. 2006 Dec;188(23):8231-43.

ABSTRACT: Legionella pneumophila, a causative agent of bacterial pneumonia, survives inside phagocytic cells by avoiding rapid targeting to the lysosome. This bacterium utilizes a type IVB secretion system, encoded by the dot/icm genes, to replicate inside host cells. DotL, a critical component of the Dot/Icm secretion apparatus, functions as the type IV coupling protein. In contrast to most dot/icm genes, which are dispensable for growth on bacteriological media, dotL is required for the viability of wild-type L. pneumophila. Previously we reported that DeltadotL lethality could be suppressed by inactivation of the Dot/Icm complex via mutations in other dot/icm genes. Here we report the isolation of non-dot/icm suppressors of this phenotype. These DeltadotL suppressors include insertions that disrupt the function of the L. pneumophila homologs of cpxR, djlA, lysS, and two novel open reading frames, lpg0742 and lpg1594, that we have named ldsA and ldsB for lethality of DeltadotL suppressor. In addition to suppressing DeltadotL lethality, inactivation of these genes in a wild-type strain background causes a range of defects in L. pneumophila virulence traits, including intracellular growth, implicating these factors in the proper function of the Dot/Icm complex. Consistent with previous data showing a role for the cpx system in regulating expression of several dot/icm genes, the cpxR insertion mutant produced decreased levels of three Dot/Icm proteins, DotA, IcmV, and IcmW. The remaining four suppressors did not affect the steady-state levels of any Dot/Icm protein and are likely to represent the first identified factors necessary for assembly and/or activation of the Dot/Icm secretion complex.

 

Type IV pili and type II secretion play a limited role in Legionella pneumophila biofilm colonization and retention

Lucas CE, Brown E, Fields BS.

Respiratory Disease Branch, Division of Bacterial and Mycotic Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, GA 30333, USA. CHL9@cdc.gov

Microbiology. 2006 Dec;152(Pt 12):3569-73.

ABSTRACT: Legionellae colonize biofilms in building water systems, yet little is known about their interaction with the organisms in these microbial communities. The role of Legionella pneumophila type IV pili and the type II secretion pre-pilin peptidase was evaluated in a model biofilm system. L. pneumophila strains 130b (wild-type), BS100 (a type IV pili mutant) and NU243 (a pre-pilin peptidase mutant) were assessed for attachment and retention in an established biofilm. Strains 130b and NU243 colonized the biofilm at a similar level while BS100 attached at a tenfold lower level. Over time, NU243 dropped below the level of detection while BS100 remained in the biofilm throughout the course of the experiment. The wild-type strain decreased but remained at a considerably higher level than either of the mutants. Inclusion of amoebae with BS100 allowed for attachment and retention at a level similar to 130b. NU243, wh