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        Studi sperimentali e molecolari (2020 -2017)

        [ultimo aggiornamento 25/11/2020]


Identification of differentially expressed Legionella genes during its intracellular growth in Acanthamoeba

Quan FS, Kong HH, Lee HA, Chu KB, Moon EK.

Department of Biomedical Science, Graduate School, Kyung Hee University, Seoul, Republic of Korea.

Heliyon 2020 Oct 12;6(10):e05238.

Abstract: Legionella grows intracellularly in free-living amoeba as well as in mammalian macrophages. Until now, the overall gene expression pattern of intracellular Legionella in Acanthamoeba was not fully explained. Intracellular bacteria are capable of not only altering the gene expression of its host, but it can also regulate the expression of its own genes for survival. In this study, differentially expressed Legionella genes within Acanthamoeba during the 24 h intracellular growth period were investigated for comparative analysis. RNA sequencing analysis revealed 3,003 genes from the intracellular Legionella. Among them, 115 genes were upregulated and 1,676 genes were downregulated more than 2 fold compared to the free Legionella. Gene ontology (GO) analysis revealed the suppression of multiple genes within the intracellular Legionella, which were categorized under 'ATP binding' and 'DNA binding' in the molecular function domain. Gene expression of alkylhydroperoxidase, an enzyme involved in virulence and anti-oxidative stress response, was strongly enhanced 24 h post-intracellular growth. Amino acid ABC transporter substrate-binding protein that utilizes energy generation was also highly expressed. Genes associated with alkylhydroperoxidase, glucose pathway, and Dot/Icm type IV secretion system were shown to be differentially expressed. These results contribute to a better understanding of the survival strategies of intracellular Legionella within Acanthamoeba.


Molecular Mimicry: a Paradigm of Host-Microbe Coevolution Illustrated by Legionella

Mondino S, Schmidt S, Buchrieser C.

Institut Pasteur, Biologie des Bactéries Intracellulaires, Paris, France.

mBio 2020 Oct 6;11(5):e01201-20.

Abstract: Through coevolution with host cells, microorganisms have acquired mechanisms to avoid the detection by the host surveillance system and to use the cell's supplies to establish themselves. Indeed, certain pathogens have evolved proteins that imitate specific eukaryotic cell proteins, allowing them to manipulate host pathways, a phenomenon termed molecular mimicry. Bacterial "eukaryotic-like proteins" are a remarkable example of molecular mimicry. They are defined as proteins that strongly resemble eukaryotic proteins or that carry domains that are predominantly present in eukaryotes and that are generally absent from prokaryotes. The widest diversity of eukaryotic-like proteins known to date can be found in members of the bacterial genus Legionella, some of which cause a severe pneumonia in humans. The characterization of a number of these proteins shed light on their importance during infection. The subsequent identification of eukaryotic-like genes in the genomes of other amoeba-associated bacteria and bacterial symbionts suggested that eukaryotic-like proteins are a common means of bacterial evasion and communication, shaped by the continuous interactions between bacteria and their protozoan hosts. In this review, we discuss the concept of molecular mimicry using Legionella as an example and show that eukaryotic-like proteins effectively manipulate host cell pathways. The study of the function and evolution of such proteins is an exciting field of research that is leading us toward a better understanding of the complex world of bacterium-host interactions. Ultimately, this knowledge will teach us how host pathways are manipulated and how infections may possibly be tackled.


Modeling of the Transmission of Coronaviruses, Measles Virus, Influenza Virus, Mycobacterium tuberculosis, and Legionella pneumophila in Dental Clinics

Zemouri C, Awad SF, Volgenant CMC, Crielaard W, Laheij AMGA, de Soet JJ.

Department of Preventive Dentistry, Academic Centre for Dentistry Amsterdam, University of Amsterdam and Vrije Universiteit Amsterdam, Amsterdam, the Netherlands.

J Dent Res 2020 Sep;99(10):1192-1198.

Abstract: Dental health care workers are in close contact to their patients and are therefore at higher risk for contracting airborne infectious diseases. The transmission rates of airborne pathogens from patient to dental health care workers are unknown. With the outbreaks of infectious diseases, such as seasonal influenza, occasional outbreaks of measles and tuberculosis, and the current pandemic of the coronavirus disease COVID-19, it is important to estimate the risks for dental health care workers. Therefore, the transmission probability of these airborne infectious diseases was estimated via mathematical modeling. The transmission probability was modeled for Mycobacterium tuberculosis, Legionella pneumophila, measles virus, influenza virus, and coronaviruses per a modified version of the Wells-Riley equation. This equation incorporated the indoor air quality by using carbon dioxide as a proxy and added the respiratory protection rate from medical face masks and N95 respirators. Scenario-specific analyses, uncertainty analyses, and sensitivity analyses were run to produce probability rates. A high transmission probability was characterized by high patient infectiousness, the absence of respiratory protection, and poor indoor air quality. The highest transmission probabilities were estimated for measles virus (100%), coronaviruses (99.4%), influenza virus (89.4%), and M. tuberculosis (84.0%). The low-risk scenario leads to transmission probabilities of 4.5% for measles virus and 0% for the other pathogens. From the sensitivity analysis, it shows that the transmission probability is strongly driven by indoor air quality, followed by patient infectiousness, and the least by respiratory protection from medical face mask use. Airborne infection transmission of pathogens such as measles virus and coronaviruses is likely to occur in the dental practice. The risk magnitude, however, is highly dependent on specific conditions in each dental clinic. Improved indoor air quality by ventilation, which reduces carbon dioxide, is the most important factor that will either strongly increase or decrease the probability of the transmission of a pathogen.


Legionella Manipulates Non-canonical SNARE Pairing Using a Bacterial Deubiquitinase

Kitao T, Taguchi K, Seto S, Arasaki K, Ando H, Nagai H, Kubori T.

Department of Microbiology, Graduate School of Medicine, Gifu University, Gifu, Japan;

Cell Rep 2020 Sep 8;32(10):108107.

Abstract: The intracellular bacterial pathogen Legionella pneumophila uses many effector proteins delivered by the bacterial type IV secretion system (T4SS) to hijack the early secretory pathway to establish its replicative niche, known as the Legionella-containing vacuole (LCV). On LCV biogenesis, the endoplasmic reticulum (ER) vesicular soluble N-ethylmaleimide-sensitive factor attachment protein receptors (v-SNARE) Sec22b is recruited to the bacterial phagosome and forms non-canonical pairings with target membrane SNAREs (t-SNAREs) from the plasma membrane. Here, we identify a Legionella deubiquitinase (DUB), LotB, that can modulate the early secretory pathway by interacting with coatomer protein complex I (COPI) vesicles when ectopically expressed. We show that Sec22b is ubiquitinated upon L. pneumophila infection in a T4SS-dependent manner and that, subsequently, LotB deconjugates K63-linked ubiquitins from Sec22b. The DUB activity of LotB stimulates dissociation of the t-SNARE syntaxin 3 (Stx3) from Sec22b, which resides on the LCV. Our study highlights a bacterial strategy manipulating the dynamics of infection-induced SNARE pairing using a bacterial DUB.


Structural analysis of the Legionella pneumophila Dot/Icm type IV secretion system core complex

Durie CL, Sheedlo MJ, Chung JM, Byrne BG, Su M, Knight T, Swanson M, Lacy DB, Ohi MD.

Life Sciences Institute, University of Michigan, Ann Arbor, USA.

Elife 2020 Sep 2;9:e59530.

Abstract: Legionella pneumophila is an opportunistic pathogen that causes the potentially fatal pneumonia Legionnaires' Disease. This infection and subsequent pathology require the Dot/Icm Type IV Secretion System (T4SS) to deliver effector proteins into host cells. Compared to prototypical T4SSs, the Dot/Icm assembly is much larger, containing ~27 different components including a core complex reported to be composed of five proteins: DotC, DotD, DotF, DotG, and DotH. Using single particle cryo-electron microscopy (cryo-EM), we report reconstructions of the core complex of the Dot/Icm T4SS that includes a symmetry mismatch between distinct structural features of the outer membrane cap (OMC) and periplasmic ring (PR). We present models of known core complex proteins, DotC, DotD, and DotH, and two structurally similar proteins within the core complex, DotK and Lpg0657. This analysis reveals the stoichiometry and contact interfaces between the key proteins of the Dot/Icm T4SS core complex and provides a framework for understanding a complex molecular machine.


Structural insight into the membrane targeting domain of the Legionella deAMPylase SidD

Tascón I, Li X, Lucas M, Nelson D, Vidaurrazaga A, Lin YH, Rojas AL, Hierro A, Machner MP.

Division of Molecular and Cellular Biology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, USA.

PLoS Pathog 2020 Aug 27;16(8):e1008734.

Abstract: AMPylation, the post-translational modification with adenosine monophosphate (AMP), is catalyzed by effector proteins from a variety of pathogens. Legionella pneumophila is thus far the only known pathogen that, in addition to encoding an AMPylase (SidM/DrrA), also encodes a deAMPylase, called SidD, that reverses SidM-mediated AMPylation of the vesicle transport GTPase Rab1. DeAMPylation is catalyzed by the N-terminal phosphatase-like domain of SidD. Here, we determined the crystal structure of full length SidD including the uncharacterized C-terminal domain (CTD). A flexible loop rich in aromatic residues within the CTD was required to target SidD to model membranes in vitro and to the Golgi apparatus within mammalian cells. Deletion of the loop (Δloop) or substitution of its aromatic phenylalanine residues rendered SidD cytosolic, showing that the hydrophobic loop is the primary membrane-targeting determinant of SidD. Notably, deletion of the two terminal alpha helices resulted in a CTD variant incapable of discriminating between membranes of different composition. Moreover, a L. pneumophila strain producing SidDΔloop phenocopied a L. pneumophila ΔsidD strain during growth in mouse macrophages and displayed prolonged co-localization of AMPylated Rab1 with LCVs, thus revealing that membrane targeting of SidD via its CTD is a critical prerequisite for its ability to catalyze Rab1 deAMPylation during L. pneumophila infection.


Divergence of Legionella Effectors Reversing Conventional and Unconventional Ubiquitination

Kitao T, Nagai H, Kubori T.

Department of Microbiology, Graduate School of Medicine, Gifu University, Japan.

Front Cell Infect Microbiol 2020 Aug 21;10:448.

Abstract: The intracellular bacterial pathogen Legionella pneumophila employs bacteria-derived effector proteins in a variety of functions to exploit host cellular systems. The ubiquitination machinery constitutes a crucial eukaryotic system for the regulation of numerous cellular processes, and is a representative target for effector-mediated bacterial manipulation. Lpneumophila transports over 300 effector proteins into host cells through its Dot/Icm type IV secretion system. Among these, several effector proteins have been found to function as ubiquitin ligases, including unprecedented enzymes that catalyze ubiquitination through unconventional mechanisms. Recent studies have identified many Lpneumophila effector proteins that can interfere with ubiquitination. These effectors include proteins that are distantly related to the ovarian tumor protein superfamily described as deubiquitinases (DUBs), which regulate important signaling cascades in human cells. Intriguingly, Lpneumophila DUBs are not limited to enzymes that exhibit canonical DUB activity. Some Lpneumophila DUBs can catalyze the cleavage of the unconventional linkage between ubiquitin and substrates. Furthermore, novel mechanisms have been found that adversely affect the function of specific ubiquitin ligases; for instance, effector-mediated posttranslational modifications of ubiquitin ligases result in the inhibition of their activity. In the context of Lpneumophila infection, the existence of enzymes that reverse ubiquitination primarily relates to a fine tuning of biogenesis and remodeling of the Legionella-containing vacuole as a replicative niche. The complexity of the effector arrays reflects sophisticated strategies that bacteria have adopted to adapt their host environment and enable their survival in host cells. This review summarizes the current state of knowledge on the divergent mechanisms of the Lpneumophila effectors that can reverse ubiquitination, which is mediated by other effectors as well as the host ubiquitin machinery.


Legionella pneumophila Infection Rewires the Acanthamoeba castellanii Transcriptome, Highlighting a Class of Sirtuin Genes

Li P, Vassiliadis D, Ong SY, Bennett-Wood V, Sugimoto C, Yamagishi J, Hartland EL, Pasricha S.

Centre for Innate Immunity and Infectious Diseases, Hudson Institute of Medical Research, Clayton, VIC, Australia.

Front Cell Infect Microbiol 2020 Aug 20;10:428.

Abstract: Legionella pneumophila is an environmental bacterium that has evolved to survive predation by soil and water amoebae such as Acanthamoeba castellanii, and this has inadvertently led to the ability of L. pneumophila to survive and replicate in human cells. L. pneumophila causes Legionnaire's Disease, with human exposure occurring via the inhalation of water aerosols containing both amoebae and the bacteria. These aerosols originate from aquatic biofilms found in artifical water sources, such as air-conditioning cooling towers and humidifiers. In these man-made environments, A. castellanii supports L. pneumophila intracellular replication, thereby promoting persistence and dissemination of the bacteria and providing protection from external stress. Despite this close evolutionary relationship, very little is known about how A. castellanii responds to L. pneumophila infection. In this study, we examined the global transcriptional response of A. castellanii to L. pneumophila infection. We compared A. castellanii infected with wild type L. pneumophila to A. castellanii infected with an isogenic ΔdotA mutant strain, which is unable to replicate intracellularly. We showed that A. castellanii underwent clear morphological and transcriptional rewiring over the course of L. pneumophila infection. Through improved annotation of the A. castellanii genome, we determined that these transcriptional changes primarily involved biological processes utilizing small GTPases, including cellular transport, signaling, metabolism and replication. In addition, a number of sirtuin-encoding genes in A. castellanii were found to be conserved and upregulated during L. pneumophila infection. Silencing of sirtuin gene, sir6f (ACA1_153540) resulted in the inhibition of A. castellanii cell proliferation during infection and reduced L. pneumophila replication. Overall our findings identified several biological pathways in amoebae that may support L. pneumophila replication and A. castellanii proliferation in environmental conditions.


Choline Supplementation Sensitizes Legionella dumoffii to Galleria mellonella Apolipophorin III

Palusińska-Szysz M, Zdybicka-Barabas A, Luchowski R, Reszczyńska E, Śmiałek J, Mak P, Gruszecki WI, Cytryńska M.

Department of Genetics and Microbiology, Institute of Biological Sciences, Faculty of Biology and Biotechnology, Maria Curie-Sklodowska University, Lublin, Poland.

Int J Mol Sci 2020 Aug 13;21(16):5818.

Abstract: The growth of Legionella dumoffii can be inhibited by Galleria mellonella apolipophorin III (apoLp-III) which is an insect homologue of human apolipoprotein E., and choline-cultured L. dumoffii cells are considerably more susceptible to apoLp-III than bacteria grown without choline supplementation. In the present study, the interactions of apoLp-III with intact L. dumoffii cells cultured without and with exogenous choline were analyzed to explain the basis of this difference. Fluorescently labeled apoLp-III (FITC-apoLp-III) bound more efficiently to choline-grown L. dumoffii, as revealed by laser scanning confocal microscopy. The cell envelope of these bacteria was penetrated more deeply by FITC-apoLp-III, as demonstrated by fluorescence lifetime imaging microscopy analyses. The increased susceptibility of the choline-cultured L. dumoffii to apoLp-III was also accompanied by alterations in the cell surface topography and nanomechanical properties. A detailed analysis of the interaction of apoLp-III with components of the L. dumoffii cells was carried out using both purified lipopolysaccharide (LPS) and liposomes composed of L. dumoffii phospholipids and LPS. A single micelle of L. dumoffii LPS was formed from 12 to 29 monomeric LPS molecules and one L. dumoffii LPS micelle bound two molecules of apoLp-III. ApoLp-III exhibited the strongest interactions with liposomes with incorporated LPS formed of phospholipids isolated from bacteria cultured on exogenous choline. These results indicated that the differences in the phospholipid content in the cell membrane, especially PC, and LPS affected the interactions of apoLp-III with bacterial cells and suggested that these differences contributed to the increased susceptibility of the choline-cultured L. dumoffii to G. mellonella apoLp-III.


Glucosylation by the Legionella Effector SetA Promotes the Nuclear Localization of the Transcription Factor TFEB

Beck WHJ, Kim D, Das J, Yu H, Smolka MB, Mao Y.

Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY, USA.

iScience 2020 Jul 24;23(7):101300.

Abstract: Legionella pneumophila is an intracellular pathogen that requires nutrients from the host for its replication. It has been shown that replicating L. pneumophila prefers amino acids as main sources of carbon and energy. The homeostasis of amino acids in eukaryotic cells is regulated by the transcription factor EB (TFEB), which translocates into the nucleus and activates genes for autophagy and lysosomal biogenesis. Here we show that the Legionella effector SetA causes a robust nuclear translocation of TFEB when exogenously expressed in mammalian cells and that the translocation is dependent on the glucosyltransferase activity of SetA. We further show that SetA directly glucosylates TFEB at multiple sites. Our findings of TFEB glucosylation by SetA may suggest an alternative strategy for exploiting host nutrients in addition to the control of host mTORC1 signaling by L. pneumophila. Our results provide further insight into the molecular mechanism of the delicate TFEB nuclear shuttling.


Cationic Porphyrins as Effective Agents in Photodynamic Inactivation of Opportunistic Plumbing Pathogen Legionella pneumophila

Lesar A, Mušković M, Begić G, Lončarić M, Tomić Linšak D, Malatesti N, Gobin I.

Department of Biotechnology, University of Rijeka, Rijeka, Croatia.

Int J Mol Sci 2020 Jul 28;21(15):5367.

Abstract: Legionella pneumophila is an environmental bacterium, an opportunistic premise plumbing pathogen that causes the Legionnaires' disease. L. pneumophila presents a serious health hazard in building water systems, due to its high resistance to standard water disinfection methods. Our aim was to study the use of photodynamic inactivation (PDI) against Legionella. We investigated and compared the photobactericidal potential of five cationic dyes. We tested toluidine blue (TBO) and methylene blue (MB), and three 3-N-methylpyridylporphyrins, one tetra-cationic and two tri-cationic, one with a short (CH3) and the other with a long (C17H35) alkyl chain, against L. pneumophila in tap water and after irradiation with violet light. All tested dyes demonstrated a certain dark toxicity against L. pneumophila; porphyrins with lower minimal effective concentration (MEC) values than TBO and MB. Nanomolar MEC values, significantly lower than with TBO and MB, were obtained with all three porphyrins in PDI experiments, with amphiphilic porphyrin demonstrating the highest PDI activity. All tested dyes showed increasing PDI with longer irradiation (0-108 J/cm2), especially the two hydrophilic porphyrins. All three porphyrins caused significant changes in cell membrane permeability after irradiation and L. pneumophila, co-cultivated with Acanthamoeba castellanii after treatment with all three porphyrins and irradiation, did not recover in amoeba. We believe our results indicate the considerable potential of cationic porphyrins as effective anti-Legionella agents.


Mitigation of Expression of Virulence Genes in Legionella pneumophila Internalized in the Free-Living Amoeba Willaertia magna C2c Maky

Mameri RM, Bodennec J, Bezin L, Demanèche S.

R&D Department, Amoéba, Chassieu, France.

Pathogens 2020 Jun 5;9(6):447.

Abstract: Legionella pneumophila is a human pathogen responsible for a severe form of pneumonia named Legionnaire disease. Its natural habitat is aquatic environments, being in a free state or intracellular parasites of free-living amoebae, such as Acanthamoeba castellanii. This pathogen is able to replicate within some amoebae. Willaertia magna C2c Maky, a non-pathogenic amoeba, was previously demonstrated to resist to L. pneumophila and even to be able to eliminate the L. pneumophila strains Philadelphia, Lens, and Paris. Here, we studied the induction of seven virulence genes of three L. pneumophila strains (Paris, Philadelphia, and Lens) within W. magna C2c Maky in comparison within A. castellanii and with the gene expression level of L. pneumophila strains alone used as controls. We defined a gene expression-based virulence index to compare easily and without bias the transcript levels in different conditions and demonstrated that W. magna C2c Maky did not increase the virulence of L. pneumophila strains in contrast to A. castellanii. These results confirmed the non-permissiveness of W. magna C2c Maky toward L. pneumophila strains.


Mechanism of effector capture and delivery by the type IV secretion system from Legionella pneumophila

Meir A, Macé K, Lukoyanova N, Chetrit D, Hospenthal MK, Redzej A, Roy C, Waksman G.

Institute of Structural and Molecular Biology, Birkbeck and UCL, Malet Street, London, UK.

Nat Commun 2020 Jun 8;11(1):2864.

Abstract: Legionella pneumophila is a bacterial pathogen that utilises a Type IV secretion (T4S) system to inject effector proteins into human macrophages. Essential to the recruitment and delivery of effectors to the T4S machinery is the membrane-embedded T4 coupling complex (T4CC). Here, we purify an intact T4CC from the Legionella membrane. It contains the DotL ATPase, the DotM and DotN proteins, the chaperone module IcmSW, and two previously uncharacterised proteins, DotY and DotZ. The atomic resolution structure reveals a DotLMNYZ hetero-pentameric core from which the flexible IcmSW module protrudes. Six of these hetero-pentameric complexes may assemble into a 1.6-MDa hexameric nanomachine, forming an inner membrane channel for effectors to pass through. Analysis of multiple cryo EM maps, further modelling and mutagenesis provide working models for the mechanism for binding and delivery of two essential classes of Legionella effectors, depending on IcmSW or DotM, respectively.


Structure, Dynamics and Cellular Insight Into Novel Substrates of the Legionella pneumophila Type II Secretion System

Portlock TJ, Tyson JY, Dantu SC, Rehman S, White RC, McIntire IE, Sewell L, Richardson K, Shaw R, Pandini A, Cianciotto NP, Garnett JA.

Centre for Host-Microbiome Interactions, Dental Institute, King’s College London, London, UK.

Front Mol Biosci 2020 Jun 11;7:112.

Abstract: Legionella pneumophila is a Gram-negative bacterium that is able to replicate within a broad range of aquatic protozoan hosts. L. pneumophila is also an opportunistic human pathogen that can infect macrophages and epithelia in the lung and lead to Legionnaires' disease. The type II secretion system is a key virulence factor of L. pneumophila and is used to promote bacterial growth at low temperatures, regulate biofilm formation, modulate host responses to infection, facilitate bacterial penetration of mucin gels and is necessary for intracellular growth during the initial stages of infection. The L. pneumophila type II secretion system exports at least 25 substrates out of the bacterium and several of these, including NttA to NttG, contain unique amino acid sequences that are generally not observed outside of the Legionella genus. NttA, NttC, and NttD are required for infection of several amoebal species but it is unclear what influence other novel substrates have within their host. In this study, we show that NttE is required for optimal infection of Acanthamoeba castellanii and Vermamoeba vermiformis amoeba and is essential for the typical colony morphology of L. pneumophila. In addition, we report the atomic structures of NttA, NttC, and NttE and through a combined biophysical and biochemical hypothesis driven approach we propose novel functions for these substrates during infection. This work lays the foundation for future studies into the mechanistic understanding of novel type II substrate functions and how these relate to L. pneumophila ecology and disease.


Structure and functional analysis of the Legionella pneumophila chitinase ChiA reveals a novel mechanism of metal-dependent mucin degradation

Rehman S, Grigoryeva LS, Richardson KH, Corsini P, White RC, Shaw R, Portlock TJ, Dorgan B, Zanjani ZS, Fornili A, Cianciotto NP, Garnett JA.

Department of Microbiology and Immunology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.

PLoS Pathog 2020 May 4;16(5):e1008342.

Abstract: Chitinases are important enzymes that contribute to the generation of carbon and nitrogen from chitin, a long chain polymer of N-acetylglucosamine that is abundant in insects, fungi, invertebrates and fish. Although mammals do not produce chitin, chitinases have been identified in bacteria that are key virulence factors in severe respiratory, gastrointestinal and urinary diseases. However, it is unclear how these enzymes are able to carry out this dual function. Legionella pneumophila is the causative agent of Legionnaires' disease, an often-fatal pneumonia and its chitinase ChiA is essential for the survival of L. pneumophila in the lung. Here we report the first atomic resolution insight into the pathogenic mechanism of a bacterial chitinase. We derive an experimental model of intact ChiA and show how its N-terminal region targets ChiA to the bacterial surface after its secretion. We provide the first evidence that L. pneumophila can bind mucins on its surface, but this is not dependent on ChiA. This demonstrates that additional peripheral mucin binding proteins are also expressed in L. pneumophila. We also show that the ChiA C-terminal chitinase domain has novel Zn2+-dependent peptidase activity against mammalian mucin-like proteins, namely MUC5AC and the C1-esterase inhibitor, and that ChiA promotes bacterial penetration of mucin gels. Our findings suggest that ChiA can facilitate passage of L. pneumophila through the alveolar mucosa, can modulate the host complement system and that ChiA may be a promising target for vaccine development.


Legionella effector MavC targets the Ube2N~Ub conjugate for noncanonical ubiquitination

Puvar K, Iyer S, Fu J, Kenny S, Negrón Terón KI, Luo ZQ, Brzovic PS, Klevit RE, Das C.

Department of Biochemistry, University of Washington, Seattle, WA 98195 USA.

Nat Commun 2020 May 12;11(1):2365.

Abstract: The bacterial effector MavC modulates the host immune response by blocking Ube2N activity employing an E1-independent ubiquitin ligation, catalyzing formation of a γ-glutamyl-ε-Lys (Gln40Ub-Lys92Ube2N) isopeptide crosslink using a transglutaminase mechanism. Here we provide biochemical evidence in support of MavC targeting the activated, thioester-linked Ube2N~ubiquitin conjugate, catalyzing an intramolecular transglutamination reaction, covalently crosslinking the Ube2N and Ub subunits effectively inactivating the E2~Ub conjugate. Ubiquitin exhibits weak binding to MavC alone, but shows an increase in affinity when tethered to Ube2N in a disulfide-linked substrate that mimics the charged E2~Ub conjugate. Crystal structures of MavC in complex with the substrate mimic and crosslinked product provide insights into the reaction mechanism and underlying protein dynamics that favor transamidation over deamidation, while revealing a crucial role for the structurally unique insertion domain in substrate recognition. This work provides a structural basis of ubiquitination by transglutamination and identifies this enzyme's true physiological substrate.


Legionella effector AnkX displaces the switch II region for Rab1b phosphocholination

Ernst S, Ecker F, Kaspers MS, Ochtrop P, Hedberg C, Groll M, Itzen A.

Department of Biochemistry and Signal Transduction, University Medical Centre Hamburg-Eppendorf (UKE), Hamburg, Germany.

Sci Adv 2020 May 15;6(20):eaaz8041.

Abstract: The causative agent of Legionnaires disease, Legionella pneumophila, translocates the phosphocholine transferase AnkX during infection and thereby posttranslationally modifies the small guanosine triphosphatase (GTPase) Rab1 with a phosphocholine moiety at S76 using cytidine diphosphate (CDP)-choline as a cosubstrate. The molecular basis for Rab1 binding and enzymatic modification have remained elusive because of lack of structural information of the low-affinity complex with AnkX. We combined thiol-reactive CDP-choline derivatives with recombinantly introduced cysteines in the AnkX active site to covalently capture the heterocomplex. The resulting crystal structure revealed that AnkX induces displacement of important regulatory elements of Rab1 by placing a β sheet into a conserved hydrophobic pocket, thereby permitting phosphocholine transfer to the active and inactive states of the GTPase. Together, the combination of chemical biology and structural analysis reveals the enzymatic mechanism of AnkX and the family of filamentation induced by cyclic adenosine monophosphate (FIC) proteins.


Recombinant PAL/PilE/FlaA DNA vaccine provides protective immunity against Legionella pneumophila in BALB/c mice

Chen Y, Yang Z, Dong Y, Chen Y.

Department of Pulmonary and Critical Care Medicine, Shengjing Hospital of China Medical University, Shenyang, China.

BMC Biotechnol 2020 May 18;20(1):28.

Abstract: Background: Legionella pneumophila (L. pneumophila), a Gram-negative small microorganism, causes hospital-acquired pneumonia especially in immunocompromised patients. Vaccination may be an effective method for preventing L .pneumophila infection. Therefore, it is necessary to develop a better vaccine against this disease. In this study, we developed a recombinant peptidoglycan-associated lipoprotein (PAL)/type IV pilin (PilE)/lagellin (FlaA) DNA vaccine and evaluated its immunogenicity and efficacy to protect against L. pneumophila infection. Results: According to the results, the expression of PAL, PilE, FlaA proteins and PAL/PilE/FlaA fusion protein in 293 cells was confirmed. Immunization with PAL/PilE/FlaA DNA vaccine resulted in highest IgG titer and strongest cytotoxic T-lymphocyte (CTL) response. Furthermore, the histopathological changes in lung tissues of mice challenged with a lethal dose of L. pneumophila were alleviated by PAL/PilE/FlaA DNA vaccine immunization. The production of T-helper-1 (Th1) cytokines (IFNγ, TGF-α, and IL-12), and Th2 cytokines (IL-4 and IL-10) were promoted in PAL/PilE/FlaA DNA vaccine group. Finally, immunization with PAL/PilE/FlaA vaccine raised the survival rate of mice to 100% after challenging with a lethal dose of L. pneumophila for 10 consecutive days. Conclusions: Our study suggests that the newly developed PAL/PilE/FlaA DNA vaccine stimulates strong humoral and cellular immune responses and may be a potential intervention on L. pneumophila infection.


Structural insights into the mechanism and inhibition of transglutaminase-induced ubiquitination by the Legionella effector MavC

Mu Y, Wang Y, Huang Y, Li D, Han Y, Chang M, Fu J, Xie Y, Ren J, Wang H, Zhang Y, Luo ZQ, Feng Y.

Beijing Advanced Innovation Center for Soft Matter Science and Engineering, Beijing Key Laboratory of Bioprocess, State Key Laboratory of Chemical Resource Engineering, College of Life Science and Technology, Beijing University of Chemical Technology, Beijing, China.

Nat Commun 2020 Apr 14;11(1):1774.

Abstract: Protein ubiquitination is one of the most prevalent post-translational modifications, controlling virtually every process in eukaryotic cells. Recently, the Legionella effector MavC was found to mediate a unique ubiquitination through transglutamination, linking ubiquitin (Ub) to UBE2N through UbGln40 in a process that can be inhibited by another Legionella effector, Lpg2149. Here, we report the structures of MavC/UBE2N/Ub ternary complex, MavC/UBE2N-Ub (product) binary complex, and MavC/Lpg2149 binary complex. During the ubiquitination, the loop containing the modification site K92 of UBE2N undergoes marked conformational change, and Lpg2149 inhibits this ubiquitination through competing with Ub to bind MavC. Moreover, we found that MavC itself also exhibits weak deubiquitinase activity towards this non-canonical ubiquitination. Together, our study not only provides insights into the mechanism and inhibition of this transglutaminase-induced ubiquitination by MavC, but also sheds light on the future studies into UBE2N inhibition by this modification and deubiquitinases of this unique ubiquitination.


Complete Genome Sequence of Novel Psychrotolerant Legionella Strain TUM19329, Isolated from Antarctic Lake Sediment

Shimada S, Nakai R, Aoki K, Shimoeda N, Ohno G, Miyazaki Y, Kudoh S, Imura S, Watanabe K, Ishii Y, Tateda K.

Department of Microbiology and Infectious Diseases, Toho University School of Medicine, Tokyo, Japan.

Microbiol Resour Announc 2020 Apr 16;9(16):e00253-20.

Abstract: Here, we report the complete genome sequence characteristics of Legionella strain TUM19329, a candidate for a novel psychrotolerant species isolated from Antarctic lake sediment. The genome assembly contains a single 3,750,805-bp contig with a G+C content of 39.1% and is predicted to encode 3,538 proteins.


The Legionella pneumophila Metaeffector Lpg2505 (MesI) Regulates SidI-Mediated Translation Inhibition and Novel Glycosyl Hydrolase Activity

Joseph AM, Pohl AE, Ball TJ, Abram TG, Johnson DK, Geisbrecht BV, Shames SR.

Division of Biology, Kansas State University, Manhattan, Kansas, USA.

Infect Immun 2020 Apr 20;88(5):e00853-19.

Abstract: Legionella pneumophila, the etiological agent of Legionnaires' disease, employs an arsenal of hundreds of Dot/Icm-translocated effector proteins to facilitate replication within eukaryotic phagocytes. Several effectors, called metaeffectors, function to regulate the activity of other Dot/Icm-translocated effectors during infection. The metaeffector Lpg2505 is essential for L. pneumophila intracellular replication only when its cognate effector, SidI, is present. SidI is a cytotoxic effector that interacts with the host translation factor eEF1A and potently inhibits eukaryotic protein translation by an unknown mechanism. Here, we evaluated the impact of Lpg2505 on SidI-mediated phenotypes and investigated the mechanism of SidI function. We determined that Lpg2505 binds with nanomolar affinity to SidI and suppresses SidI-mediated inhibition of protein translation. SidI binding to eEF1A and Lpg2505 is not mutually exclusive, and the proteins bind distinct regions of SidI. We also discovered that SidI possesses GDP-dependent glycosyl hydrolase activity and that this activity is regulated by Lpg2505. We have therefore renamed Lpg2505 MesI (metaeffector of SidI). This work reveals novel enzymatic activity for SidI and provides insight into how intracellular replication of L. pneumophila is regulated by a metaeffector.


Components of the endocytic and recycling trafficking pathways interfere with the integrity of the Legionella-containing vacuole

Anand IS, Choi W, Isberg RR.

Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts.

Cell Microbiol 2020 Apr;22(4):e13151.

Abstract: Legionella pneumophila requires the Dot/Icm translocation system to replicate in a vacuolar compartment within host cells. Strains lacking the translocated substrate SdhA form a permeable vacuole during residence in the host cell, exposing bacteria to the host cytoplasm. In primary macrophages, mutants are defective for intracellular growth, with a pyroptotic cell death response mounted due to bacterial exposure to the cytosol. To understand how SdhA maintains vacuole integrity during intracellular growth, we performed high-throughput RNAi screens against host membrane trafficking genes to identify factors that antagonise vacuole integrity in the absence of SdhA. Depletion of host proteins involved in endocytic uptake and recycling resulted in enhanced intracellular growth and lower levels of permeable vacuoles surrounding the ΔsdhA mutant. Of interest were three different Rab GTPases involved in these processes: Rab11b, Rab8b and Rab5 isoforms, that when depleted resulted in enhanced vacuole integrity surrounding the sdhA mutant. Proteins regulated by these Rabs are responsible for interfering with proper vacuole membrane maintenance, as depletion of the downstream effectors EEA1, Rab11FIP1, or VAMP3 rescued vacuole integrity and intracellular growth of the sdhA mutant. To test the model that specific vesicular components associated with these effectors could act to destabilise the replication vacuole, EEA1 and Rab11FIP1 showed increased density about the sdhA mutant vacuole compared with the wild type (WT) vacuole. Depletion of Rab5 isoforms or Rab11b reduced this aberrant redistribution. These findings are consistent with SdhA interfering with both endocytic and recycling membrane trafficking events that act to destabilise vacuole integrity during infection.


Targeting Eukaryotic mRNA Translation by Legionella pneumophila

Belyi Y.

Gamaleya Research Centre for Epidemiology and Microbiology, Moscow, Russia.

Front Mol Biosci 2020 Apr 29;7:80.

Abstract: Legionella is a gram-negative microorganism and an infectious agent of pneumonia in humans. It is an intracellular pathogen and multiplies in different eukaryotic cells like amoebae, ciliated protozoa, macrophages, monocytes, and lung epithelial cells. Proliferation of L. pneumophila in eukaryotic cells depends on its type 4 secretion system, which delivers an arsenal of bacterial effector proteins to cytoplasm of its host. Once within the cytoplasm, effectors modify a broad range of host activities, including mRNA translation. Translation is inhibited by Legionella through the action of several effector proteins including Lgt1, Lgt2, Lgt3, SidI, LegK4, SidL, and RavX. Lgt1-3 and SidI target elongation factors: Lgt1-3 mono-glucosylate elongation factor eEF1A, while SidI binds eEF1A, and eEF1Bγ. Effector LegK4 inhibits protein synthesis by phosphorylating Hsp70 proteins, while SidL and RavX have no defined targets in protein synthesis machinery thus far. In addition to direct inhibition of protein synthesis, SidI also affects the stress response, whereas Lgt1-3 - unfolded protein response and cell-cycle progression of host cells. Whether manipulation of these processes is linked to canonical or non-canonical function(s) of targeted elongation factors remains unknown.


In vitro and intracellular activities of frog skin temporins against Legionella pneumophila and its eukaryotic hosts

Crépin A, Jégou JF, André S, Ecale F, Croitoru A, Cantereau A, Berjeaud JM, Ladram A, Verdon J.

Laboratoire Ecologie & Biologie des Interactions, UMR CNRS 7267, Université de Poitiers, POITIERS, France.

Sci Rep 2020 Mar 4;10(1):3978.

Abstract: Temporin-SHa (SHa) is a small cationic host defence peptide (HDP) produced in skin secretions of the Sahara frog Pelophylax saharicus. This peptide has a broad-spectrum activity, efficiently targeting bacteria, parasites and viruses. Noticeably, SHa has demonstrated an ability to kill Leishmania infantum parasites (amastigotes) within macrophages. Recently, an analog of SHa with an increased net positive charge, named [K3]SHa, has been designed to improve those activities. SHa and [K3]SHa were both shown to exhibit leishmanicidal activity mainly by permeabilization of cell membranes but could also induce apoptotis-like death. Temporins are usually poorly active against Gram-negative bacteria whereas many of these species are of public health interest. Among them, Legionella pneumophila, the etiological agent of Legionnaire's disease, is of major concern. Indeed, this bacterium adopts an intracellular lifestyle and replicate inside alveolar macrophages likewise inside its numerous protozoan hosts. Despite several authors have studied the antimicrobial activity of many compounds on L. pneumophila released from host cells, nothing is known about activity on intracellular L. pneumophila within their hosts, and subsequently mechanisms of action that could be involved. Here, we showed for the first time that SHa and [K3]SHa were active towards several species of Legionella. Both peptides displayed bactericidal activity and caused a loss of the bacterial envelope integrity leading to a rapid drop in cell viability. Regarding amoebae and THP-1-derived macrophages, SHa was less toxic than [K3]SHa and exhibited low half maximal lethal concentrations (LC50). When used at non-toxic concentration (6.25 µM), SHa killed more than 90% L. pneumophila within amoebae and around 50% within macrophages. Using SHa labeled with the fluorescent dye Cy5, we showed an evenly diffusion within cells except in vacuoles. Moreover, SHa was able to enter the nucleus of amoebae and accumulate in the nucleolus. This subcellular localization seemed specific as macrophages nucleoli remained unlabeled. Finally, no modifications in the expression of cytokines and HDPs were recorded when macrophages were treated with 6.25 µM SHa. By combining all data, we showed that temporin-SHa decreases the intracellular L. pneumophila load within amoebae and macrophages without being toxic for eukaryotic cells. This peptide was also able to reach the nucleolus of amoebae but was not capable to penetrate inside vacuoles. These data are in favor of an indirect action of SHa towards intracellular Legionella and make this peptide a promising template for further developments.


Divergent Evolution of Legionella RCC1 Repeat Effectors Defines the Range of Ran GTPase Cycle Targets

Swart AL, Steiner B, Gomez-Valero L, Schütz S, Hannemann M, Janning P, Irminger M, Rothmeier E, Buchrieser C, Itzen A, Panse VG, Hilbi H.

Institute of Medical Microbiology, University of Zurich, Zurich, Switzerland.

mBio 2020 Mar 24;11(2):e00405-20.

Abstract: Legionella pneumophila governs its interactions with host cells by secreting >300 different "effector" proteins. Some of these effectors contain eukaryotic domains such as the RCC1 (regulator of chromosome condensation 1) repeats promoting the activation of the small GTPase Ran. In this report, we reveal a conserved pattern of L. pneumophila RCC1 repeat genes, which are distributed in two main clusters of strains. Accordingly, strain Philadelphia-1 contains two RCC1 genes implicated in bacterial virulence, legG1 (Legionella eukaryotic gene 1), and ppgA, while strain Paris contains only one, pieG The RCC1 repeat effectors localize to different cellular compartments and bind distinct components of the Ran GTPase cycle, including Ran modulators and the small GTPase itself, and yet they all promote the activation of Ran. The pieG gene spans the corresponding open reading frames of legG1 and a separate adjacent upstream gene, lpg1975 legG1 and lpg1975 are fused upon addition of a single nucleotide to encode a protein that adopts the binding specificity of PieG. Thus, a point mutation in pieG splits the gene, altering the effector target. These results indicate that divergent evolution of RCC1 repeat effectors defines the Ran GTPase cycle targets and that modulation of different components of the cycle might fine-tune Ran activation during Legionella infection. IMPORTANCE: Legionella pneumophila is a ubiquitous environmental bacterium which, upon inhalation, causes a life-threatening pneumonia termed Legionnaires' disease. The opportunistic pathogen grows in amoebae and macrophages by employing a "type IV" secretion system, which secretes more than 300 different "effector" proteins into the host cell, where they subvert pivotal processes. The function of many of these effector proteins is unknown, and their evolution has not been studied. L. pneumophila RCC1 repeat effectors target the small GTPase Ran, a molecular switch implicated in different cellular processes such as nucleocytoplasmic transport and microtubule cytoskeleton dynamics. We provide evidence that one or more RCC1 repeat genes are distributed in two main clusters of L. pneumophila strains and have divergently evolved to target different components of the Ran GTPase activation cycle at different subcellular sites. Thus, L. pneumophila employs a sophisticated strategy to subvert host cell Ran GTPase during infection.


A MicroRNA Network Controls Legionella pneumophila Replication in Human Macrophages via LGALS8 and MX1

Herkt CE, Caffrey BE, Surmann K, Blankenburg S, Gesell Salazar M, Jung AL, Herbel SM, Hoffmann K, Schulte LN, Chen W, Sittka-Stark A, Völker U, Vingron M, Marsico A, Bertrams W, Schmeck B.

Institute for Lung Research, Universities of Giessen and Marburg Lung Center, Philipps University Marburg, Marburg, Germany.

mBio 2020 Mar 24;11(2):e03155-19.

Abstract: Legionella pneumophila is an important cause of pneumonia. It invades alveolar macrophages and manipulates the immune response by interfering with signaling pathways and gene transcription to support its own replication. MicroRNAs (miRNAs) are critical posttranscriptional regulators of gene expression and are involved in defense against bacterial infections. Several pathogens have been shown to exploit the host miRNA machinery to their advantage. We therefore hypothesize that macrophage miRNAs exert positive or negative control over Legionella intracellular replication. We found significant regulation of 85 miRNAs in human macrophages upon L. pneumophila infection. Chromatin immunoprecipitation and sequencing revealed concordant changes of histone acetylation at the putative promoters. Interestingly, a trio of miRNAs (miR-125b, miR-221, and miR-579) was found to significantly affect intracellular L. pneumophila replication in a cooperative manner. Using proteome-analysis, we pinpointed this effect to a concerted downregulation of galectin-8 (LGALS8), DExD/H-box helicase 58 (DDX58), tumor protein P53 (TP53), and then MX dynamin-like GTPase 1 (MX1) by the three miRNAs. In summary, our results demonstrate a new miRNA-controlled immune network restricting Legionella replication in human macrophages. IMPORTANCE. Cases of Legionella pneumophila pneumonia occur worldwide, with potentially fatal outcome. When causing human disease, Legionella injects a plethora of virulence factors to reprogram macrophages to circumvent immune defense and create a replication niche. By analyzing Legionella-induced changes in miRNA expression and genomewide chromatin modifications in primary human macrophages, we identified a cell-autonomous immune network restricting Legionella growth. This network comprises three miRNAs governing expression of the cytosolic RNA receptor DDX58/RIG-I, the tumor suppressor TP53, the antibacterial effector LGALS8, and MX1, which has been described as an antiviral factor. Our findings for the first time link TP53, LGALS8, DDX58, and MX1 in one miRNA-regulated network and integrate them into a functional node in the defense against L. pneumophila.


Identification of Anti- Mycobacterium and Anti- Legionella Compounds With Potential Distinctive Structural Scaffolds From an HD-PBL Using Phenotypic Screens in Amoebae Host Models

Hanna N, Kicka S, Chiriano G, Harrison C, Sakouhi HO, Trofimov V, Kranjc A, Nitschke J, Pagni M, Cosson P, Hilbi H, Scapozza L, Soldati T.

Department of Biochemistry, Faculty of Sciences, University of Geneva, Geneva, Switzerland.

Front Microbiol 2020 Feb 21;11:266.

Abstract: Tubercular Mycobacteria and Legionella pneumophila are the causative agents of potentially fatal respiratory diseases due to their intrinsic pathogenesis but also due to the emergence of antibiotic resistance that limits treatment options. The aim of our study was to explore the antimicrobial activity of a small ligand-based chemical library of 1255 structurally diverse compounds. These compounds were screened in a combination of three assays, two monitoring the intracellular growth of the pathogenic bacteria, Mycobacterium marinum and L. pneumophila, and one assessing virulence of M. marinum. We set up these assays using two amoeba strains, the genetically tractable social amoeba Dictyostelium discoideum and the free-living amoeba Acanthamoeba castellanii. In summary, 64 (5.1%) compounds showed anti-infective/anti-virulence activity in at least one of the three assays. The intracellular assays hit rate varied between 1.7% (n = 22) for M. marinum and 2.8% (n = 35) for L. pneumophila with seven compounds in common for both pathogens. In parallel, 1.2% (n = 15) of the tested compounds were able to restore D. discoideum growth in the presence of M. marinum spiked in a lawn of food bacteria. We also validated the generality of the hits identified in the A. castellanii-M. marinum anti-infective screen using the D. discoideum-M. marinum host-pathogen model. The characterization of anti-infective and antibacterial hits in the latter infection model revealed compounds able to reduce intracellular growth more than 50% at 30 μM. Moreover, the chemical space and physico-chemical properties of the anti-M. marinum hits were compared to standard and candidate Mycobacterium tuberculosis (Mtb) drugs using ChemGPS-NP. A principle component analysis identified separate clusters for anti-M. marinum and anti-L. pneumophila hits unveiling the potentially new physico-chemical properties of these hits compared to standard and candidate M. tuberculosis drugs. Our studies underscore the relevance of using a combination of low-cost and low-complexity assays with full 3R compliance in concert with a rationalized focused library of compounds to identify new chemical scaffolds and to dissect some of their properties prior to taking further steps toward compound development.



Type I-F CRISPR-Cas Distribution and Array Dynamics in Legionella pneumophila

Deecker SR, Ensminger AW.

Department of Molecular Genetics, University of Toronto, Ontario, Canada.

G3 (Bethesda) 2020 Mar;10(3):1039-1050.

Abstract: In bacteria and archaea, several distinct types of CRISPR-Cas systems provide adaptive immunity through broadly similar mechanisms: short nucleic acid sequences derived from foreign DNA, known as spacers, engage in complementary base pairing with invasive genetic elements setting the stage for nucleases to degrade the target DNA. A hallmark of type I CRISPR-Cas systems is their ability to acquire spacers in response to both new and previously encountered invaders (naïve and primed acquisition, respectively). Our phylogenetic analyses of 43 L. pneumophila type I-F CRISPR-Cas systems and their resident genomes suggest that many of these systems have been horizontally acquired. These systems are frequently encoded on plasmids and can co-occur with nearly identical chromosomal loci. We show that two such co-occurring systems are highly protective and undergo efficient primed acquisition in the lab. Furthermore, we observe that targeting by one system's array can prime spacer acquisition in the other. Lastly, we provide experimental and genomic evidence for a model in which primed acquisition can efficiently replenish a depleted type I CRISPR array following a mass spacer deletion event.


Legionella pneumophila Regulates the Activity of UBE2N by Deamidase-Mediated Deubiquitination

Gan N, Guan H, Huang Y, Yu T, Fu J, Nakayasu ES, Puvar K, Das C, Wang D, Ouyang S, Luo ZQ.

College of Life Sciences, Fujian Normal University, Fuzhou, China.

EMBO J 2020 Feb;39(4):e102806.

Abstract: The Legionella pneumophila effector MavC induces ubiquitination of the E2 ubiquitin-conjugating enzyme UBE2N by transglutamination, thereby abolishing its function in the synthesis of K63 -type polyubiquitin chains. The inhibition of UBE2N activity creates a conundrum because this E2 enzyme is important in multiple signaling pathways, including some that are important for intracellular L. pneumophila replication. Here, we show that prolonged inhibition of UBE2N activity by MavC restricts intracellular bacterial replication and that the activity of UBE2N is restored by MvcA, an ortholog of MavC (50% identity) with ubiquitin deamidase activity. MvcA functions to deubiquitinate UBE2N-Ub using the same catalytic triad required for its deamidase activity. Structural analysis of the MvcA-UBE2N-Ub complex reveals a crucial role of the insertion domain in MvcA in substrate recognition. Our study establishes a deubiquitination mechanism catalyzed by a deamidase, which, together with MavC, imposes temporal regulation of the activity of UBE2N during L. pneumophila infection.

IFNγ Receptor Down-Regulation Facilitates Legionella Survival in Alveolar Macrophages

Yang C, McDermot DS, Pasricha S, Brown AS, Bedoui S, Lenz LL, van Driel IR, Hartland EL.

Department of Microbiology and Immunology, University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne, Victoria, Australia.

J Leukoc Biol 2020 Feb;107(2):273-284.

Abstract: Legionella pneumophila is an opportunistic human pathogen and causative agent of the acute pneumonia known as Legionnaire's disease. Upon inhalation, the bacteria replicate in alveolar macrophages (AM), within an intracellular vacuole termed the Legionella-containing vacuole. We recently found that, in vivo, IFNγ was required for optimal clearance of intracellular L. pneumophila by monocyte-derived cells (MC), but the cytokine did not appear to influence clearance by AM. Here, we report that during L. pneumophila lung infection, expression of the IFNγ receptor subunit 1 (IFNGR1) is down regulated in AM and neutrophils, but not MC, offering a possible explanation for why AM are unable to effectively restrict L. pneumophila replication in vivo. To test this, we used mice that constitutively express IFNGR1 in AM and found that prevention of IFNGR1 down-regulation enhanced the ability of AM to restrict L. pneumophila intracellular replication. IFNGR1 down-regulation was independent of the type IV Dot/Icm secretion system of L. pneumophila indicating that bacterial effector proteins were not involved. In contrast to previous work, we found that signaling via type I IFN receptors was not required for IFNGR1 down-regulation in macrophages but rather that MyD88- or Trif- mediated NF-κB activation was required. This work has uncovered an alternative signaling pathway responsible for IFNGR1 down-regulation in macrophages during bacterial infection.

Legionella pneumophila balances ubiquitin transglutamination.

Yan F, Zhu Y, Zhou Y.

Institute of Microbiology, College of Life Sciences, Zhejiang University, Hangzhou, Zhejiang, China.

EMBO J 2020 Feb;39(4):e104258.

Abstract: The effector MavC of the bacterial pathogen Legionella pneumophila catalyzes a noncanonical ubiquitination of the host ubiquitin-conjugating E2 enzyme UBE2N by crosslinking a glutamine residue of ubiquitin to UBE2N lysine residues via its transglutaminase activity. A new study by Gan et al. (2020) reveals that L. pneumophila reverses this noncanonical ubiquitination via its ubiquitin deamidase effector MvcA to allow precise temporal regulation of host signaling during infection.


Intracellular Behaviour of Three Legionella pneumophila Strains within Three Amoeba Strains, Including Willaertia magna C2c Maky

Hasni I, Jarry A, Quelard B, Carlino A, Eberst JB, Abbe O, Demanèche S.

R&D department, Amoéba, 38 Avenue des Frères Montgolfier, Chassieu, France.

Pathogens 2020 Feb;9(2):E105.

ABSTRACT: Legionella pneumophila is a facultative intracellular pathogen found in aquatic environments as planktonic cells within biofilms and as intracellular parasites of free-living amoebae such as Acanthamoeba castellanii. This pathogen bypasses the elimination mechanism to replicate within amoebae; however, not all amoeba species support the growth of L. pneumophilaWillaertia magna C2c Maky, a non-pathogenic amoeba, was previously demonstrated to possess the ability to eliminate the L. pneumophila strain Paris. Here, we study the intracellular behavior of three L. pneumophila strains (Paris, Philadelphia, and Lens) within W. magna C2c Maky and compare this strain to A. castellanii and W. magna Z503, which are used as controls. We observe the intracellular growth of strain Lens within W. magna Z503 and A. castellanii at 22°C and 37°C. Strain Paris grows within A. castellanii at any temperature, while it only grows at 22°C within W. magna Z503. Strain Philadelphia proliferates only within A. castellanii at 37°C. Within W. magna C2c Maky, none of the three legionella strains exhibit intracellular growth. Additionally, the ability of W. magna C2c Maky to decrease the number of internalized L. pneumophila is confirmed. These results support the idea that W. magna C2c Maky possesses unique behavior in regard to L. pneumophila strains.


Crystal Structure of a Hypothetical T2SS Effector Lpg0189 from Legionella pneumophila Reveals a Novel Protein Fold

Chen X, Liu S, Jiang S, Zhang X, Zhang N, Ma J, Ge H.

Institute of Physical Science and Information Technology, Anhui University, Hefei, Anhui, China.

Biochem Biophys Res Commun 2020 Jan;521(3):799-805.

Abstract: Lpg0189 is a type II secretion system-dependent extracellular protein with unknown function from Legionella pneumophila. Herein, we determined the crystal structure of Lpg0189 at 1.98 Å resolution by using single-wavelength anomalous diffraction (SAD). Lpg0189 folds into a novel chair-shaped architecture, with two sheets roughly perpendicular to each other. Bioinformatics analysis suggests Lpg0189 and its homologues are unique to Legionellales and evolved divergently. The interlinking structural and bioinformatics studies provide a better understanding of this hypothetical protein.


Methylomic Changes of Autophagy-Related Genes by Legionella Effector Lpg2936 in Infected Macrophages

Abd El Maksoud AI, Elebeedy D, Abass NH, Awad AM, Nasr GM, Roshdy T, Khalil H.

Department of Molecular Biology, Genetic Engineering and Biotechnology Research Institute, University of Sadat City, Sadat City, Egypt.

Front Cell Dev Biol 2020 Jan;7:390.

ABSTRACT: Legionella pneumophila (L. pneumophila) is a Gram-negative bacterium that infects the human respiratory tract causing Legionnaires' disease, a severe form of pneumonia. Recently, rising evidence indicated the ability of Legionella to regulate host defense via its type 4 secretion system including hundreds of effectors that promote intracellular bacterial replication. The host defense against such invaders includes autophagic machinery that is responsible for degradation events of invading pathogens and recycling of cell components. The interplay between host autophagy and Legionella infection has been reported, indicating the role of bacterial effectors in the regulation of autophagy during intracellular replication. Here, we investigated the potential impact of Legionella effector Lpg2936 in the regulation of host autophagy and its role in bacterial replication using mice-derived macrophages and human lung epithelial cells (A549 cells). First, monitoring of autophagic flux following infection revealed a marked reduction of Atg7 and LC3B expression profile and low accumulation levels of autophagy-related LC3-I, LC3-II, and the Atg12-Atg5 protein complex. A novel methyladenine alteration was observed due to irreversible changes of GATC motif to G (6 mA) TC in the promoter region of Atg7 and LC3B indicated by cleaved genomic-DNA using the N6 methyladenine-sensitive restriction enzyme DpnI. Interestingly, RNA interference (RNAi) of Lpg2936 in infected macrophages showed dramatic inhibition of bacterial replication by restoring the expression of autophagy-related proteins. This is accompanied by low production levels of bacterial-associated pro-inflammatory cytokines. Furthermore, a constructed Lpg2936 segment in the GFP expression vector was translocated in the host nucleus and successfully induced methyladenine changes in Atg7 and LC3B promoter region and subsequently regulated autophagy in A549 cells independent of infection. Finally, treatment with methylation inhibitors 5-AZA and (2)-Epigallocatechin-3-gallate (EGCG) was able to restore autophagy-related gene expression and to disrupt bacterial replication in infected macrophages. This cumulative evidence indicates the methylation effect of Legionella effector Lpg2936 on the host autophagy-related molecules Atg7 and LC3B and subsequent reduction in the expression levels of autophagy effectors during intracellular replication of L. pneumophila.


Complete Genome Sequence of a Legionella longbeachae Serogroup 2 Isolate Derived from a Patient with Legionnaires' Disease.

Haviernik J, Dawson K, Anderson T, Murdoch D, Chambers S, Biggs P, Cree S, Slow S.

Department of Pathology and Biomedical Science, University of Otago, Christchurch, New Zealand.

Microbiol Resour Announc 2020 Jan;9(5):e01563-19. 

Abstract: Legionella longbeachae is the predominant cause of Legionnaires' disease (LD) in New Zealand. Although serogroup 2 (sg2) does not contain the most clinically significant strain, it is an important cause of disease. Here, we report the complete genome sequence of a sg2 isolate from a patient who was hospitalized with LD.


A Novel Legionella Genomic Island Encodes a Copper-Responsive Regulatory System and a Single Icm/Dot Effector Protein Transcriptionally Activated by Copper

Linsky M, Vitkin Y, Segal G.

Department of Molecular Microbiology and Biotechnology, School of Molecular Cell Biology and Biotechnology, George S. Wise Faculty of Life Sciences, Tel-Aviv University, Tel-Aviv, Israel.

mBio 2020 Jan;11(1):e03232-19. 

Abstract: The intracellular pathogen Legionella pneumophila utilizes the Icm/Dot type IV secretion system to translocate >300 effector proteins into host cells during infection. The regulation of some of these effector-encoding genes was previously shown to be coordinated by several global regulators, including three two-component systems (TCSs) found in all the Legionella species examined. Here, we describe the first Legionella genomic island encoding a single Icm/Dot effector and a dedicated TCS, which regulates its expression. This genomic island, which we named Lci, undergoes horizontal gene transfer in the Legionella genus, and the TCS encoded from this island (LciRS) is homologous to TCSs that control the expression of various metal resistance systems found in other bacteria. We found that the L. pneumophila sensor histidine kinase LciS is specifically activated by copper via a unique, small periplasmic sensing domain. Upon activation by LciS, the response regulator LciR directly binds to a conserved regulatory element and activates the expression of the adjacently located lciE effector-encoding gene. Thus, LciR represents the first local regulator of effectors identified in L. pneumophila Moreover, we found that the expression of the lciRS operon is repressed by the Fis1 and Fis3 regulators, leading to Fis-mediated effects on copper induction of LciE and silencing of the expression of this genomic island in the absence of copper. This island represents a novel type of effector regulation in Legionella, shedding new light on the ways by which the Legionella pathogenesis system evolves its effector repertoire and expands its activating signals. IMPORTANCE: Legionella pneumophila is an intracellular human pathogen that utilizes amoebae as its environmental host. The adaptation of L. pneumophila to the intracellular environment requires coordination of expression of its multicomponent pathogenesis system, which is composed of a secretion system and effector proteins. However, the regulatory factors controlling the expression of this pathogenesis system are only partially uncovered. Here, we discovered a novel regulatory system that is activated by copper and controls the expression of a single effector protein. The genes encoding both the regulatory system and the effector protein are located on a genomic island that undergoes horizontal gene transfer within the Legionella genus. This regulator-effector genomic island represents the first reported case of local regulation of effectors in Legionella The discovery of this regulatory mechanism is an important step forward in the understanding of how the regulatory network of effectors functions and evolves in the Legionella genus.


Whole genome sequence analysis reveals the broad distribution of the RtxA type 1 secretion system and four novel putative type 1 secretion systems throughout the Legionella genus

Brown CL, Garner E, Jospin G, Coil DA, Schwake DO, Eisen JA, Mukhopadhyay B, Pruden AJ.

Department of Civil and Environmental Engineering, Virginia Tech, Blacksburg, VA, USA.

PLoS One 2020 Jan;15(1):e0223033.

Abstract: Type 1 secretion systems (T1SSs) are broadly distributed among bacteria and translocate effectors with diverse function across the bacterial cell membrane. Legionella pneumophila, the species most commonly associated with Legionellosis, encodes a T1SS at the lssXYZABD locus which is responsible for the secretion of the virulence factor RtxA. Many investigations have failed to detect lssD, the gene encoding the membrane fusion protein of the RtxA T1SS, in non-pneumophila Legionella, which has led to the assumption that this system is a virulence factor exclusively possessed by L. pneumophila. Here we discovered RtxA and its associated T1SS in a novel Legionella taurinensis strain, leading us to question whether this system may be more widespread than previously thought. Through a bioinformatic analysis of publicly available data, we classified and determined the distribution of four T1SSs including the RtxA T1SS and four novel T1SSs among diverse Legionella spp. The ABC transporter of the novel Legionella T1SS Legionella repeat protein secretion system shares structural similarity to those of diverse T1SS families, including the alkaline protease T1SS in Pseudomonas aeruginosa. The Legionella bacteriocin (1-3) secretion systems T1SSs are novel putative bacteriocin transporting T1SSs as their ABC transporters include C-39 peptidase domains in their N-terminal regions, with LB2SS and LB3SS likely constituting a nitrile hydratase leader peptide transport T1SSs. The LB1SS is more closely related to the colicin V T1SS in Escherichia coli. Of 45 Legionella spp. whole genomes examined, 19 (42%) were determined to possess lssB and lssD homologs. Of these 19, only 7 (37%) are known pathogens. There was no difference in the proportions of disease associated and non-disease associated species that possessed the RtxA T1SS (p=0.4), contrary to the current consensus regarding the RtxA T1SS. These results draw into question the nature of RtxA and its T1SS as a singular virulence factor. Future studies should investigate mechanistic explanations for the association of RtxA with virulence.


L. pneumophila CMP-5,7-di-N-acetyllegionaminic Acid Synthetase (LpCLS)-involved Chemoenzymatic Synthesis of Sialosides and Analogues

McArthur JB, Santra A, Li W, Kooner AS, Liu Z, Yu H, Chen X.

Department of Chemistry, University of California, CA, USA.

Org Biomol Chem 2020 Jan;18(4):738-744.

Abstract: 5,7-Di-N-acetyllegionaminic acid (Leg5,7Ac2) is a bacterial nonulosonic acid (NulO) analogue of sialic acids, an important class of monosaccharides in mammals and in some bacteria. To develop efficient one-pot multienzyme (OPME) glycosylation systems for synthesizing Leg5,7Ac2-glycosides, Legionella pneumophila cytidine 5'-monophosphate (CMP)-Leg5,7Ac2 synthetase (LpCLS) was cloned and characterized. It was successfully used in producing Leg5,7Ac2-glycosides from chemoenzymatically synthesized Leg5,7Ac2 using a one-pot two-enzyme system or from its chemically synthesized six-carbon monosaccharide precursor 2,4-diacetamido-2,4,6-trideoxymannose (6deoxyMan2,4diNAc) in a one-pot three-enzyme system. In addition, LpCLS was shown to tolerate Neu5Ac7NAc, a C9-hydroxyl analogue of Leg5,7Ac2 and also a stable analogue of 7-O-acetylneuraminic acid (Neu5,7Ac2), to allow OPME synthesis of the corresponding α2-3-linked sialosides, from chemically synthesized six-carbon monosaccharide precursor 4-N-acetyl-4-deoxy-N-acetylmannosamine (ManNAc7NAc).


The Bacterial Deubiquitinase Ceg23 Regulates the Association of Lys-63-linked Polyubiquitin Molecules on the Legionella Phagosome

Ma K, Zhen X, Zhou B, Gan N, Cao Y, Fan C, Ouyang S, Luo ZQ, Qiu J.

Key Laboratory of Zoonosis, Ministry of Education, College of Veterinary Medicine, Jilin University, Changchun, China.

Org Biomol Chem 2020 Jan;18(4):738-744.

Abstract: Legionella pneumophila is the causative agent of the lung malady Legionnaires' disease, it modulates host function to create a niche termed the Legionella-containing vacuole (LCV) that permits intracellular L. pneumophila replication. One important aspect of such modulation is the co-option of the host ubiquitin network with a panel of effector proteins. Here, using recombinantly expressed and purified proteins, analytic ultracentrifugation, structural analysis, and computational modeling, along with deubiquitinase (DUB), and bacterial infection assays, we found that the bacterial defective in organelle trafficking/intracellular multiplication effector Ceg23 is a member of the ovarian tumor (OTU) DUB family. We found that Ceg23 displays high specificity toward Lys-63-linked polyubiquitin chains and is localized on the LCV, where it removes ubiquitin moieties from proteins ubiquitinated by the Lys-63-chain type. Analysis of the crystal structure of a Ceg23 variant lacking two putative transmembrane domains at 2.80 Å resolution revealed that despite very limited homology to established members of the OTU family at the primary sequence level, Ceg23 harbors a catalytic motif resembling those associated with typical OTU-type DUBs. Ceg23 deletion increased the association of Lys-63-linked polyubiquitin with the bacterial phagosome, indicating that Ceg23 regulates Lys-63-linked ubiquitin signaling on the LCV. In summary, our findings indicate that Ceg23 contributes to the regulation of the association of Lys-63 type polyubiquitin with the Legionella phagosome. Future identification of host substrates targeted by Ceg23 could clarify the roles of these polyubiquitin chains in the intracellular life cycle of L. pneumophila and Ceg23's role in bacterial virulence.


A valuable experimental setup to model exposure to Legionella's aerosols generated by shower-like systems

Allegra S, Riffard S, Leclerc L, Girardot F, Stauffert M, Forest V, Pourchez J.

University of Lyon, University Jean Monnet of Saint-Etienne, CNRS, EVS-ISTHME UMR 5600, F-42023, Saint-Etienne, France.

Water Res 2020 Jan;172:115496.

Abstract: The mechanism underlying Legionella aerosolization and entry into the respiratory tract remains poorly documented. In previous studies, we characterized the aerodynamic behaviour of Legionella aerosols and assessed their regional deposition within the respiratory tract using a human-like anatomical model. The aim of this study was to assess whether this experimental setup could mimic the exposure to bioaerosols generated by showers. To achieve this objective, we performed experiments to measure the mass median aerodynamic diameter (MMAD) as well as the emitted dose and the physiological state of the airborne bacteria generated by a shower and two nebulizers (vibrating-mesh and jet nebulizers). The MMADs of the dispersed bioaerosols were characterized using a 12-stage cascade low-pressure impactor. The amount of dispersed airborne bacteria from a shower was quantified using a Coriolis® Delta air sampler and compared to the airborne bacteria reaching the thoracic region in the experimental setup. The physiological state and concentration of airborne Legionella were assessed by qPCR for total cells, culture for viable and cultivable Legionella (VC), and flow cytometry for viable but non-cultivable Legionella (VBNC). In summary, the experimental setup developed appears to mimic the bioaerosol emission of a shower in terms of aerodynamic size distribution. Compared to the specific case of a shower used as a reference in this study, the experimental setup developed underestimates by 2 times (when the jet nebulizer is used) or overestimates by 43 times (when the vibrating-mesh nebulizer is used) the total emitted dose of airborne bacteria. To our knowledge, this report is the first showing that an experimental model mimics so closely an exposure to Legionella aerosols produced by showers to assess human lung deposition and infection in well-controlled and safe conditions.


IRG1 and Inducible Nitric Oxide Synthase Act Redundantly with Other Interferon-Gamma-Induced Factors to Restrict Intracellular Replication of Legionella pneumophila

Price JV, Russo D, Ji DX, Chavez RA, DiPeso L, Lee AY, Coers J, Vance RE.

Department of Biology, Oberlin College, Oberlin, Ohio, USA. e

mBio 2019 Nov;10(6):e02629-19.

Abstract: Interferon gamma (IFN-γ) restricts the intracellular replication of many pathogens, but the mechanism by which IFN-γ confers cell-intrinsic pathogen resistance remains unclear. For example, intracellular replication of the bacterial pathogen Legionella pneumophila in macrophages is potently curtailed by IFN-γ. However, consistent with prior studies, no individual genetic deficiency that we tested completely abolished IFN-γ-mediated control. Intriguingly, we observed that the glycolysis inhibitor 2-deoxyglucose (2DG) partially rescued L. pneumophila replication in IFN-γ-treated macrophages. 2DG inhibits glycolysis and triggers the unfolded protein response, but unexpectedly, it appears these effects are not responsible for perturbing the antimicrobial activity of IFN-γ. Instead, we found that 2DG rescues bacterial replication by inhibiting the expression of two key antimicrobial factors, inducible nitric oxide synthase (iNOS) and immune-responsive gene 1 (IRG1). Using immortalized and primary macrophages deficient in iNOS and IRG1, we confirmed that loss of both iNOS and IRG1, but not individual deficiency in either gene, partially reduced IFN-γ-mediated restriction of L. pneumophila Further, using a combinatorial CRISPR/Cas9 mutagenesis approach, we found that mutation of iNOS and IRG1 in combination with four other genes (CASP11, IRGM1, IRGM3, and NOX2) resulted in a total loss of L. pneumophila restriction by IFN-γ in primary bone marrow macrophages. Our study defines a complete set of cell-intrinsic factors required for IFN-γ-mediated restriction of an intracellular bacterial pathogen and highlights the combinatorial strategy used by hosts to block bacterial replication in macrophages. IMPORTANCE: Legionella pneumophila is one example among many species of pathogenic bacteria that replicate within mammalian macrophages during infection. The immune signaling factor interferon gamma (IFN-γ) blocks L. pneumophila replication in macrophages and is an essential component of the immune response to L. pneumophila and other intracellular pathogens. However, to date, no study has identified the exact molecular factors induced by IFN-γ that are required for its activity. We generated macrophages lacking different combinations of IFN-γ-induced genes in an attempt to find a genetic background in which there is a complete loss of IFN-γ-mediated restriction of L. pneumophila We identified six genes that comprise the totality of the IFN-γ-dependent restriction of L. pneumophila replication in macrophages. Our results clarify the molecular basis underlying the potent effects of IFN-γ and highlight how redundancy downstream of IFN-γ is key to prevent exploitation of macrophages by pathogens.


The Temporal Expression of Global Regulator Protein CsrA Is Dually Regulated by ClpP During the Biphasic Life Cycle of Legionella pneumophila

Ge ZH, Long QS, Yuan PB, Pan X, Shen D, Lu YJ.

School of Life Sciences, Sun Yat-sen University, Guangzhou, China.

Front Microbiol 2019 Nov;10:2495.

Abstract: Legionella pneumophila, an environmental bacterium that parasitizes protozoa, is the causative pathogen of Legionnaires' disease. L. pneumophila adopts a distinct biphasic life cycle that allows it to adapt to environmental conditions for survival, replication, and transmission. This cycle consists of a non-virulent replicative phase (RP) and a virulent transmissive phase (TP). Timely and fine-tuned expression of growth and virulence factors in a life cycle-dependent manner is crucial. Herein, we report evidence that CsrA, a key regulator of the switch between the RP and the TP, is dually regulated in a ClpP-dependent manner during the biphasic life cycle of L. pneumophila. First, we show that the protein level of CsrA is temporal during the life cycle and is degraded by ClpP during the TP. The ectopic expression of CsrA in a ΔclpP mutant, but not in the wild type, inhibits both the initiation of the RP in vitro and the invasiveness to Acanthamoeba castellanii, indicating that the ClpP-mediated proteolytic pathway regulates the CsrA protein level. We further show that the temporally expressed IHFB is the transcriptional inhibitor of csrA and is degraded via a ClpP-dependent manner during the RP. During the RP, the level of CsrA is increased by promoting the degradation of IHFB and reducing the degradation of the accumulated CsrA via a ClpP-dependent manner. During the TP, the level of CsrA is decreased by inhibiting the degradation of IHFB and promoting the degradation of the accumulated CsrA via a ClpP-dependent manner as well. In conclusion, our results show that the growth-stage-specific expression level of CsrA is dually regulated by ClpP-dependent proteolysis at both the transcription and protein levels during the biphasic life cycle of L. pneumophila.


Deubiquitination of Phosphoribosyl-Ubiquitin Conjugates by Phosphodiesterase-Domain-Containing Legionella Effectors

Wan M, Sulpizio AG, Akturk A, Beck WHJ, Lanz M, Faça VM, Smolka MB, Vogel JP, Mao Y.

Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, NY, USA.

Proc Natl Acad Sci USA 2019 Nov;116(47):23518-23526.

Abstract: Posttranslational protein modification by ubiquitin (Ub) is a central eukaryotic mechanism that regulates a plethora of physiological processes. Recent studies unveiled an unconventional type of ubiquitination mediated by the SidE family of Legionella pneumophila effectors, such as SdeA, that catalyzes the conjugation of Ub to a serine residue of target proteins via a phosphoribosyl linker (hence named PR-ubiquitination). Comparable to the deubiquitinases in the canonical ubiquitination pathway, here we show that 2 paralogous Legionella effectors, Lpg2154 (DupA; deubiquitinase for PR-ubiquitination) and Lpg2509 (DupB), reverse PR-ubiquitination by specific removal of phosphoribosyl-Ub from substrates. Both DupA and DupB are fully capable of rescuing the Golgi fragmentation phenotype caused by exogenous expression of SdeA in mammalian cells. We further show that deletion of these 2 genes results in significant accumulation of PR-ubiquitinated species in host cells infected with Legionella In addition, we have identified a list of specific PR-ubiquitinated host targets and show that DupA and DupB play a role in modulating the association of PR-ubiquitinated host targets with Legionella-containing vacuoles. Together, our data establish a complete PR-ubiquitination and deubiquitination cycle and demonstrate the intricate control that Legionella has over this unusual Ub-dependent posttranslational modification.


Screening Legionella Effectors for Antiviral Effects Reveals Rab1 GTPase as a Proviral Factor Coopted for Tombusvirus Replication

Inaba JI, Xu K, Kovalev N, Ramanathan H, Roy CR, Lindenbach BD, Nagy PD.

Department of Plant Pathology, University of Kentucky, Lexington, KY, USA.

Proc Natl Acad Sci USA 2019 Oct;116(43):21739-21747.

Abstract: Bacterial virulence factors or effectors are proteins targeted into host cells to coopt or interfere with cellular proteins and pathways. Viruses often coopt the same cellular proteins and pathways to support their replication in infected cells. Therefore, we screened the Legionella pneumophila effectors to probe virus-host interactions and identify factors that modulate tomato bushy stunt virus (TBSV) replication in yeast surrogate host. Among 302 Legionella effectors tested, 28 effectors affected TBSV replication. To unravel a coopted cellular pathway in TBSV replication, the identified DrrA effector from Legionella was further exploited. We find that expression of DrrA in yeast or plants blocks TBSV replication through inhibiting the recruitment of Rab1 small GTPase and endoplasmic reticulum derived COPII vesicles into the viral replication compartment. TBSV hijacks Rab1 and COPII vesicles to create enlarged membrane surfaces and optimal lipid composition within the viral replication compartment. To further validate our Legionella effector screen, we used the Legionella effector LepB lipid kinase to confirm the critical proviral function of PI (3)P phosphoinositide and the early endosomal compartment in TBSV replication. We demonstrate the direct inhibitory activity of LegC8 effector on TBSV replication using a cell-free replicase reconstitution assay. LegC8 inhibits the function of eEF1A, a coopted proviral host factor. Altogether, the identified bacterial effectors with anti-TBSV activity could be powerful reagents in cell biology and virus-host interaction studies. This study provides important proof of concept that bacterial effector proteins can be a useful toolbox to identify host factors and cellular pathways coopted by (+) RNA viruses.


IroT/MavN Is a Legionella Transmembrane Fe (II) Transporter: Metal Selectivity and Translocation Kinetics Revealed by in Vitro Real-Time Transport

Abeyrathna SS, Abeyrathna NS, Thai NK, Sarkar P, D'Arcy S, Meloni G.

Department of Chemistry and Biochemistry, The University of Texas at Dallas, Richardson, Texas, USA.

Biochemistry 2019 Oct;58(43):4337-4342.

Abstract: In intravacuolar pathogens, iron is essential for growth and virulence. In Legionella pneumophila, a putative transmembrane protein inserted on the surface of the host pathogen-containing vacuole, IroT/MavN, facilitates intravacuolar iron acquisition from the host by an unknown mechanism, bypassing the problem of Fe (III) insolubility and mobilization. We developed a platform for purification and reconstitution of IroT in artificial lipid bilayer vesicles (proteoliposomes). By encapsulating the fluorescent reporter probe Fluozin-3, we reveal, by real-time metal transport assays, that IroT is a high-affinity iron transporter selective for Fe (II) over other essential transition metals. Mutational analysis reveals important residues in the transmembrane helices, soluble domains, and loops important for substrate recognition and translocation. The work establishes the substrate transport properties in a novel transporter family important for iron acquisition at the host-pathogen intravacuolar interface and provides chemical tools for a comparative investigation of the translocation properties in other iron transporter families.


NosP Modulates Cyclic-di-GMP Signaling in Legionella pneumophila

Fischer JT, Hossain S, Boon EM.

Department of Chemistry and Institute of Chemical Biology & Drug Discovery, Stony Brook University, Stony Brook, New York, USA.

Biochemistry 2019 Oct;58(42):4325-4334.

Abstract: Biofilms form when bacteria adhere to a surface and secrete an extracellular polymeric substance. Bacteria embedded within a biofilm benefit from increased resistance to antibiotics, host immune responses, and harsh environmental factors. Nitric oxide (NO) is a signaling molecule that can modulate communal behavior, including biofilm formation, in many bacteria. In many cases, NO-induced biofilm dispersal is accomplished through signal transduction pathways that ultimately lead to a decrease in intracellular cyclic-di-GMP levels. H-NOX (heme nitric oxide/oxygen binding domain) proteins are the best characterized bacterial NO sensors and have been implicated in NO-mediated cyclic-di-GMP signaling, but we have recently discovered a second family of NO-sensitive proteins in bacteria named NosP (NO sensing protein); to date, a clear link between NosP signaling and cyclic-di-GMP metabolism has not been established. Here we present evidence that NosP (Lpg0279) binds to NO and directly affects cyclic-di-GMP production from two-component signaling proteins Lpg0278 and Lpg0277 encoded within the NosP operon. Lpg0278 and Lpg0277 are a histidine kinase and cyclic-di-GMP synthase/phosphodiesterase, respectively, that have already been established as being important in regulating Legionella pneumophila cyclic-di-GMP levels; NosP is thus implicated in regulating cyclic-di-GMP in L. pneumophila.


Systematic Identification of Host Cell Regulators of Legionella pneumophila Pathogenesis Using a Genome-wide CRISPR Screen

Jeng EE, Bhadkamkar V, Ibe NU, Gause H, Jiang L, Chan J, Jian R, Jimenez-Morales D, Stevenson E, Krogan NJ, Swaney DL, Snyder MP, Mukherjee S, Bassik MC.

Department of Microbiology and Immunology, University of California, San Francisco, CA, USA.

Department of Genetics, Stanford University School of Medicine, Stanford, CA, USA.

Cell Host Microbe 2019 Oct;26(4):551-563.

Abstract: During infection, Legionella pneumophila translocates over 300 effector proteins into the host cytosol, allowing the pathogen to establish an endoplasmic reticulum (ER)-like Legionella-containing vacuole (LCV) that supports bacterial replication. Here, we perform a genome-wide CRISPR-Cas9 screen and secondary targeted screens in U937 human monocyte/macrophage-like cells to systematically identify host factors that regulate killing by L. pneumophila. The screens reveal known host factors hijacked by L. pneumophila, as well as genes spanning diverse trafficking and signaling pathways previously not linked to L. pneumophila pathogenesis. We further characterize C1orf43 and KIAA1109 as regulators of phagocytosis and show that RAB10 and its chaperone RABIF are required for optimal L. pneumophila replication and ER recruitment to the LCV. Finally, we show that Rab10 protein is recruited to the LCV and ubiquitinated by the effectors SidC/SdcA. Collectively, our results provide a wealth of previously undescribed insights into L. pneumophila pathogenesis and mammalian cell function.


Genetic Diversity of Legionella Pcs and pmtA Genes and the Effect of Utilization of Choline by Legionella Spp. On Induction of Proinflammatory Cytokines

Palusińska-Szysz M, Szuster-Ciesielska A, Janczarek M, Wdowiak-Wróbel S, Schiller J, Reszczyńska E, Gruszecki WI, Fuchs B.

Department of Genetics and Microbiology, Institute of Biological Sciences, Faculty of Biology and Biotechnology, Maria Curie-Sklodowska University, Lublin, Poland.

Pathog Dis 2019 Oct;77(7):ftz065.

Abstract: Legionella species synthesize phosphatidylcholine (PC) in two independent pathways: the three-step methylation of phosphatidylethanolamine PMT pathway and the one-step PCS pathway, in which the Pcs enzyme catalyzes the reaction between choline and CDP-diacylglycerol to form PC. Legionella pcs genes encode highly hydrophobic proteins with phosphatidylcholine synthase activity, which contain up to eight transmembrane helices with N- and C-termini located inside the bacterial cell. The comparative analysis of nucleotide sequences of pcs showed that these genes share high sequence identity among members of the Legionellaceae family. Legionella pmtA genes involved in the PMT pathway encoded small cytosolic proteins with putative phosphatidylethanolamine N-methyltransferase activity. The pmtA genes identified in Legionella species had lower sequence identity to each other than the pcs genes. The phylogenetic tree constructed based on the pcs and pmtA gene sequences showed phylogenetic relatedness between Legionella spp. and other bacteria. The utilization of extracellular choline by the four Legionella species leads to changes not only in the lipid components but also in proteins, and the interactions between these components lead to changes in cell surface properties, which result in a decline in induction of proinflammatory cytokines (TNF-α and IL-6).


Type II Secretion Promotes Bacterial Growth Within the Legionella-Containing Vacuole in Infected Amoebae

White RC, Truchan HK, Zheng H, Tyson JY, Cianciotto NP.

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

Infect Immun 2019 Oct;87(11):e00374-19.

Abstract: It was previously determined that the type II secretion system (T2SS) promotes the ability of Legionella pneumophila to grow in coculture with amoebae. Here, we discerned the stage of intracellular infection that is potentiated by comparing the wild-type and T2SS mutant legionellae for their capacity to parasitize Acanthamoeba castellanii Whereas the mutant behaved normally for entry into the host cells and subsequent evasion of degradative lysosomes, it was impaired in the ability to replicate, with that defect being first evident at approximately 9 h postentry. The replication defect was initially documented in three ways: by determining the numbers of CFU recovered from the lysates of the infected monolayers, by monitoring the levels of fluorescence associated with amoebal monolayers infected with green fluorescent protein (GFP)-expressing bacteria, and by utilizing flow cytometry to quantitate the amounts of GFP-expressing bacteria in individual amoebae. By employing confocal microscopy and newer imaging techniques, we further determined the progression in volume and shape of the bacterial vacuoles and found that the T2SS mutant grows at a decreased rate and does not attain maximally sized phagosomes. Overall, the entire infection cycle (i.e., entry to egress) was considerably slower for the T2SS mutant than it was for the wild-type strain, and the mutant's defect was maintained over multiple rounds of infection. Thus, the T2SS is absolutely required for L. pneumophila to grow to larger numbers in its intravacuolar niche within amoebae. Combining these results with those of our recent analysis of macrophage infection, T2SS is clearly a major component of L. pneumophila intracellular infection.


Legionnaires' Disease Mortality in Guinea Pigs Involves the p45 Mobile Genomic Element

Christensen LM, Sule P, Cirillo SLG, Strain M, Plumlee Q, Adams LG, Cirillo JD.

Department of Microbial Pathogenesis and Immunology, College of Medicine, Texas A&M University Health Science Center, Bryan.

J Infect Dis 2019 Oct;220(10):1700-1710.

Abstract: Background: Legionella can cause Legionnaires' disease, a potentially fatal form of pneumonia that occurs as sporadic epidemics. Not all strains display the same propensity to cause disease in humans. Because Legionella pneumophila serogroup 1 is responsible for >85% of infections, the majority of studies have examined this serogroup, but there are 3 commonly used laboratory strains: L pneumophila serogroup 1 Philadelphia (Phil-1)-derived strains JR32 and Lp01 and 130b-derived strain AA100. Methods: We evaluated the ability of Phil-1, JR32, Lp01, and AA100 to cause disease in guinea pigs. Results: We found that, although Phil-1, JR32, and AA100 cause an acute pneumonia and death by 4 days postinfection (100%), strain Lp01 does not cause mortality (0%). We also noted that Lp01 lacks a mobile element, designated p45, whose presence correlates with virulence. Transfer of p45 into Lp01 results in recovery of the ability of this strain to cause mortality, leads to more pronounced disease, and correlates with increased interferon-γ levels in the lungs and spleens before death. Conclusions: These observations suggest a mechanism of Legionnaires' disease pathogenesis due to the presence of type IVA secretion systems that cause higher mortality due to overinduction of a proinflammatory response in the host.


Viewing Legionella pneumophila Pathogenesis Through an Immunological Lens

Liu X, Shin S.

Department of Microbiology, University of Pennsylvania Perelman School of Medicine, 3610 Hamilton Walk, Johnson Pavilion 201B, Philadelphia, PA 19104, USA.

J Mol Biol 2019 Oct;431(21):4321-4344.

Abstract: Legionella pneumophila is the causative agent of the severe pneumonia Legionnaires' disease. L. pneumophila is ubiquitously found in freshwater environments, where it replicates within free-living protozoa. Aerosolization of contaminated water supplies allows the bacteria to be inhaled into the human lung, where L. pneumophila can be phagocytosed by alveolar macrophages and replicate intracellularly. The Dot/Icm type IV secretion system (T4SS) is one of the key virulence factors required for intracellular bacterial replication and subsequent disease. The Dot/Icm apparatus translocates more than 300 effector proteins into the host cell cytosol. These effectors interfere with a variety of cellular processes, thus enabling the bacterium to evade phagosome-lysosome fusion and establish an endoplasmic reticulum-derived Legionella-containing vacuole, which facilitates bacterial replication. In turn, the immune system has evolved numerous strategies to recognize intracellular bacteria such as L. pneumophila, leading to potent inflammatory responses that aid in eliminating infection. This review aims to provide an overview of L. pneumophila pathogenesis in the context of the host immune response.


Diverse Conjugative Elements Silence Natural Transformation in Legionella Species

Durieux I, Ginevra C, Attaiech L, Picq K, Juan PA, Jarraud S, Charpentier X.

Team "Horizontal gene transfer in bacterial pathogens" Centre International de Recherche en Infectiologie, INSERM U1111, Université Claude Bernard Lyon 1, CNRS, UMR 5308, École Normale Supérieure de Lyon, Université de Lyon, Villeurbanne, France.

Proc Natl Acad Sci USA 2019 Sep;116(37):18613-18618.

Abstract: Natural transformation (i.e., the uptake of DNA and its stable integration in the chromosome) is a major mechanism of horizontal gene transfer in bacteria. Although the vast majority of bacterial genomes carry the genes involved in natural transformation, close relatives of naturally transformable species often appear not competent for natural transformation. In addition, unexplained extensive variations in the natural transformation phenotype have been reported in several species. Here, we addressed this phenomenon by conducting a genome-wide association study (GWAS) on a panel of isolates of the opportunistic pathogen Legionella pneumophila GWAS revealed that the absence of the transformation phenotype is associated with the conjugative plasmid pLPL. The plasmid inhibits transformation by simultaneously silencing the genes required for DNA uptake and recombination. We identified a small RNA (sRNA), RocRp, as the sole plasmid-encoded factor responsible for the silencing of natural transformation. RocRp is homologous to the highly conserved and chromosome-encoded sRNA RocR which controls the transient expression of the DNA uptake system. Assisted by the ProQ/FinO-domain RNA chaperone RocC, RocRp acts as a substitute of RocR, ensuring that the bacterial host of the conjugative plasmid does not become naturally transformable. Distinct homologs of this plasmid-encoded sRNA are found in diverse conjugative elements in other Legionella species. Their low to high prevalence may result in the lack of transformability of some isolates up to the apparent absence of natural transformation in the species. Generally, our work suggests that conjugative elements obscure the widespread occurrence of natural transformability in bacteria.


Evolutionary Dissection of the Dot/Icm System Based on Comparative Genomics of 58 Legionella Species

Gomez-Valero L, Chiner-Oms A, Comas I, Buchrieser C.

Institut Pasteur, Departement of Microbiology, Biologie des Bactéries Intracellulaires, Paris, France. e

Genome Biol Evol 2019 Sep;11(9):2619-2632.

Abstract: The Dot/Icm type IVB secretion system of Legionella pneumophila is essential for its pathogenesis by delivering >300 effector proteins into the host cell. However, their precise secretion mechanism and which components interact with the host cell is only partly understood. Here, we undertook evolutionary analyses of the Dot/Icm system of 58 Legionella species to identify those components that interact with the host and/or the substrates. We show that high recombination rates are acting on DotA, DotG, and IcmX, supporting exposure of these proteins to the host. Specific amino acids under positive selection on the periplasmic region of DotF, and the cytoplasmic domain of DotM, support a role of these regions in substrate binding. Diversifying selection acting on the signal peptide of DotC suggests its interaction with the host after cleavage. Positive selection acts on IcmR, IcmQ, and DotL revealing that these components are probably participating in effector recognition and/or translocation. Furthermore, our results predict the participation in host/effector interaction of DotV and IcmF. In contrast, DotB, DotO, most of the core subcomplex elements, and the chaperones IcmS-W show a high degree of conservation and not signs of recombination or positive selection suggesting that these proteins are under strong structural constraints and have an important role in maintaining the architecture/function of the system. Thus, our analyses of recombination and positive selection acting on the Dot/Icm secretion system predicted specific Dot/Icm components and regions implicated in host interaction and/or substrate recognition and translocation, which will guide further functional analyses.


Two Sequential Layers of Antibody-Mediated Control of Legionella pneumophila Infection

Weber SS, Stoycheva D, Nimmerjahn F, Oxenius A.

Institute of Microbiology, Zürich, Switzerland.

Eur J Immunol 2019 Sep;49(9):1415-1420.

Abstract: Protective immunity against intracellular pathogens, including bacteria, usually relies on cellular immunity. However, antibodies are also implicated in mediating protection against intracellular bacteria. In case of airway infection with Legionella pneumophila (Lpn), the causative agent of Legionnaires' disease, pre-existing Lpn-specific antibodies were shown to afford protection within two days of infection. Here we dissected the early kinetics of Ab-mediated protection against airway Lpn infection and observed two kinetically and mechanistically distinct phases of protection by passively administered antibodies. Within the first hour of infection, Lpn-opsonizing antibodies provided almost 10-fold protection in an antibody Fc-dependent, but FcR-independent manner. Later on, by two days post infection, Lpn-specific Ab-mediated protection strictly involved FcγR, Syk kinase activity in alveolar macrophages and induction of reactive oxygen species (ROS). The findings presented here contribute to the understanding of the mechanisms of Ab-mediated control of Lpn infection in actively or passively immunized individuals.


Bactericidal Efficacies of Food Additive Grade Calcium Hydroxide Toward Legionella pneumophila

Alam MS, Takahashi S, Ito M, Komura M, Kabir MH, Shoham D, Sakai K, Suzuki M, Takehara K.

Laboratory of Animal Health, Department of Veterinary Medicine, Faculty of Agriculture, Tokyo University of Agriculture and Technology, Tokyo, Japan.

J Vet Med Sci 2019 Sep;81(9):1318-1325.

Abstract: Food additive grade calcium hydroxide (FdCa(OH)2) in the solution of 0.17% was evaluated for its bactericidal efficacies toward Legionella pneumophila with or without sodium hypochlorite (NaOCl) at a concentration of 200 ppm total residual chlorine, at room temperature (RT) (25°C±2°C) and 42°C, either with or without 5% fetal bovine serum (FBS). Besides, FdCa(OH)2 in different concentration solutions were prepared in field water samples (hot spring and bath tab water) and evaluated for their bactericidal efficacies at 42°C. FdCa(OH)2 (0.17%) inactivated the L. pneumophila to the undetectable level (<2.6 log CFU/ml) within 5 min and 3 min, respectively, at RT and 42°C, with 5% FBS. At RT and 42°C, NaOCl inactivated L. pneumophila to the undetectable level within 5 min, without 5% FBS, but with 5% FBS, it could only inactivate this bacterium effectively (≥3 log reductions). Conversely, at RT and 42°C, the mixture of 0.17% FdCa(OH)2 and 200 ppm NaOCl could inactivate L. pneumophila to the undetectable level, respectively, within 3 min and 1 min, even with 5% FBS, and it was elucidated that FdCa(OH)2 has a synergistic bactericidal effect together with NaOCl. FdCa(OH)2 0.05% solution prepared in hot spring water could inactivate L. pneumophila to the undetectable within 3 min at 42°C. So, FdCa(OH)2 alone could show nice bactericidal efficacy at 42°C, even with 5% FBS, as well as in field water samples.


Interaction of the Ankyrin H Core Effector of Legionella with the Host LARP7 Component of the 7SK snRNP Complex

Von Dwingelo J, Chung IYW, Price CT, Li L, Jones S, Cygler M, Abu Kwaik Y.

Department of Microbiology and Immunology, University of Louisville, Louisville, Kentucky, USA.

mBio 2019 Aug;10(4):e01942-19.

Abstract: Species of the Legionella genus encode at least 18,000 effector proteins that are translocated through the Dot/Icm type IVB translocation system into macrophages and protist hosts to enable intracellular growth. Eight effectors, including ankyrin H (AnkH), are common to all Legionella species. The AnkH effector is also present in Coxiella and Rickettsiella. To date, no pathogenic effectors have ever been described that directly interfere with host cell transcription. We determined that the host nuclear protein La-related protein 7 (LARP7), which is a component of the 7SK small nuclear ribonucleoprotein (snRNP) complex, interacts with AnkH in the host cell nucleus. The AnkH-LARP7 interaction partially impedes interactions of the 7SK snRNP components with LARP7, interfering with transcriptional elongation by polymerase (Pol) II. Consistent with that, our data show AnkH-dependent global reprogramming of transcription of macrophages infected by Legionella pneumophila. The crystal structure of AnkH shows that it contains four N-terminal ankyrin repeats, followed by a cysteine protease-like domain and an α-helical C-terminal domain. A substitution within the β-hairpin loop of the third ankyrin repeat results in diminishment of LARP7-AnkH interactions and phenocopies the ankH null mutant defect in intracellular growth. LARP7 knockdown partially suppresses intracellular proliferation of wild-type (WT) bacteria and increases the severity of the defect of the ΔankH mutant, indicating a role for LARP7 in permissiveness of host cells to intracellular bacterial infection. We conclude that the AnkH-LARP7 interaction impedes interaction of LARP7 with 7SK snRNP, which would block transcriptional elongation by Pol II, leading to host global transcriptional reprogramming and permissiveness to L. pneumophila. IMPORTANCE: For intracellular pathogens to thrive in host cells, an environment that supports survival and replication needs to be established. L. pneumophila accomplishes this through the activity of the 330 effector proteins that are injected into host cells during infection. Effector functions range from hijacking host trafficking pathways to altering host cell machinery, resulting in altered cell biology and innate immunity. One such pathway is the host protein synthesis pathway. Five L. pneumophila effectors have been identified that alter host cell translation, and 2 effectors have been identified that indirectly affect host cell transcription. No pathogenic effectors have been described that directly interfere with host cell transcription. Here we show a direct interaction of the AnkH effector with a host cell transcription complex involved in transcriptional elongation. We identify a novel process by which AnkH interferes with host transcriptional elongation through interference with formation of a functional complex and show that this interference is required for pathogen proliferation.


Distribution of lag-1 Alleles, ORF7, and ORF8 Genes of Lipopolysaccharide and Sequence-Based Types Among Legionella pneumophila Serogroup 1 Isolates in Japan and China

Jiang L, Amemura-Maekawa J, Ren H, Li Y, Sakata M, Zhou H, Murai M, Chang B, Ohnishi M, Qin T.

State Key Laboratory for Infectious Disease Prevention and Control, Chinese Centre for Disease Control and Prevention, National Institute for Communicable Disease Control and Prevention, Beijing, China.

Front Cell Infect Microbiol 2019 Aug;9:274.

Abstract: Approximately 85% of cases of Legionnaires' disease are caused by Legionella pneumophila serogroup 1. In this study, we analyzed the distribution of lag-1 alleles, ORF 7 and ORF 8 genes of lipopolysaccharide (LPS) and sequence-based types of 616 L. pneumophila serogroup 1 strains isolated in Japan (206 clinical, 225 environmental) and China (13 clinical and 172 environmental). The lag-1 gene was harbored by significantly more of the clinical isolates compared with the environmental isolates (90.3 vs. 19.1% and 61.6 vs. 3.0%, respectively; both P<0.001). ORF 7 genes were detected in 51.0% of Japanese clinical and 36.0% of Japanese environmental (P=0.001) isolates, as well as 15.3% of Chinese clinical and 9.9% of Chinese environmental isolates (P=0.544). ORF 8 genes were detected in 12.1% of Japanese clinical and 5.8% of Japanese environmental (P=0.017) isolates, as well as 7.7% of Chinese clinical and 3.4% of Chinese environmental isolates (P=0.388). The Japanese and Chinese isolates were assigned to 203 and 36 different sequence-types (ST), respectively. ST1 was predominant. Most isolates with the same ST also had the same lag 1, ORF 7, and ORF 8 gene subgroups. In conclusion, the lag-1 was present in most of the clinical isolates but was absent from most of the environmental isolates from both China and Japan, regardless of the water source and SBT type. PCR-based serotyping and subgrouping methods can be used to define a hierarchy of virulence genotypes that require stringent surveillance to prevent human disease.


A Two-Component System That Modulates Cyclic di-GMP Metabolism Promotes Legionella pneumophila Differentiation and Viability in Low-Nutrient Conditions

Hughes ED, Byrne BG, Swanson MS.

Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, Michigan, USA.

J Bacteriol 2019 Aug;201(17):e00253-19.

Abstract: During its life cycle, the environmental pathogen Legionella pneumophila alternates between a replicative and transmissive cell type when cultured in broth, macrophages, or amoebae. Within a protozoan host, L. pneumophila further differentiates into the hardy cell type known as the mature infectious form (MIF). The second messenger cyclic di-GMP coordinates lifestyle changes in many bacterial species, but its role in the L. pneumophila life cycle is less understood. Using an in vitro broth culture model that approximates the intracellular transition from the replicative to the transmissive form, here we investigate the contribution to L. pneumophila differentiation of a two-component system (TCS) that regulates cyclic di-GMP metabolism. The TCS is encoded by lpg0278-lpg0277 and is cotranscribed with lpg0279, which encodes a protein upregulated in MIF cells. The promoter for this operon is RpoS dependent and induced in nutrient-limiting conditions that do not support replication, as demonstrated using a gfp reporter and quantitative PCR (qPCR). The response regulator of the TCS (Lpg0277) is a bifunctional enzyme that both synthesizes and degrades cyclic di-GMP. Using a panel of site-directed point mutants, we show that cyclic di-GMP synthesis mediated by a conserved GGDEF domain promotes growth arrest of replicative L. pneumophila, accumulation of pigment and poly-3-hydroxybutyrate storage granules, and viability in nutrient-limiting conditions. Genetic epistasis tests predict that the MIF protein Lpg0279 acts as a negative regulator of the TCS. Thus, L. pneumophila is equipped with a regulatory network in which cyclic di-GMP stimulates the switch from a replicative to a resilient state equipped to survive in low-nutrient environments. IMPORTANCE: Although an intracellular pathogen, L. pneumophila has developed mechanisms to ensure long-term survival in low-nutrient aqueous conditions. Eradication of L. pneumophila from contaminated water supplies has proven challenging, as outbreaks have been traced to previously remediated systems. Understanding the genetic determinants that support L. pneumophila persistence in low-nutrient environments can inform design and assessment of remediation strategies. Here we characterize a genetic locus that encodes a two-component signaling system (lpg0278-lpg0277) and a putative regulator protein (lpg0279) that modulates the production of the messenger molecule cyclic di-GMP. We show that this locus promotes both L. pneumophila cell differentiation and survival in nutrient-limiting conditions, thus advancing the understanding of the mechanisms that contribute to L. pneumophila environmental resilience.


Molecular Architecture, Polar Targeting and Biogenesis of the Legionella Dot/Icm T4SS

Ghosal D, Jeong KC, Chang YW, Gyore J, Teng L, Gardner A, Vogel JP, Jensen GJ.

Howard Hughes Medical Institute, California Institute of Technology, Pasadena, CA, USA.

Nat Microbiol 2019 Jul;4(7):1173-1182.

Abstract: Legionella pneumophila survives and replicates inside host cells by secreting ~300 effectors through the defective in organelle trafficking (Dot)/intracellular multiplication (Icm) type IVB secretion system (T4BSS). Here, we used complementary electron cryotomography and immunofluorescence microscopy to investigate the molecular architecture and biogenesis of the Dot/Icm secretion apparatus. Electron cryotomography mapped the location of the core and accessory components of the Legionella core transmembrane subcomplex, revealing a well-ordered central channel that opens into a large, windowed secretion chamber with an unusual 13-fold symmetry. Immunofluorescence microscopy deciphered an early-stage assembly process that begins with the targeting of Dot/Icm components to the bacterial poles. Polar targeting of this T4BSS is mediated by two Dot/Icm proteins, DotU and IcmF, that, interestingly, are homologues of the T6SS membrane complex components TssL and TssM, suggesting that the Dot/Icm T4BSS is a hybrid system. Together, these results revealed that the Dot/Icm complex assembles in an 'axial-to-peripheral' pattern.


An Investigation on the Molecular Characteristics and Intracellular Growth Ability Among Environmental and Clinical Isolates of Legionella pneumophila in Sichuan Province, China

Zeng LZ, Liao HY, Luo LZ, He SS, Qin T, Zhou HJ, Li HX, Chen DL, Chen JP.

Department of Parasitology, West China School of Basic Medical Sciences & Forensic Medicine, Sichuan University, Chengdu, Sichuan, China; Sichuan Center for Disease Control and Prevention, Chengdu, Sichuan, China. e

Biomed Environ Sci 2019 Jul;32(7):520-530.

Abstract: Objective: To investigate the molecular characteristics and intracellular growth ability of Legionella pneumophila (L. pneumophila) strains from 1989 to 2016 in Sichuan Province, China. Methods: Seventy-nine isolates of L. pneumophila were collected from environmental and clinical sources, including cooling towers, hot springs, bath water, fountains, and patients, and identified with 16S rRNA gene analysis and serum agglutination assay. The isolates were then typed by Sequence-Based Typing (SBT), and Genotyping of forty-two LP1 strains were analyzed by means of multiple-locus VNTR analysis with 8 loci (MLVA-8). All strains were further analyzed for two virulence genes: Legionella vir homologue (lvh) and repeats in structural toxin (rtxA). The intracellular growth ability of 33 selected isolates was determined by examining their interaction with J774 cells. Results: All isolates were identified to L. pneumophila including 11 serogroups, among which the main serogroup were LP1, accounting for 54.43%. Thirty-three different sequence types (STs) from five main clonal groups and five singletons were identified, along with 8 different MLVA patterns. Both the lvh and rtxA loci were found in all 79 strains. Thirty isolates showed high intracellular growth ability in J774 cells. Conclusion: L. pneumophila is a potential threat to public health, and effective control and prevention strategies are urgently needed.


Intracellular Parasitism, the Driving Force of Evolution of Legionella pneumophila and the Genus Legionella

Gomez-Valero L, Buchrieser C.

Institut Pasteur, Biologie des Bactéries Intracellulaires and CNRS UMR 3525, 75724, Paris, France.

Microbes Infect 2019 Jun-Jul;21(5-6):230-236.

Abstract: Legionella pneumophila is an intracellular pathogen that causes a severe pneumonia called Legionnaires' disease that is often fatal when not promptly diagnosed and treated. Legionella parasitize aquatic protozoa with which it co-evolved over an evolutionary long time. The close relationship between hosts and pathogens, their co-evolution, led to molecular interactions such as the exchange of genetic material through horizontal gene transfer (HGT). Genome sequencing of L. pneumophila and of the entire genus Legionella that comprises over 60 species revealed that Legionellae have co-opted genes and thus cellular functions from their eukaryotic hosts to a surprisingly high extent. Acquisition and loss of these eukaryotic-like genes and domains is an on-going process underlining the highly dynamic nature of the Legionella genomes. Although the large amount and diversity of HGT in Legionella seems to be unique in the prokaryotic world the analyses of more and more genomes from environmental organisms and symbionts of amoeba revealed that such genetic exchanges occur among all amoeba associated bacteria and also among the different microorganisms that infect amoeba. This dynamic reshuffling and gene-acquisition has led to the emergence of Legionella as human pathogen and may lead to the emergence of new human pathogens from the environment.


Metabolic Characterization of Supernatants Produced by Lactobacillus Spp. with in vitro Anti- Legionella Activity

Fuochi V, Coniglio MA, Laghi L, Rescifina A, Caruso M, Stivala A, Furneri PM.

Department of Biomedical and Biotechnological Sciences (BIOMETEC), University of Catania, Catania, Italy.

Front Microbiol 2019 Jun;10:1403.

Abstract: Legionella pneumophila is an organism of public health interest for its presence in water supply systems and other humid thermal habitats. In this study, ten cell-free supernatants produced by Lactobacillus strains were evaluated for their ability to inhibit L. pneumophila strains isolated from hot tap water. Production of antimicrobial substances by Lactobacillus strains were assessed by agar well diffusion test on BCYE agar plates pre-inoculated with L. pneumophila. Cell-free supernatants (CFS) showed antimicrobial activity against all Legionella strains tested: L. rhamnosus and L. salivarius showed the highest activity. By means of a proton-based nuclear magnetic resonance (1H-NMR) spectroscopy, we detected and quantified the Lactobacillus metabolites of these CFSs, so to gain information about which metabolic pathway was likely to be connected to the observed inhibition activity. A panel of metabolites with variations in concentration were revealed, but considerable differences among inter-species were not showed as reported in a similar work by Foschi et al. (2018). More than fifty molecules belonging mainly to the groups of amino acids, organic acids, monosaccharides, ketones, and alcohols were identified in the metabolome. Significant differences were recorded comparing the metabolites found in the supernatants of strains grown in MRS with glycerol and the same strains grown in MRS without supplements. Indeed, pathway analysis revealed that glycine, serine and threonine, pyruvate, and sulfur metabolic pathways had a higher impact when strains were grown in MRS medium with a supplement such as glycerol. Among the metabolites identified, many were amino acids, suggesting the possible presence of bacteriocins which could be linked to the anti-Legionella activity shown by cell-free supernatants.


Legionella pneumophila p45 Element Influences Host Cell Entry and Sensitivity to Sodium

Christensen LM, Sule P, Strain M, Cirillo JD.

Department of Microbial Pathogenesis and Immunology, College of Medicine, Texas A&M University Health Science Center, Bryan, Texas, USA.

PLoS One 2019 Jun;14(6):e0218941.

Abstract: Legionella pneumophila are environmental bacteria found ubiquitously in both natural and man-made water reservoirs, sometimes as constituents of biofilm communities, but mostly intracellularly within protozoal hosts. In the event that Legionella become aerosolized in water droplets and inhaled by humans, they can cause a potentially fatal form of pneumonia called Legionnaires' disease. Strains of L. pneumophila have highly plastic genomes that harbor numerous inter- and intra-genomic elements, enhancing their ability to live under diverse environmental conditions. One such mobile genomic element, p45 carries ~45 kbp of genes, including the Lvh (Legionella Vir homolog) type IVa secretion system. This element was evaluated for its contribution to L. pneumophila environmental resilience and virulence-related characteristics by comparing clinically isolated strain Philadelphia-1 that carries p45, Lp01 that lacks p45, and Lp01 with p45 reintroduced, Lp01+p45. We found that the p45 element impacts host cell entry and resistance to sodium, both virulence-related characteristics in Legionella species.


Gasdermin-D and Caspase-7 Are the Key Caspase-1/8 Substrates Downstream of the NAIP5/NLRC4 Inflammasome Required for Restriction of Legionella pneumophila

Gonçalves AV, Margolis SR, Quirino GFS, Mascarenhas DPA, Rauch I, Nichols RD, Ansaldo E, Fontana MF, Vance RE, Zamboni DS.

Department of Cell Biology, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, São Paulo, Brazil.

PLoS Pathog 2019 Jun;15(6):e1007886.

Abstract: Inflammasomes are cytosolic multi-protein complexes that detect infection or cellular damage and activate the Caspase-1 (CASP1) protease. The NAIP5/NLRC4 inflammasome detects bacterial flagellin and is essential for resistance to the flagellated intracellular bacterium Legionella pneumophila. The effectors required downstream of NAIP5/NLRC4 to restrict bacterial replication remain unclear. Upon NAIP5/NLRC4 activation, CASP1 cleaves and activates the pore-forming protein Gasdermin-D (GSDMD) and the effector caspase-7 (CASP7). However, Casp1-/- (and Casp1/11-/-) mice are only partially susceptible to L. pneumophila and do not phenocopy Nlrc4-/-mice, because NAIP5/NLRC4 also activates CASP8 for restriction of L. pneumophila infection. Here we show that CASP8 promotes the activation of CASP7 and that Casp7/1/11-/- and Casp8/1/11-/- mice recapitulate the full susceptibility of Nlrc4-/- mice. Gsdmd-/- mice exhibit only mild susceptibility to L. pneumophila, but Gsdmd-/-Casp7-/- mice are as susceptible as the Nlrc4-/- mice. These results demonstrate that GSDMD and CASP7 are the key substrates downstream of NAIP5/NLRC4/CASP1/8 required for resistance to L. pneumophila.


Potentiation of Cytokine-Mediated Restriction of Legionella Intracellular Replication by a Dot/Icm-Translocated Effector

Ngwaga T, Hydock AJ, Ganesan S, Shames SR.

Division of Biology, Kansas State University, Manhattan, Kansas, USA.

J Bacteriol 2019 Jun;201(14):e00755-18.

Abstract: Legionella pneumophila is ubiquitous in freshwater environments, where it replicates within unicellular protozoa. However, L. pneumophila is also an accidental human pathogen that can cause Legionnaires' disease in immunocompromised individuals by uncontrolled replication within alveolar macrophages. To replicate within eukaryotic phagocytes, L. pneumophila utilizes a Dot/Icm type IV secretion system to translocate a large arsenal of over 300 effector proteins directly into host cells. In mammals, translocated effectors contribute to innate immune restriction of L. pneumophila. We found previously that the effector LegC4 is important for L. pneumophila replication within a natural host protist but is deleterious to replication in a mouse model of Legionnaires' disease. In the present study, we used cultured mouse primary macrophages to investigate how LegC4 attenuates L. pneumophila replication. We found that LegC4 enhanced restriction of L. pneumophila replication within macrophages activated with tumor necrosis factor (TNF) or interferon gamma (IFN-γ). In addition, expression of legC4 was sufficient to restrict Legionella longbeachae replication within TNF- or IFN-γ-activated macrophages. Thus, this study demonstrates that LegC4 contributes to L. pneumophila clearance from healthy hosts by potentiating cytokine-mediated host defense mechanisms. IMPORTANCE: Legionella spp. are natural pathogens of protozoa and accidental pathogens of humans. Innate immunity in healthy individuals effectively controls Legionella infection due in part to rapid and robust production of proinflammatory cytokines resulting from detection of Dot/Icm-translocated substrates, including effectors. Here, we demonstrate that the effector LegC4 enhances proinflammatory host restriction of Legionella by macrophages. These data suggest that LegC4 may augment proinflammatory signaling or antimicrobial activity of macrophages, a function that has not previously been observed for another bacterial effector. Further insight into LegC4 function will likely reveal novel mechanisms to enhance immunity against pathogens.


Assessing the Impact, Genomics and Evolution of Type II Secretion Across a Large, Medically Important Genus: The Legionella Type II Secretion Paradigm

White RC, Cianciotto NP.

Department of Microbiology and Immunology, Northwestern University Medical School, Chicago, IL 60611, USA.

Microb Genom 2019 Jun;5(6):e000273.

Abstract: The type II secretion system (T2SS) plays a major role in promoting bacterial survival in the environment and in human hosts. One of the best characterized T2SS is that of Legionella pneumophila, the agent of Legionnaires' disease. Secreting at least 25 proteins, including degradative enzymes, eukaryotic-like proteins and novel effectors, this T2SS contributes to the ability of L. pneumophila to grow at low temperatures, infect amoebal and macrophage hosts, damage lung tissue, evade the immune system, and undergo sliding motility. The genes encoding the T2SS are conserved across the genus Legionella, which includes 62 species and >30 pathogens in addition to L. pneumophila. The vast majority of effectors associated with L. pneumophila are shared by a large number of Legionella species, hinting at a critical role for them in the ecology of Legionella as a whole. However, no other species has the same repertoire as L. pneumophila, with, as a general rule, phylogenetically more closely related species sharing similar sets of effectors. T2SS effectors that are involved in infection of a eukaryotic host(s) are more prevalent throughout Legionella, indicating that they are under stronger selective pressure. The Legionella T2SS apparatus is closest to that of Aquicella (another parasite of amoebae), and a significant number of L. pneumophila effectors have their closest homologues in Aquicella. Thus, the T2SS of L. pneumophila probably originated within the order Legionellales, with some of its effectors having arisen within that Aquicella-like progenitor, while other effectors derived from the amoebal host, mimiviruses, fungi and less closely related bacteria.


The Legionella pneumophila Effector WipA Disrupts Host F-actin Polymerisation by Hijacking Phosphotyrosine Signalling

He L, Lin Y, Ge ZH, He SY, Zhao BB, Shen D, He JG, Lu YJ.

School of life sciences, Sun Yat-sen University, Guangzhou, China.

Cell Microbiol Jun 2019;21(6):e13014.

Abstract: The major virulence determinant of Legionella pneumophila is the type IVB secretion system (T4BSS), which delivers approximately 330 effector proteins into the host cell to modulate various cellular processes. However, the functions of most effector proteins remain unclear. WipA, an effector, was the first phosphotyrosine phosphatase of Legionella with unknown function. In this study, we found that WipA induced relatively strong growth defects in yeast in a phosphatase activity-dependent manner. Phosphoproteomics data showed that WipA was likely involved into endocytosis, FcγR-mediated phagocytosis, tight junction, and regulation of actin cytoskeleton pathways. Western blotting further confirmed WipA dephosphorylates several proteins associated with actin polymerisation, such as p-N-WASP, p-ARP3, p-ACK1, and p-NCK1. Thus, we hypothesised that WipA targets N-WASP/ARP2/3 complex signalling pathway, leading to disturbance of actin polymerisation. Indeed, we demonstrated that WipA inhibits host F-actin polymerisation by reducing the G-actin to F-actin transition during L. pneumophila infection. Furthermore, the intracellular proliferation of wipA/legK2 double mutant was significantly impaired at the late stage of infection, although the absence of WipA does not confer any further effect on actin polymerisation to the legK2 mutant. Collectively, this study provides unique insights into the WipA-mediated regulation of host actin polymerisation and assists us to elucidate the pathogenic mechanisms of L. pneumophila infection.


Analysis of Genetic Characterization and Clonality of Legionella pneumophila Isolated from Cooling Towers in Japan

Nakanishi N, Nomoto R, Tanaka S, Arikawa K, Iwamoto T.

Department of Infectious Diseases, Kobe Institute of Health, Kobe, Japan.

Int J Environ Res Public Health 2019 May;16(9):1664.

Abstract: We investigated the genetic characteristics of 161 Legionella pneumophila strains isolated over a period of 10 years from cooling towers in Japan. Minimum spanning tree analysis based on the sequence-based typing (SBT) of them identified three clonal complexes (CCs); CC1 (105/161, 65.2%), CC2 (22/161, 13.7%), and CC3 (20/161, 12.4%). CC1 was formed by serogroup (SG) 1 and SG7, whereas CC2 was mainly formed by SG1. All of the CC3 isolates except two strains were SG13. The major sequence types (STs) in CC1 and CC2 were ST1 (88/105, 83.8%) and ST154 (15/22, 68.2%), respectively. These STs are known as typical types of L. pneumophila SG1 in Japanese cooling tower. Additionally, we identified 15 strains of ST2603 as the major type in CC3. This ST has not been reported in Japanese cooling tower. Whole genome sequencing (WGS) analysis of the representative strains in the three CCs, which were isolated from various cooling towers over the 10 years, elucidated high clonal population of L. pneumophila in Japanese cooling tower. Moreover, it revealed that the strains of CC2 are phylogenetically distant compared to those of CC1 and CC3 and belonged to L. pneumophila subsp. fraseri.


Intracellular Parasitism, the Driving Force of Evolution of Legionella pneumophila and the Genus Legionella

Gomez-Valero L, Buchrieser C.

Institut Pasteur, Biologie des Bactéries Intracellulaires and CNRS UMR 3525, 75724, Paris, France.

Genes Immun 2019 May;20(5):394-402. 

Abstract: Legionella pneumophila is an intracellular pathogen that causes a severe pneumonia called Legionnaires' disease that is often fatal when not promptly diagnosed and treated. However, L. pneumophila is mainly an environmental pathogen of protozoa. This bacterium parasitizes free-living amoeba and other aquatic protozoa with which it co-evolved over an evolutionary long time. Due to the close relationship between hosts and pathogens, their co-evolution leads to molecular interactions such as the exchange of genetic material through horizontal gene transfer (HGT). Those genes that confer an advantage to the bacteria were fixed in their genomes and help these pathogens to subvert host functions to their advantage. Genome sequencing of L. pneumophila and recently of the entire genus Legionella that comprises over 60 species revealed that Legionellae have co-opted genes and thus cellular functions from their eukaryotic hosts to a surprisingly high extent never observed before for a prokaryotic organism. Acquisition and loss of these eukaryotic-like genes and eukaryotic domains is an ongoing process underlining the highly dynamic nature of the Legionella genomes. Although the large amount and diversity of HGT that occurred between Legionella and their protozoan hosts seems to be unique in the prokaryotic world, the analyses of more and more genomes from environmental organisms and symbionts of amoeba revealed that such genetic exchanges occur among all amoeba-associated bacteria and also among the different microorganisms that infect amoeba such as viruses. This dynamic reshuffling and gene-acquisition has led to the emergence of major human pathogens such as Legionella and may lead to the emergence of new human pathogens from the environment.


Draft Genome Sequences of Seven Legionella pneumophila Isolates from a Hot Water System of a Large Building

Gomez-Alvarez V, Boczek L, King D, Pemberton A, Pfaller S, Rodgers M, SantoDomingo J, Revetta RP.

Office of Research and Development, U.S. Environmental Protection Agency, Cincinnati, Ohio, USA.

Microbiol Resour Announc 2019 May;8(18):e00384-19.

Abstract: Public health data show that a significant fraction of the nation's waterborne disease outbreaks is attributable to premise plumbing. We report the draft genome sequences of seven Legionella pneumophila serogroup 1 isolates from hot water lines of a large building. Genomic analysis identified the isolates as belonging to sequence type 1.


In Vitro Activities of Antimicrobial Peptides and Ceragenins Against Legionella pneumophila

Birteksöz-Tan AS, Zeybek Z.

Department of Pharmaceutical Microbiology, Faculty of Pharmacy, Istanbul University, Beyazit, Istanbul, Turkey.

J Antibiot (Tokyo) 2019 May;72(5):291-297.

Abstract: Legionella pneumophila is a waterborne intracellular pathogenic bacterium, the most frequent cause of human legionellosis and a relatively common cause of community-acquired and nosocomial pneumonia. Some legionellosis outbreaks are related to the presence of biofilms, which provide a reservoir for L. pneumophila strains. We investigated the in vitro activities of antibiotics; erythromycin and doxycycline, antimicrobial peptides AMPs; melittin, LL-37 and CAMA (cecropin A (1-7)-Melittin A (2-9) and ceragenins; CSA-8, CSA-13, CSA-44, CSA-131 and CSA-138 against L. pneumophila. Isolation of Legionella strains was conducted according to ISO 1998. Minimum inhibitory concentrations (MICs), minimum bactericidal concentrations (MBCs) and minimum biofilm eradication concentrations (MBECs) were determined using microbroth dilution techniques. MIC ranges for melittin, LL-37, and CAMA were 0.25-1, 1-4, and 2-8 µg ml-1, respectively. MIC ranges for CSA-8, 13, 44, 131, and 138 were 0.5-2, 0.5-1, 1-4, 0.5-2, and 1-2 µg ml-1, respectively, and MBEC values for the ceragenins were 10-160 µg ml-1. These results demonstrate that AMPs and ceragenins display broad-spectrum, in vitro activity against L. pneumophila. In particular, CSA-8, CSA-13 and melittin gave the lowest MICs and MBCs. We also observed that ceragenins are active against established L. pneumophila biofilms.


Symbiont-Mediated Defense Against Legionella pneumophila in Amoebae

König L, Wentrup C, Schulz F, Wascher F, Escola S, Swanson MS, Buchrieser C, Horn M.

Centre for Microbiology and Environmental Systems Science, University of Vienna, Vienna, Austria.

mBio 2019 May;10(3):e00333-19.

Abstract: Legionella pneumophila is an important opportunistic pathogen for which environmental reservoirs are crucial for the infection of humans. In the environment, free-living amoebae represent key hosts providing nutrients and shelter for highly efficient intracellular proliferation of L. pneumophila, which eventually leads to lysis of the protist. However, the significance of other bacterial players for L. pneumophila ecology is poorly understood. In this study, we used a ubiquitous amoeba and bacterial endosymbiont to investigate the impact of this common association on L. pneumophila infection. We demonstrate that L. pneumophila proliferation was severely suppressed in Acanthamoeba castellanii harboring the chlamydial symbiont Protochlamydia amoebophila. The amoebae survived the infection and were able to resume growth. Different environmental amoeba isolates containing the symbiont were equally well protected as different L. pneumophila isolates were diminished, suggesting ecological relevance of this symbiont-mediated defense. Furthermore, protection was not mediated by impaired L. pneumophila uptake. Instead, we observed reduced virulence of L. pneumophila released from symbiont-containing amoebae. Pronounced gene expression changes in the presence of the symbiont indicate that interference with the transition to the transmissive phase impedes the L. pneumophila infection. Finally, our data show that the defensive response of amoebae harboring P. amoebophila leaves the amoebae with superior fitness reminiscent of immunological memory. Given that mutualistic associations between bacteria and amoebae are widely distributed, P. amoebophila and potentially other amoeba endosymbionts could be key in shaping environmental survival, abundance, and virulence of this important pathogen, thereby affecting the frequency of human infection. IMPORTANCE: Bacterial pathogens are generally investigated in the context of disease. To prevent outbreaks, it is essential to understand their lifestyle and interactions with other microbes in their natural environment. Legionella pneumophila is an important human respiratory pathogen that survives and multiplies in biofilms or intracellularly within protists, such as amoebae. Importantly, transmission to humans occurs from these environmental sources. Legionella infection generally leads to rapid host cell lysis. It was therefore surprising to observe that amoebae, including fresh environmental isolates, were well protected during Legionella infection when the bacterial symbiont Protochlamydia amoebophila was also present. Legionella was not prevented from invading amoebae but was impeded in its ability to develop fully virulent progeny and were ultimately cleared in the presence of the symbiont. This study highlights how ecology and virulence of an important human pathogen is affected by a defensive amoeba symbiont, with possibly major consequences for public health.


Density-dependent Resistance Protects Legionella pneumophila From Its Own Antimicrobial Metabolite, HGA

Levin TC, Goldspiel BP, Malik HS.

Howard Hughes Medical Institute, Fred Hutchinson Cancer Research Center, Seattle, USA.

Elife 2019 May;8:e46086.

Abstract: To persist in microbial communities, the bacterial pathogen Legionella pneumophila must withstand competition from neighboring bacteria. Here, we find that L. pneumophila can antagonize the growth of other Legionella species using a secreted inhibitor: HGA (homogentisic acid). Unexpectedly, L. pneumophila can itself be inhibited by HGA secreted from neighboring, isogenic strains. Our genetic approaches further identify lpg1681 as a gene that modulates L. pneumophila susceptibility to HGA. We find that L. pneumophila sensitivity to HGA is density-dependent and cell intrinsic. Resistance is not mediated by the stringent response nor the previously described Legionella quorum-sensing pathway. Instead, L. pneumophila cells secrete HGA only when they are conditionally HGA-resistant, which allows these bacteria to produce a potentially self-toxic molecule while restricting the opportunity for self-harm. We propose that established Legionella communities may deploy molecules such as HGA as an unusual public good that can protect against invasion by low-density competitors.


Polymorphisms of a Collagen-Like Adhesin Contributes to Legionella pneumophila Adhesion, Biofilm Formation Capacity and Clinical Prevalence

Abdel-Nour M, Su H, Duncan C, Li S, Raju D, Shamoun F, Valton M, Ginevra C, Jarraud S, Guyard C, Kerman K, Terebiznik MR.

Department of Biological Sciences, University of Toronto at Scarborough, Toronto, ON, Canada.

Front Microbiol 2019 Apr;10:604.

Abstract: Legionellosis is a severe respiratory illness caused by the inhalation of aerosolized water droplets contaminated with the opportunistic pathogen Legionella pneumophila. The ability of L. pneumophila to produce biofilms has been associated with its capacity to colonize and persist in human-made water reservoirs and distribution systems, which are the source of legionellosis outbreaks. Nevertheless, the factors that mediate L. pneumophila biofilm formation are largely unknown. In previous studies we reported that the adhesin Legionella collagen-like protein (Lcl), is required for auto-aggregation, attachment to multiple surfaces and the formation of biofilms. Lcl structure contains three distinguishable regions: An N-terminal region with a predicted signal sequence, a central region containing tandem collagen-like repeats (R-domain) and a C-terminal region (C-domain) with no significant homology to other known proteins. Lcl R-domain encodes tandem repeats of the collagenous tripeptide Gly-Xaa-Yaa (GXY), a motif that is key for the molecular organization of mammalian collagen and mediates the binding of collagenous proteins to different cellular and environmental ligands. Interestingly, Lcl is polymorphic in the number of GXY tandem repeats. In this study, we combined diverse biochemical, genetic, and cellular approaches to determine the role of Lcl domains and GXY repeats polymorphisms on the structural and functional properties of Lcl, as well as on bacterial attachment, aggregation and biofilm formation. Our results indicate that the R-domain is key for assembling Lcl collagenous triple-helices and has a more preponderate role over the C-domain in Lcl adhesin binding properties. We show that Lcl molecules oligomerize to form large supramolecular complexes to which both, R and C-domains are required. Furthermore, we found that the number of GXY tandem repeats encoded in Lcl R-domain correlates positively with the binding capabilities of Lcl and with the attachment and biofilm production capacity of L. pneumophila strains. Accordingly, the number of GXY tandem repeats in Lcl influences the clinical prevalence of L. pneumophila strains. Therefore, the number of Lcl tandem repeats could be considered as a potential predictor for virulence in L. pneumophila isolates.


Antimicrobial Agent Susceptibilities of Legionella pneumophila MLVA-8 Genotypes

Sharaby Y, Nitzan O, Brettar I, Höfle MG, Peretz A, Halpern M.

Department of Evolutionary and Environmental Biology, Faculty of Natural Sciences, University of Haifa, Haifa, Israel.

Sci Rep 2019 Apr;9(1):6138.

Abstract: Legionella pneumophila causes human lung infections resulting in severe pneumonia. High-resolution genotyping of L. pneumophila isolates can be achieved by multiple-locus variable-number tandem-repeat analysis (MLVA-8). Legionella infections in humans occur as a result of inhalation of bacteria-containing aerosols, thus, our aim was to study the antimicrobial susceptibilities of different MLVA-8 genotypes to ten commonly used antimicrobial agents in legionellosis therapy. Epidemiological cut-off values were determined for all antibiotics. Significant differences were found between the antimicrobial agents' susceptibilities of the three studied environmental genotypes (Gt4, Gt6, and Gt15). Each genotype exhibited a significantly different susceptibility profile, with Gt4 strains (Sequence Type 1) significantly more resistant towards most studied antimicrobial agents. In contrast, Gt6 strains (also Sequence Type 1) were more susceptible to six of the ten studied antimicrobial agents compared to the other genotypes. Our findings show that environmental strains isolated from adjacent points of the same water system, exhibit distinct antimicrobial resistance profiles. These differences highlight the importance of susceptibility testing of Legionella strains. In Israel, the most extensively used macrolide for pneumonia is azithromycin. Our results point at the fact that clarithromycin (another macrolide) and trimethoprim with sulfamethoxazole (SXT) were the most effective antimicrobial agents towards L. pneumophila strains. Moreover, legionellosis can be caused by multiple L. pneumophila genotypes, thus, the treatment approach should be the use of combined antibiotic therapy. Further studies are needed to evaluate specific antimicrobial combinations for legionellosis therapy.


Synthesis of a 3,4-Disubstituted 1,8-Naphthalimide-Based DNA Intercalator for Direct Imaging of Legionella pneumophila

Sharma H, Sidhu JS, Hassen WM, Singh N, Dubowski JJ.

Laboratory for Quantum Semiconductors and Photon-Based BioNanotechnology, Interdisciplinary Institute for Technological Innovation (3IT), CNRS UMI-3463, Department of Electrical and Computer Engineering, Université de Sherbrooke, Sherbrooke, Québec J1K 0A5, Canada.

ACS Omega 2019 Mar;4(3):5829-5838.

Abstract: The development of organic molecules to target nucleic acid is an active area of research at the interface of chemistry and biochemistry, which involves DNA binding, nuclear imaging, and antitumor studies. These molecules bind with DNA through covalent interactions, electrostatic interactions, or intercalation. However, they are less permeable to membrane, and they have a significant cytotoxicity, which limits their application under in vivo conditions. In the present work, various mono- and disubstituted 1,8-naphthalimides-based derivatives (S-12, S-13, S-15, and S-21) have been synthesized and characterized through various spectroscopic techniques. Among these, 3-amino-4-bromo-1,8-naphthalimide (S-15) was found to have an attractive water solubility and act as a nuclear imaging agent. The spectroscopic absorption and emission data showed that S-15 has a strong affinity for salmon sperm DNA with a binding constant of 6.61×104 M-1, and the ratiometric fluorescence intensity (I 489/I 552) of S-15 has a linear relationship in the 0-50 μM range of DNA concentrations. It intercalates with DNA through the hydrophobic planar naphthalimide core as confirmed through cyclic voltammetry, circular dichroism, 1H NMR titration, and thermal denaturation studies. Positively charged amine groups also participate in H-bonding with the bases and backbone of DNA. The S-15 intercalator showed a large Stokes shift and photostability, which made it attractive for direct imaging of Legionella pneumophila, without the need for a prior membrane permeabilization.


A Legionella pneumophila Kinase Phosphorylates the Hsp70 Chaperone Family to Inhibit Eukaryotic Protein Synthesis

Moss SM, Taylor IR, Ruggero D, Gestwicki JE, Shokat KM, Mukherjee S.

Department of Microbiology and Immunology, University of California, San Francisco, CA, USA.

Cell Host Microbe 2019 Mar;25(3):454-462.

Abstract: Legionella pneumophila (L.p.), the microbe responsible for Legionnaires' disease, secretes 300 bacterial proteins into the host cell cytosol. A subset of these proteins affects a wide range of post-translational modifications (PTMs) to disrupt host cellular pathways. L.p. has 5 conserved eukaryotic-like Ser/Thr effector kinases, LegK1-4 and LegK7, which are translocated during infection. Using a chemical genetic screen, we identified the Hsp70 chaperone family as a direct host target of LegK4. Phosphorylation of Hsp70s at T495 in the substrate-binding domain disrupted Hsp70's ATPase activity and greatly inhibited its protein folding capacity. Phosphorylation of cytosolic Hsp70 by LegK4 resulted in global translation inhibition and an increase in the amount of Hsp70 on highly translating polysomes. LegK4's ability to inhibit host translation via a single PTM uncovers a role for Hsp70 in protein synthesis and directly links it to the cellular translational machinery.


The Pleiotropic Legionella Transcription Factor LvbR Links the Lqs and c-di-GMP Regulatory Networks to Control Biofilm Architecture and Virulence

Hochstrasser R, Kessler A, Sahr T, Simon S, Schell U, Gomez-Valero L, Buchrieser C, Hilbi H.

Institute of Medical Microbiology, Faculty of Medicine, University of Zürich, Zürich, Switzerland.

Environ Microbiol 2019 Mar;21(3):1035-1053.

Abstract: The causative agent of Legionnaires' disease, Legionella pneumophila, colonizes amoebae and biofilms in the environment. The opportunistic pathogen employs the Lqs (Legionella quorum sensing) system and the signalling molecule LAI-1 (Legionella autoinducer-1) to regulate virulence, motility, natural competence and expression of a 133 kb genomic "fitness island", including a putative novel regulator. Here, we show that the regulator termed LvbR is a LqsS-regulated transcription factor that binds to the promoter of lpg1056/hnox1 (encoding an inhibitor of the diguanylate cyclase Lpg1057), and thus, regulates proteins involved in c-di-GMP metabolism. LvbR determines biofilm architecture, since L. pneumophila lacking lvbR accumulates less sessile biomass and forms homogeneous mat-like structures, while the parental strain develops more compact bacterial aggregates. Comparative transcriptomics of sessile and planktonic ΔlvbR or ΔlqsR mutant strains revealed concerted (virulence, fitness island, metabolism) and reciprocally (motility) regulated genes in biofilm and broth respectively. Moreover, ΔlvbR is hyper-competent for DNA uptake, defective for phagocyte infection, outcompeted by the parental strain in amoebae co-infections and impaired for cell migration inhibition. Taken together, our results indicate that L. pneumophila LvbR is a novel pleiotropic transcription factor, which links the Lqs and c-di-GMP regulatory networks to control biofilm architecture and pathogen-host cell interactions.


Legionella Effector SetA as a General O-glucosyltransferase for Eukaryotic Proteins

Gao L, Song Q, Liang H, Zhu Y, Wei T, Dong N, Xiao J, Shao F, Lai L, Chen X.

College of Chemistry and Molecular Engineering, Peking University, Beijing, China.

Nat Chem Biol 2019 Mar;15(3):213-216.

Abstract: The identification of host protein substrates is key to understanding effector glycosyltransferases secreted by pathogenic bacteria and to using them for glycoprotein engineering. Here we report a chemical method for tagging, enrichment, and site-specific proteomic profiling of effector-modified proteins in host cells. Using this method, we discover that Legionella effector SetA α-O-glucosylates various eukaryotic proteins by recognizing a S/T-X-L-P/G sequence motif, which can be exploited to site-specifically introduce O-glucose on recombinant proteins.


Induction of Protective Immunity by Recombinant Peptidoglycan Associated Lipoprotein (rPAL) Protein of Legionella pneumophila in a BALB/c Mouse Model

Mobarez AM, Rajabi RA, Salmanian AH, Khoramabadi N, Hosseini Doust SR.

Department of Bacteriology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran.

Microb Pathog 2019 Mar;128:100-105.

Abstract: Legionella pneumophila causes a severe form of pneumonia known as Legionnaires' disease especially in patients with impaired cellular immune response. In order to prevent the disease, immunogenicity and the level of the induction of protective immunity from the recombinant peptidoglycan-associated lipoprotein (rPAL) against Legionella pneumophila in BALB/c mice was examined. Mice immunized with (rPAL) rapidly increased an antibody response in serum and also displayed a strong activation of both innate and adaptive cell-mediated immunity as determined by antigen-specific splenocyte proliferation, an early production of pro-inflammatory cytokines in the serum and in the splenocyte cultures. Infection with a primary sublethal does of Legionella pneumophila serogroup 1, strain paris, caused resistance to a lethal challenge infection in the animals with 100% survival rate. However, mice treated with rPAL survived with 60% rate in 10 days after a lethal i.v challenge with L. pneumophila. All of the control animals receiving PBS died within 24 h. The present study indicates that recombinant protein PAL of Legionella pneumophila is strongly immunogenic and capable to elicit early innate and adaptive immune responses and lasting immunity against a lethal dose of Legionella pneumophila challenge. Antigenic characterization and immune protection of recombinant protein PAL would be of considerable value in comprehension the immune pathogenesis of the disease and in development possible vaccine against the Legionella.


More Than 18,000 Effectors in the Legionella Genus Genome Provide Multiple, Independent Combinations for Replication in Human Cells

Gomez-Valero L, Rusniok C, Carson D, Mondino S, Pérez-Cobas AE, Rolando M, Pasricha S, Reuter S, Demirtas J, Crumbach J, Descorps-Declere S, Hartland EL, Jarraud S, Dougan G, Schroeder GN, Frankel G, Buchrieser C.

Institut Pasteur, Biologie des Bactéries Intracellulaires, 75724 Paris, France. e

Proc Natl Acad Sci USA 2019 Feb;116(6):2265-2273.

Abstract: The genus Legionella comprises 65 species, among which Legionella pneumophila is a human pathogen causing severe pneumonia. To understand the evolution of an environmental to an accidental human pathogen, we have functionally analyzed 80 Legionella genomes spanning 58 species. Uniquely, an immense repository of 18,000 secreted proteins encoding 137 different eukaryotic-like domains and over 200 eukaryotic-like proteins is paired with a highly conserved type IV secretion system (T4SS). Specifically, we show that eukaryotic Rho- and Rab-GTPase domains are found nearly exclusively in eukaryotes and Legionella Translocation assays for selected Rab-GTPase proteins revealed that they are indeed T4SS secreted substrates. Furthermore, F-box, U-box, and SET domains were present in >70% of all species, suggesting that manipulation of host signal transduction, protein turnover, and chromatin modification pathways are fundamental intracellular replication strategies for legionellae. In contrast, the Sec-7 domain was restricted to L. pneumophila and seven other species, indicating effector repertoire tailoring within different amoebae. Functional screening of 47 species revealed 60% were competent for intracellular replication in THP-1 cells, but interestingly, this phenotype was associated with diverse effector assemblages. These data, combined with evolutionary analysis, indicate that the capacity to infect eukaryotic cells has been acquired independently many times within the genus and that a highly conserved yet versatile T4SS secretes an exceptional number of different proteins shaped by interdomain gene transfer. Furthermore, we revealed the surprising extent to which legionellae have coopted genes and thus cellular functions from their eukaryotic hosts, providing an understanding of how dynamic reshuffling and gene acquisition have led to the emergence of major human pathogens.


The Legionella Effector LtpM Is a New Type of Phosphoinositide-Activated Glucosyltransferase

Levanova N, Mattheis C, Carson D, To KN, Jank T, Frankel G, Aktories K, Schroeder GN.

MRC Centre for Molecular Bacteriology and Infection, Department of Life Sciences, Imperial College London, London, United Kingdom.

J Biol Chem 2019 Feb;294(8):2862-2879.

Abstract: Legionella pneumophila causes Legionnaires' disease, a severe form of pneumonia. L. pneumophila translocates more than 300 effectors into host cells via its Dot/Icm (Defective in organelle trafficking/Intracellular multiplication) type IV secretion system to enable its replication in target cells. Here, we studied the effector LtpM, which is encoded in a recombination hot spot in L. pneumophila Paris. We show that a C-terminal phosphoinositol 3-phosphate (PI3P)-binding domain, also found in otherwise unrelated effectors, targets LtpM to the Legionella-containing vacuole and to early and late endosomes. LtpM expression in yeast caused cytotoxicity. Sequence comparison and structural homology modeling of the N-terminal domain of LtpM uncovered a remote similarity to the glycosyltransferase (GT) toxin PaTox from the bacterium Photorhabdus asymbiotica; however, instead of the canonical DxD motif of GT-A type glycosyltransferases, essential for enzyme activity and divalent cation coordination, we found that a DxN motif is present in LtpM. Using UDP-glucose as sugar donor, we show that purified LtpM nevertheless exhibits glucohydrolase and autoglucosylation activity in vitro and demonstrate that PI3P binding activates LtpM's glucosyltransferase activity toward protein substrates. Substitution of the aspartate or the asparagine in the DxN motif abolished the activity of LtpM. Moreover, whereas all glycosyltransferase toxins and effectors identified so far depend on the presence of divalent cations, LtpM is active in their absence. Proteins containing LtpM-like GT domains are encoded in the genomes of other L. pneumophila isolates and species, suggesting that LtpM is the first member of a novel family of glycosyltransferase effectors employed to subvert hosts.


A Versatile De Novo Synthesis of Legionaminic Acid and 4-epi-legionaminic Acid Starting From D-Serine

Gintner M, Yoneda Y, Schmölzer C, Denner C, Kählig H, Schmid W.

AG Schmid, Institute of Organic Chemistry, University of Vienna, Vienna, Austria.

Carbohydr Res 2019 Feb;474:34-42. 

Abstract: Legionaminic acid and 4-epi-legionaminic acid are 5,7-diacetamido nonulosonic acids and are assumed to play a crucial role in the virulence of Legionella pneumophila, the causative agent of Legionnaires' disease. Moreover, they are ideal target motifs for the development of vaccines and pathogen detection. Herein, we present a versatile de novo synthesis of legionaminic acid and 4-epi-legionaminic acid. Starting from simple d-serine, the C9-backbone is built up by two CC-bond formation reactions. First, the protected d-serine motif is elongated utilizing a highly stereoselective nitroaldol reaction to give a C6-precursor of desired d-rhamno configuration. Second, an indium-mediated allylation is employed to further elongate the carbon backbone and introduce a masked α-keto acid function.


Legionella feeleii: Pneumonia or Pontiac Fever? Bacterial Virulence Traits and Host Immune Response

Wang C, Chuai X, Liang M.

Department of Pathogenic Biology, Hebei Medical University, Shijiazhuang, China.

Med Microbiol Immunol 2019 Feb;208(1):25-32.

Abstract: Gram-negative bacterium Legionella is able to proliferate intracellularly in mammalian host cells and amoeba, which became known in 1976 since they caused a large outbreak of pneumonia. It had been reported that different strains of Legionella pneumophila, Legionella micdadei, Legionella longbeachae, and Legionella feeleii caused human respiratory diseases, which were known as Pontiac fever or Legionnaires' disease. However, the differences of the virulence traits among the strains of the single species and the pathogenesis of the two diseases that were due to the bacterial virulence factors had not been well elucidated. L. feeleii is an important pathogenic organism in Legionellae, which attracted attention due to cause an outbreak of Pontiac fever in 1981 in Canada. In published researches, it has been found that L. feeleii serogroup 2 (ATCC 35849, LfLD) possess mono-polar flagellum, and L. feeleii serogroup 1 (ATCC 35072, WRLf) could secrete some exopolysaccharide (EPS) materials to the surrounding. Although the virulence of the L. feeleii strain was evidenced that could be promoted, the EPS might be dispensable for the bacteria that caused Pontiac fever. Based on the current knowledge, we focused on bacterial infection in human and murine host cells, intracellular growth, cytopathogenicity, stimulatory capacity of cytokines secretion, and pathogenic effects of the EPS of L. feeleii in this review.


PIKfyve/Fab1 Is Required for Efficient V-ATPase and Hydrolase Delivery to Phagosomes, Phagosomal Killing, and Restriction of Legionella Infection

Buckley CM, Heath VL, Guého A, Bosmani C, Knobloch P, Sikakana P, Personnic N, Dove SK, Michell RH, Meier R, Hilbi H, Soldati T, Insall RH, King JS.

Centre for Membrane Interactions and Dynamics, Department of Biomedical Sciences, University of Sheffield, Firth Court, Western Bank, Sheffield, United Kingdom.

PLoS Pathog 2019 Feb;15(2):e1007551.

Abstract: By engulfing potentially harmful microbes, professional phagocytes are continually at risk from intracellular pathogens. To avoid becoming infected, the host must kill pathogens in the phagosome before they can escape or establish a survival niche. Here, we analyse the role of the phosphoinositide (PI) 5-kinase PIKfyve in phagosome maturation and killing, using the amoeba and model phagocyte Dictyostelium discoideum. PIKfyve plays important but poorly understood roles in vesicular trafficking by catalysing formation of the lipids phosphatidylinositol (3,5)-bisphosphate (PI(3,5)2) and phosphatidylinositol-5-phosphate (PI(5)P). Here we show that its activity is essential during early phagosome maturation in Dictyostelium. Disruption of PIKfyve inhibited delivery of both the vacuolar V-ATPase and proteases, dramatically reducing the ability of cells to acidify newly formed phagosomes and digest their contents. Consequently, PIKfyve- cells were unable to generate an effective antimicrobial environment and efficiently kill captured bacteria. Moreover, we demonstrate that cells lacking PIKfyve are more susceptible to infection by the intracellular pathogen Legionella pneumophila. We conclude that PIKfyve-catalysed phosphoinositide production plays a crucial and general role in ensuring early phagosomal maturation, protecting host cells from diverse pathogenic microbes.


Legionella pneumophila Translocated Translation Inhibitors Are Required for Bacterial-Induced Host Cell Cycle Arrest

Sol A, Lipo E, de Jesús-Díaz DA, Murphy C, Devereux M, Isberg RR.

Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, MA, USA.

Proc Natl Acad Sci USA 2019 Feb;116(8):3221-3228.

Abstract: The cell cycle machinery controls diverse cellular pathways and is tightly regulated. Misregulation of cell division plays a central role in the pathogenesis of many disease processes. Various microbial pathogens interfere with the cell cycle machinery to promote host cell colonization. Although cell cycle modulation is a common theme among pathogens, the role this interference plays in promoting diseases is unclear. Previously, we demonstrated that the G1 and G2/M phases of the host cell cycle are permissive for Legionella pneumophila replication, whereas S phase provides a toxic environment for bacterial replication. In this study, we show that L. pneumophila avoids host S phase by blocking host DNA synthesis and preventing cell cycle progression into S phase. Cell cycle arrest upon Legionella contact is dependent on the Icm/Dot secretion system. In particular, we found that cell cycle arrest is dependent on the intact enzymatic activity of translocated substrates that inhibits host translation. Moreover, we show that, early in infection, the presence of these translation inhibitors is crucial to induce the degradation of the master regulator cyclin D1. Our results demonstrate that the bacterial effectors that inhibit translation are associated with preventing entry of host cells into a phase associated with restriction of L. pneumophila Furthermore, control of cyclin D1 may be a common strategy used by intracellular pathogens to manipulate the host cell cycle and promote bacterial replication.


The Structure of Legionella Effector Protein LpnE Provides Insights into Its Interaction with Oculocerebrorenal Syndrome of Lowe (OCRL) Protein

Voth KA, Chung IYW, van Straaten K, Li L, Boniecki MT, Cygler M.

Department of Biochemistry, University of Saskatchewan, Saskatoon, Canada.

FEBS J 2019 Feb;286(4):710-725.

Abstract: Legionella pneumophila is a freshwater bacterium that replicates in predatory amoeba and alveolar macrophage. The ability of L. pneumophila to thrive in eukaryotic host cells is conferred by the Legionella containing vacuole (LCV). Formation and intracellular trafficking of the LCV are governed by an arsenal of effector proteins, many of which are secreted by the Icm/Dot Type 4 Secretion System. One such effector, known as LpnE (L. pneumophila Entry), has been implicated in facilitating bacterial entry into host cells, LCV trafficking, and substrate translocation. LpnE belongs to a subfamily of tetratricopeptide repeat proteins known as Sel1-like repeats (SLRs). All eight of the predicted SLRs in LpnE are required to promote host cell invasion. Herein, we report that LpnE (1-375) localizes to cis-Golgi in HEK293 cells via its signal peptide (aa 1-22). We further verify the interaction of LpnE (73-375) and LpnE (22-375) with Oculocerebrorenal syndrome of Lowe protein (OCRL) residues 10-208, restricting the known interacting residues for both proteins. To further characterize the SLR region of LpnE, we solved the crystal structure of LpnE (73-375) to 1.75Å resolution. This construct comprises all SLRs, which are arranged in a superhelical fold. The α-helices forming the inner concave surface of the LpnE superhelix suggest a potential protein-protein interaction interface. DATABASE: Coordinates and structure factors were deposited in the Protein Data Bank with the accession number 6DEH.


Whole-Genome Sequencing of Legionella jordanis Strains NML 060502 and NML 130005, Recovered from a Lower Respiratory Tract Infection and Water, Respectively

Bernier AM, Bernard K.

National Microbiology Laboratory (NML)-CSCHAH Site, Public Health Agency of Canada, Winnipeg, Canada.

Microbiol Resour Announc 2019 Jan;8(4): e01537-18.

Abstract: Draft genome sequences of two strains of the rarely isolated organism Legionella jordanis, NML 060502 (from a patient with a lower respiratory infection) and NML 130005 (from water), were assembled and studied. Respectively, the genome sizes obtained were 2,927,328 bp and 3,101,130 bp, with G+C contents of 41.9% and 41.7%.


Draft Genome Sequences of Legionella taurinensis Recovered from a Hot Water System in Austria, 2018

Chakeri A, Allerberger F, Kundi M, Stöger A, Rehak S, Ruppitsch W, Schmid D.

Institute of Medical Microbiology and Hygiene, Austrian Agency for Health and Food Safety, Vienna, Austria.

Microbiol Resour Announc 2019 Jan;8(3):e01478-18.

Abstract: Members of the genus Legionella are widespread in natural water sources. This is the first report on the draft genome sequences of Legionella taurinensis in Austria. The isolates were recovered from a hot water system associated with a case of Legionnaires' disease caused by Legionella pneumophila in 2018.


Using an Optimal Set of Features with a Machine Learning-Based Approach to Predict Effector Proteins for Legionella pneumophila

Esna Ashari Z, Brayton KA, Broschat SL.

School of Electrical Engineering and Computer Science, Washington State University, Pullman, Washington, USA.

PLoS One 2019 Jan;14(1):e0202312.

Abstract: Type IV secretion systems exist in a number of bacterial pathogens and are used to secrete effector proteins directly into host cells in order to change their environment making the environment hospitable for the bacteria. In recent years, several machine learning algorithms have been developed to predict effector proteins, potentially facilitating experimental verification. However, inconsistencies exist between their results. Previously we analysed the disparate sets of predictive features used in these algorithms to determine an optimal set of 370 features for effector prediction. This study focuses on the best way to use these optimal features by designing three machine learning classifiers, comparing our results with those of others, and obtaining de novo results. We chose the pathogen Legionella pneumophila strain Philadelphia-1, a cause of Legionnaires' disease, because it has many validated effector proteins and others have developed machine learning prediction tools for it. While all of our models give good results indicating that our optimal features are quite robust, Model 1, which uses all 370 features with a support vector machine, has slightly better accuracy. Moreover, Model 1 predicted 472 effector proteins that are deemed highly probable to be effectors and include 94% of known effectors. Although the results of our three models agree well with those of other researchers, their models only predicted 126 and 311 candidate effectors.


Legionella pneumophila Infection-Mediated Regulation of RICTOR via miR-218 in U937 Macrophage Cells

Koriyama T, Yamakuchi M, Takenouchi K, Oyama Y, Takenaka H, Nagakura T, Masamoto I, Hashiguchi T.

Department of Laboratory and Vascular Medicine, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan.

Biochem Biophys Res Commun 2019 Jan;508(2):608-613.

Abstract: Background: Inhalation of aerosolized Legionella pneumophila, a Gram-negative bacterium, can cause severe pneumonia. During infection, L. pneumophila replicates intracellularly in macrophages. The involvement of host microRNAs (miRNAs) in L. pneumophila infection is not fully understood. Methods: The human macrophage-like cell line U937 was infected with L. pneumophila. The levels of miRNA and messenger RNA (mRNA) were measured using reverse transcriptase polymerase chain reaction. Release of lactate dehydrogenase was used to evaluate cytotoxicity. The expression of RICTOR and related proteins was examined by western blotting of cell lysates. Results: L. pneumophila infection upregulated the expression of miR-218 and the host genes SLIT2 and SLIT3 in U937 cells. The expression of RICTOR, a component of the mechanistic target of rapamycin complex 2 (mTORC2), decreased during L. pneumophila infection. RICTOR protein expression was inhibited by the overexpression of miR-218, whereas knockdown of miR-218 restored the downregulation of RICTOR by L. pneumophila. L. pneumophila infection induced the expression of the proinflammatory cytokines IL-6 and TNF-alpha, which was modulated by knockdown of miR-218 or RICTOR. Conclusions: Our study revealed the involvement of miR-218 in regulating the inflammatory response of macrophages against L. pneumophila infection. These findings suggest potential novel roles for miR-218 and RICTOR as therapeutic targets of L. pneumophila infection.


Legionella pneumophila Inhibits Immune Signalling via MavC-mediated Transglutaminase-Induced Ubiquitination of UBE2N

Gan N, Nakayasu ES, Hollenbeck PJ, Luo ZQ.

Purdue Institute for Inflammation, Immunology and Infectious Disease and Department of Biological Sciences, Purdue University, West Lafayette, IN, USA.

Nat Microbiol 2019 Jan;4(1):134-143.

Abstract: The bacterial pathogen Legionella pneumophila modulates host immunity using effectors translocated by its Dot/Icm transporter to facilitate its intracellular replication. A number of these effectors employ diverse mechanisms to interfere with protein ubiquitination, a post-translational modification essential for immunity. Here, we have found that L. pneumophila induces monoubiquitination of the E2 enzyme UBE2N by its Dot/Icm substrate MavC (Lpg2147). We demonstrate that MavC is a transglutaminase that catalyses covalent linkage of ubiquitin to Lys92 and Lys94 of UBE2N via Gln40. Similar to canonical transglutaminases, MavC possess deamidase activity that targets ubiquitin at Gln40. We identified Cys74 as the catalytic residue for both ubiquitination and deamidation activities. Furthermore, ubiquitination of UBE2N by MavC abolishes its activity in the formation of K63-type polyubiquitin chains, which dampens NF-κB signalling in the initial phase of bacterial infection. Our results reveal an unprecedented mechanism of modulating host immunity by modifying a key ubiquitination enzyme by ubiquitin transglutamination.


Evasion of Phagotrophic Predation by Protist Hosts and Innate Immunity of Metazoan Hosts by Legionella pneumophila

Best AM, Abu Kwaik Y.

Center for Predictive Medicine, College of Medicine, University of Louisville, Louisville, Kentucky.

Cell Microbiol 2019 Jan;21(1):e12971.

Abstract: Legionella pneumophila is a ubiquitous environmental bacterium that has evolved to infect and proliferate within amoebae and other protists. It is thought that accidental inhalation of contaminated water particles by humans is what has enabled this pathogen to proliferate within alveolar macrophages and cause pneumonia. However, the highly evolved macrophages are equipped with more sophisticated innate defence mechanisms than are protists, such as the evolution of phagotrophic feeding into phagocytosis with more evolved innate defence processes. Not surprisingly, the majority of proteins involved in phagosome biogenesis (~80%) have origins in the phagotrophy stage of evolution. There is a plethora of highly evolved cellular and innate metazoan processes, not represented in protist biology, that are modulated by L. pneumophila, including TLR2 signalling, NF-κB, apoptotic and inflammatory processes, histone modification, caspases, and the NLRC-Naip5 inflammasomes. Importantly, L. pneumophila infects haemocytes of the invertebrate Galleria mellonella, kill G. mellonella larvae, and proliferate in and kill Drosophila adult flies and Caenorhabditis elegans. Although coevolution with protist hosts has provided a substantial blueprint for L. pneumophila to infect macrophages, we discuss the further evolutionary aspects of coevolution of L. pneumophila and its adaptation to modulate various highly evolved innate metazoan processes prior to becoming a human pathogen.

Exploiting the Richness of Environmental Waterborne Bacterial Species to Find Natural Legionella pneumophila Competitors

Corre MH, Delafont V, Legrand A, Berjeaud JM, Verdon J.

Laboratoire Ecologie et Biologie des Interactions, UMR CNRS 7267, Université de Poitiers, Poitiers, France.

Front Microbiol 2019 Jan;9:3360.

Abstract: Legionella pneumophila is one of the most tracked waterborne pathogens and remains an important threat to human health. Despite the use of biocides, L. pneumophila is able to persist in engineered water systems with the help of multispecies biofilms and phagocytic protists. For few years now, high-throughput sequencing methods have enabled a better understanding of microbial communities in freshwater environments. Those unexplored and complex communities compete for nutrients using antagonistic molecules as war weapons. Up to now, few of these molecules were characterized in regards of L. pneumophila sensitivity. In this context, we established, from five freshwater environments, a vast collection of culturable bacteria and investigated their ability to inhibit the growth of L. pneumophila. All bacterial isolates were classified within 4 phyla, namely Proteobacteria (179/273), Bacteroidetes (48/273), Firmicutes (43/273), and Actinobacteria (3/273) according to 16S rRNA coding sequences. AeromonasBacillusFlavobacterium, and Pseudomonas were the most abundant genera (154/273). Among the 273 isolates, 178 (65.2%) were shown to be active against L. pneumophila including 137 isolates of the four previously cited main genera.

Additionally, other less represented genera depicted anti-Legionella activity such as AcinetobacterKluyveraRahnella, or Sphingobacterium.

Furthermore, various inhibition diameters were observed among active isolates, ranging from 0.4 to 9 cm. Such variability suggests the presence of numerous and diverse natural compounds in the microenvironment of L. pneumophila. These molecules include both diffusible secreted compounds and volatile organic compounds, the latter being mainly produced by Pseudomonas strains. Altogether, this work sheds light on unexplored freshwater bacterial communities that could be relevant for the biological control of L. pneumophila in manmade water systems.


Phylogenetic Characterization of Viable But-Not-Yet Cultured Legionella Groups Grown in Amoebic Cocultures: A Case Study Using Various Cooling Tower Water Samples

Inoue H, Agata K, Ohta H.

Tsukuba Research Laboratories, Aquas Corporation.

Biocontrol Sci 2019;24(1):39-45.

Abstract: Legionella spp. exist naturally in association with amoeba in water environments and are known to be the etiological agent of a severe form of pneumonia. To detect diverse Legionella populations in cooling tower water systems, amoebic coculturing was performed for 15 water samples obtained from five different kinds of facilities in six geographically different locations. The growth of Legionella in coculture with Acanthamoeba sp. cells was monitored by quantitative PCR targeting Legionella-specific 16S rRNA genes. Seven out of the 15 samples were positive for Legionella growth and subjected to clone library analysis. A total of 333 clones were classified into 14 operational taxonomic units composed of seven known species and seven previously undescribed groups. Four of the seven Legionella-growth-positive samples harbored detectable levels of free-living amoeba and were predominated by either L. drozanskii or L. lytica, by both L. bozemanii and L. longbeachae, or by a not-yet-described group named OTU 4. The Legionella-growth-positive samples contained higher ATP levels (>980 pM) than the growth-negative samples (<160 pM), suggesting that ATP content would be a good indicator of the presence of viable but nonculturable Legionella populations able to grow with amoeba.


Sorting of Phagocytic Cells Infected with Legionella pneumophila

Rolando MBuchrieser C.

CNRS UMR 3525, Paris, France.

Methods Mol Biol 2019;1921:179-189.

Abstract: The ability of Legionella pneumophila to colonize host cells and to form a replicative vacuole depends on its ability to counteract the host cell response by secreting more than 300 effectors. The host cell responds to this bacterial invasion with extensive intracellular signaling to counteract the replication of the pathogen. When studying L. pneumophila infection in vitro, only a small proportion of the cell lines or primary cells used to analyze the host response are infected; the study of such a mixed cell population leads to unprecise results. In order to study the multitude of pathogen-induced phenotypic changes occurring in the host cell, the separation of infected from uninfected cells is a top priority. Here we describe a highly efficient FACS-derived protocol to separate cells infected with a L. pneumophila strain encoding a fluorescent protein. Indeed, the highly infected, homogenous cell population obtained after sorting is the best possible starting point for the studies of infection-induced effects.


Quantitative Imaging Flow Cytometry of Legionella-Containing Vacuoles in Dually Fluorescence-Labeled Dictyostelium

Welin A, Weber S, Hilbi H.

Institute of Medical Microbiology, University of Zürich, Zürich, Switzerland.

Methods Mol Biol 2019;1921:161-177.

Abstract: Legionella pneumophila enters and replicates within protozoan and mammalian phagocytes by forming through a conserved mechanism a specialized intracellular compartment termed the Legionella-containing vacuole (LCV). This compartment avoids fusion with bactericidal lysosomes but communicates extensively with different cellular vesicle trafficking pathways and ultimately interacts closely with the endoplasmic reticulum. In order to delineate the process of pathogen vacuole formation and to better understand L. pneumophila virulence, an analysis of markers of the different trafficking pathways on the pathogen vacuole is crucial. Here, we describe a method for rapid, objective and quantitative analysis of different fluorescently tagged proteins or probes on the LCV. To this end, we employ an imaging flow cytometry approach and use the D. discoideum-L. pneumophila infection model. Imaging flow cytometry enables quantification of many different parameters by fluorescence microscopy of cells in flow, rapidly producing statistically robust data from thousands of cells. We also describe the generation of D. discoideum strains simultaneously producing two different fluorescently tagged probes that enable visualization of compartments and processes in parallel. The quantitative imaging flow technique can be corroborated and enhanced by laser scanning confocal microscopy.


The Pathometabolism of Legionella Studied by Isotopologue Profiling

Heuner K, Kunze M, Chen F, Eisenreich W.

Lehrstuhl für Biochemie, Technische Universität München, Garching, Germany.

Methods Mol Biol 2019;1921:21-44.

Abstract: Metabolic pathways and fluxes can be analyzed under in vivo conditions by incorporation experiments using general 13C-labelled precursors. On the basis of the isotopologue compositions in amino acids or other metabolites, the incorporation rates of the supplied precursors and the pathways of their utilization can be studied in considerable detail. In this chapter, the method of isotopologue profiling is illustrated with recent work on the metabolism of intracellular living Legionella pneumophila.


Selection of Legionella Virulence-Related Traits by Environmental Protozoa

Amaro F, Shuman H.

Department of Microbiology, University of Chicago, Chicago, IL, USA.

Methods Mol Biol 2019;1921:55-78.

Abstract: Predation by protozoa provides a strong selective pressure for Legionella to develop and maintain mechanisms conferring resistance to digestion and ability to replicate within both amoebae and mammalian macrophages. Here we describe how to isolate environmental protozoa that prey on virulent Legionella. These protists are extremely useful models to study the cellular mechanisms employed by Legionellae to survive and grow in its natural environment. We present here procedures that are available to study the interactions between environmental protozoa and Legionella and thus increase our current understanding of Legionella virulence and the infection process.


Infection of Human Lung Tissue Explants (HLTEs) with Legionella pneumophila

Scheithauer L, Steinert M.

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

Methods Mol Biol 2019;1921:323-331.

Abstract: Legionnaires' disease is a severe pneumonia caused by inhalation of Legionella pneumophila. Although powerful infection models ranging from monocellular host systems to mammals were developed, numerous intra- and extracellular interactions of L. pneumophila factors with human lung tissue structures remain unknown. Therefore, we developed and applied a novel infection model for Legionnaires' disease comprising living human lung tissue explants (HLTEs). This model allows analyzing Legionella infections at a unique level of complexity and narrows the gap between current infection models and postmortem histopathology analyses of infected patients. Here we describe the infection of tumor-free pulmonary tissue samples from patients undergoing lobe- or pneumectomy because of lung cancer. The method comprises bacterial cultivation, preparation of HLTEs, and infection of HLTEs. The infected tissue samples allow to characterize tissue damage, bacterial localization, dissemination and growth kinetics, and the host's molecular response.


Genetic Manipulation of Non-pneumophila Legionella: Protocols Developed for Legionella longbeachae

Korevaar E, Khoo CA, Newton HJ.

Department of Microbiology and Immunology, University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne, VIC, Australia.

Methods Mol Biol 2019;1921:145-157.

Abstract: Current biomedical research into Legionnaires' disease is dominated by studies of Legionella pneumophila, largely because this pathogen is responsible for approximately 90% of clinical disease worldwide. However, in certain geographical regions, infections with non-pneumophila species are responsible for a significant proportion of diagnosed Legionnaires' disease. Understanding the pathogenesis of these non-pneumophila species of Legionella is an important step toward clinical intervention. The capacity to genetically manipulate these pathogens is essential in order to understand the genetic factors that contribute to infection and the environmental life cycle of these bacteria. The capacity to delete, mutate, and relocate genetic regions of interest allows molecular research into gene function and importance. In this chapter, methods are outlined to introduce plasmids into Legionella by electroporation. This technique is particularly useful as it is often the essential preliminary step to experiments that observe the behavior of the bacterium under altered conditions, for example, the transformation of bacteria with reporter plasmids to monitor Dot/Icm effector translocation. Electroporation is a well-established method for transformation of competent bacteria, and here specific protocols are provided, suiting a range of materials and conditions that have been successfully applied to L. longbeachae and L. dumoffii. Additionally, a homologous recombination approach to delete genetic regions of interest in L. longbeachae is outlined. The application of these techniques allows for identification of the genetic determinants of non-pneumophila Legionella virulence and for important comparative studies with other Legionella species.


Subcellular Protein Fractionation in Legionella pneumophila and Preparation of the Derived Sub-proteomes for Analysis by Mass Spectrometry

Maaß S, Moog G, Becher D.

Institute of Microbiology, Center for Functional Genomics of Microbes, University of Greifswald, Greifswald, Germany.

Methods Mol Biol 2019;1921:445-464.

Abstract: Classical proteomic techniques are perfectly suited to reflect changes in the metabolism by detection of changed protein synthesis rates and protein abundances in a global protein-centered analysis. Although the proteome of microbes is considered as rather low complex, usually the subcellular fractionation of proteins leads to higher proteome coverage which might be important for the proteome quantification. Additionally, such fractionation provides the possibility to detect changes in the protein localization as well as the protein abundance in single sub-proteomes. Here, a workflow for subcellular fractionation of Legionella pneumophila into cytosolic, periplasmic, membrane, and extracellular proteins for global proteome analyses is provided. The methods included in this workflow can be used to analyze the adaptation of L. pneumophila to different environmental and nutritional situations during infection or during different life cycle stages including planktonic or biofilm phase.


Analysis of the Pulmonary Microbiome Composition of Legionella pneumophila-Infected Patients

Pérez-Cobas AE, Buchrieser C.

Institut Pasteur, Biologie des Bactéries Intracellulaires, Paris, France.

Methods Mol Biol 2019;1921:429-443.

Abstract: The analysis of the lung microbiome composition is a field of research that recently emerged. It gained great interest in pulmonary diseases such as pneumonia since the microbiome seems to be involved in host immune responses, inflammation, and protection against pathogens. Thus, it is possible that the microbial communities living in the lungs play a role in the outcome and severity of lung infections such as Legionella-caused pneumonia and in the response to antibiotic therapy. In this chapter, all steps necessary for the characterization of the bacterial and fungal fraction of the lung microbiome using high-throughput sequencing approaches are explained, starting from the selection of clinical samples to the analysis of the taxonomic composition, diversity, and ecology of the microbiome.


The Mouse as a Model for Pulmonary Legionella Infection

Ng GZ, Solomatina A, van Driel IR, Hartland EL.

Centre for Innate Immunity and Infectious Diseases, Hudson Institute of Medical Research, Clayton, VIC, Australia.

Methods Mol Biol 2019;1921:399-417.

Abstract: Infection of C57BL/6 mice with wild-type Legionella pneumophila typically results in very mild disease. However, in mice where the cytosolic recognition of flagellin is impaired by mutation, L. pneumophila infection results in more severe lung inflammation that is reminiscent of Legionnaires' disease. This can be replicated in wild-type mice by using aflagellated mutants of L. pneumophila. These models greatly facilitate the investigation of L. pneumophila virulence factors and the complex pulmonary immune system that is triggered by infection. Here we describe methods for infecting C57BL/6 mice with aflagellated L. pneumophila, the quantification of bacterial load in the lungs and isolation and analysis of invading immune cells. These assays enable the identification of phagocyte subsets and can determine whether phagocytic cells act as a replicative niche for L. pneumophila replication.


The Caenorhabditis Elegans Model of Legionella Infection

Brassinga AKC, Sifri CD.

Department of Microbiology, University of Manitoba, Winnipeg, MB, Canada.

Methods Mol Biol 2019;1921:371-397.

Abstract: Caenorhabditis elegans can serve as a simple genetic host to study interactions between Legionellaceae and their hosts and to examine the contribution of specific gene products to virulence and immunity. C. elegans nematodes have several appealing attributes as a host organism; they are inexpensive, have robust genetic analysis tools, have a simple anatomy yet display a wide range of complex behaviors, and, as invertebrates, do not require animal ethics protocols. Use of C. elegans as a host model complements cell-based models, providing additional support and consistency of the experimental data obtained from multiple models. The C. elegans innate immune system functions similarly to that of the alveolar macrophage including the apoptosis [a.k.a. programmed cell death (PCD)] pathway located within the germline. The digestive tract of C. elegans is a primary interface between the innate immune system and bacterial pathogens. Thus, the C. elegans host model provides an alternative approach to investigate L. pneumophila immunopathogenesis, particularly in the view of the recent discovery of Legionella-containing vacuoles within the gonadal tissues of Legionella-colonized nematodes supporting the plausible evolutionary origin of the strategies employed by L. pneumophila to counteract macrophage cellular responses.


The Galleria Mellonella Infection Model for Investigating the Molecular Mechanisms of Legionella Virulence

Frankel G, Schroeder GN.

Wellcome-Wolfson Institute for Experimental Medicine, Queen's University Belfast, Belfast, UK.

Methods Mol Biol 2019;1921:333-346.

Abstract: Legionella species evolved virulence factors to exploit protozoa as replicative niches in the environment. Cell culture infection models demonstrated that many of these factors also enable the bacteria to thrive in human macrophages; however, these models do not recapitulate the complex interactions between macrophages, lung epithelial, and additional immune cells, which are crucial to control bacterial infections. Thus, suitable infection models are required to understand which bacterial factors are important to trigger disease. Guinea pigs and, most frequently, mice have been successfully used as mammalian model hosts; however, ethical and economic considerations impede their use in high-throughput screening studies of Legionella isolates or small molecule inhibitors. Here, we describe the larvae of the lepidopteran Galleria mellonella as insect model of Legionella pathogenesis. Larvae can be obtained from commercial suppliers in large numbers, maintained without the need of specialized equipment, and infected by injection. Although lacking the complexity of a mammalian immune system, the larvae mount humoral and cellular immune responses to infection. L. pneumophila strain 130b and other prototype isolates withstand these responses and use the Defective in organelle trafficking/Intracellular multiplication (Dot/Icm) type IV secretion system (T4SS) to inject effectors enabling survival and replication in hemocytes, insect phagocytes, ultimately leading to the death of the larvae. Differences in virulence between L. pneumophila isolates or gene deletion mutants can be analyzed using indicators of larval health and immune induction, such as pigmentation, mobility, histopathology, and survival. Bacterial replication can be measured by plating hemolymph or by immunofluorescence microscopy of isolated circulating hemocytes from infected larvae. Combined, these straightforward experimental readouts make G. mellonella larvae a versatile model host to rapidly assess the virulence of different Legionella isolates and investigate the role of specific virulence factors in overcoming innate host defense mechanisms.


Inflammasome Activation in Legionella-Infected Macrophages

Mascarenhas DPA, Zamboni DS.

Department of Cell Biology, School of Medicine of Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP, Brazil.

Methods Mol Biol 2019;1921:305-319.

Abstract: Legionella pneumophila is a gram-negative bacterium that infects many species of unicellular protozoa in freshwater environments. The human infection is accidental, and the bacteria may not have evolved strategies to bypass innate immune signaling in mammalian macrophages. Thus, L. pneumophila triggers many innate immune pathways including inflammasome activation. The inflammasomes are multimolecular platforms assembled in the host cell cytoplasm and lead to activation of inflammatory caspases. Inflammasome activation leads to secretion of inflammatory cytokines, such as IL-1β and IL-18, and an inflammatory form of cell death called pyroptosis, which initiates with the induction of a pore in the macrophage membranes. In this chapter we provide detailed protocols to evaluate Legionella-induced inflammasome activation in macrophages, including real-time pore formation assay, western blotting to detect activation of inflammatory caspases (cleavage and pulldown), and the measurement of inflammatory cytokines.


Purification and Analysis of Effector Glucosyltransferase Lgt1 From Legionella pneumophila

Levanova N, Tabakova I, Jank T, Belyi Y.

Gamaleya Research Centre, Moscow, Russia.

Methods Mol Biol 2019;1921:277-287. 

Abstract: Legionella pneumophila is a facultative intracellular pathogen responsible for legionellosis, a severe lung disease in humans. This bacterium uses a type 4b secretion system to deliver various effector proteins into the cytoplasm of a eukaryotic target cell. Among those is the glucosyltransferase Lgt1. This effector modifies serine-53 in eukaryotic elongation factor 1A (eEF1A) by mono-O-glucosylation. Modification of eEF1A results in inhibition of protein synthesis and death of the eukaryotic cell, processes which are thought to contribute to Legionella infection. Here we describe a protocol for isolation of the glucosyltransferase Lgt1 from L. pneumophila culture followed by assaying its enzymatic activity using 14C-UDP-glucose autoradiography.


Methods for Noncanonical Ubiquitination and Deubiquitination Catalyzed by Legionella pneumophila Effector Proteins

Qiu J, Luo ZQ.

Department of Biological Sciences, Purdue Institute for Inflammation, Immunology and Infectious Disease, Purdue University, West Lafayette, IN, USA.

Methods Mol Biol 2019;1921:267-276.

Abstract: Protein ubiquitination is one of the most prevalent posttranslational modifications; it regulates a wide range of critical cellular processes in eukaryotes. This modification occurs by covalent attachment of the ubiquitin molecule to other proteins via an isopeptide bond in reactions typically catalyzed by sequential actions of three enzymes, including ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin ligase (E3). Ubiquitination is a reversible process catalyzed by a group of proteins known as deubiquitinase (DUB), which specifically cleaves the isopeptide bond between ubiquitin and modified proteins. Recently, a novel form of ubiquitination catalyzed by the SidE family of effectors from the bacterial pathogen Legionella pneumophila was reported. These proteins ubiquitinate structurally diverse host proteins such as reticulons and ER-associated Rab small GTPases by a two-step mechanism that uses NAD as the energy source for ubiquitin activation prior to being transferred to serine residues in target proteins. This process bypasses the need for E1 and E2 enzymes. Intriguingly, ubiquitination induced by SidEs is regulated by SidJ, another L. pneumophila effector protein which reverses the modification by functioning as an unconventional DUB. Here, we summarize the experimental details of Rab small GTPases (use Rab33b as an example) ubiquitination catalyzed by SidEs (use SdeA as an example) as well as deubiquitination catalyzed by SidJ.


Perturbation of Legionella Cell Infection by RNA Interference

Steiner B, Swart AL, Hilbi H.

Institute of Medical Microbiology, University of Zürich, Zürich, Switzerland.

Methods Mol Biol 2019;1921:221-238.

Abstract: Legionella pneumophila is a facultative intracellular bacterium, which grows in amoebae as well as in macrophages and epithelial cells. Depletion of genes of interest by RNA interference (RNAi) has proven to be a robust and economic technique to study L. pneumophila-host cell interactions. Predesigned and often validated double-stranded (ds) RNA oligonucleotides that silence specific genes are commercially available. RNAi results in a reduced level of distinct proteins, which allows studying the specific role of host cell components involved in L. pneumophila infection. Here, we describe how to assess RNAi-mediated protein depletion efficiency and cytotoxic effects in human A549 lung epithelial cells and murine RAW 264.7 macrophages. Moreover, we demonstrate how RNAi can be used to screen for novel host cell proteins involved in the formation of the Legionella-containing vacuole and intracellular replication of the pathogen.


Screening Targeted Legionella Pneumophila Mutant Libraries In Vivo Using INSeq

Shames SR.

Division of Biology, Kansas State University, Manhattan, KS, USA.

Methods Mol Biol 2019;1921:123-144.

Abstract: Legionella pneumophila is an intracellular bacterial pathogen that can cause a severe inflammatory pneumonia in humans called Legionnaires' disease, which results from bacterial replication within alveolar macrophages. L. pneumophila replication within macrophages is dependent on hundreds of individual protein virulence factors. Understanding how these virulence factors contribute to disease in an animal model is important to reveal aspects of host-pathogen interactions. High-throughput sequencing (HTS)-based screens using transposon (Tn) mutagenesis are powerful approaches to identify bacterial genes important for host-pathogen interactions. Since large libraries of Tn mutants are at risk of bottleneck effects, phenotypic screening of smaller numbers of targeted mutants is an effective alternative. Insertion sequencing (INSeq) is a method that enables production of targeted Tn mutant libraries and has been used successfully to identify L. pneumophila virulence phenotypes. In this chapter, a protocol is described for using INSeq to generate an arrayed L. pneumophila Tn mutant library and for subsequent screening of targeted mutant pools in a mouse model of infection.


Querying Legionella Genomes Using Transposition-Sequencing

Hardy LCharpentier X.

CIRI, Centre International de Recherche en Infectiologie, Team "Horizontal gene transfer in bacterial pathogens", Inserm, U1111, Université Claude Bernard Lyon 1, CNRS, UMR5308, École Normale Supérieure de Lyon, Univ Lyon, Villeurbanne, France.

Methods Mol Biol 2019;1921:107-122. 

Abstract: Transposition-sequencing (Tn-seq) has recently emerged as a powerful technique to query bacterial genomes. Tn-seq can be used to query the bacterial genome with unprecedented resolution, allowing the identification of small genes (e.g., noncoding RNA) that may be missed in conventional screening approaches. Tn-seq can be used to predict genes essential for in vitro growth and to directly identify genetic requirements for survival under multiple conditions. For instance, Tn-seq can be applied to determine the genes, and cellular processes, required to resist an antibacterial treatment or to acquire new resistance genes, to adapt to intracellular life or to compete with other bacteria. Virtually any assay that involves a selection pressure can be used to identify the associated genetic determinants. So far, genome-wide Tn-seq has not been applied to Legionella species. Here, we provide a protocol covering all the different steps to conduct a Tn-seq analysis in L. pneumophila. This includes generating a high-density library of insertional mutants, setting up a selection screen, sequencing the libraries, mapping the insertion sites, and analyzing the data to obtain the list of genes involved in surviving the applied selection.


Scar-Free Genome Editing in Legionella pneumophila

Bailo N, Kanaan H, Kay E, Charpentier X, Doublet P, Gilbert C.

CIRI, International Center for Infectiology Research, CNRS, UMR5308, Inserm, U1111, Université Lyon 1, Université de Lyon, Lyon, France.

Methods Mol Biol 2019;1921:93-105.

Abstract: Studying bacterial physiology and pathogenesis often requires isolation of targeted mutants. From the early days of bacterial genetics, many genetic tools have been developed to achieve this goal in a lot of bacteria species, and a major key is to be able to manipulate the targeted genome region with a minimum impact on the rest of the genome. Here, we described a two-step protocol relevant in Legionella pneumophila. This efficient two-step protocol uses the natural transformability of L. pneumophila and linear DNA fragments as substrates for recombination without the necessity of intermediate hosts to amplify targeted DNA. Based on a suicide cassette strategy, this genetic toolbox enables to generate clean scar-free deletions, single-nucleotide mutation, transcriptional or translational fusions, as well as insertion at any chosen place in L. pneumophila chromosome, therefore enabling multiple mutations with no need of multiple selection markers.


Legionella-Containing Vacuoles Capture PtdIns(4) P-Rich Vesicles Derived from the Golgi Apparatus

Weber S, Steiner B, Welin A, Hilbi H.

Institute of Medical Microbiology, University of Zürich, Zürich, Switzerland.

mBio 2018 Dec;9(6):e02420-18.

Abstract: Legionella pneumophila is the causative agent of a pneumonia termed Legionnaires' disease. The facultative intracellular bacterium employs the Icm/Dot type IV secretion system (T4SS) and a plethora of translocated "effector" proteins to interfere with host vesicle trafficking pathways and establish a replicative niche, the Legionella-containing vacuole (LCV). Internalization of the pathogen and the events immediately ensuing are accompanied by host cell-mediated phosphoinositide (PI) lipid changes and the Icm/Dot-controlled conversion of the LCV from a PtdIns(3)P-positive vacuole into a PtdIns(4)P-positive replication-permissive compartment, which tightly associates with the endoplasmic reticulum. The source and formation of PtdIns(4)P are ill-defined. Using dually labeled Dictyostelium discoideum amoebae and real-time high-resolution confocal laser scanning microscopy (CLSM), we show here that nascent LCVs continuously capture and accumulate PtdIns(4)P-positive vesicles from the host cell. Trafficking of these PtdIns(4)P-positive vesicles to LCVs occurs independently of the Icm/Dot system, but their sustained association requires a functional T4SS. During the infection, PtdIns(3)P-positive membranes become compacted and segregated from the LCV, and PtdIns(3)P-positive vesicles traffic to the LCV but do not fuse. Moreover, using eukaryotic and prokaryotic PtdIns(4)P probes (2×PHFAPP-green fluorescent protein [2×PHFAPP-GFP] and P4CSidC-GFP, respectively) along with Arf1-GFP, we show that PtdIns(4)P-rich membranes of the trans-Golgi network associate with the LCV. Intriguingly, the interaction dynamics of 2×PHFAPP-GFP and P4CSidC-GFP are spatially separable and reveal the specific PtdIns(4)P pool from which the LCV PI originates. These findings provide high-resolution real-time insights into how L. pneumophila exploits the cellular dynamics of membrane-bound PtdIns(4)P for LCV formation. IMPORTANCE: The environmental bacterium Legionella pneumophila causes a life-threatening pneumonia termed Legionnaires' disease. The bacteria grow intracellularly in free-living amoebae as well as in respiratory tract macrophages. To this end, L. pneumophila forms a distinct membrane-bound compartment called the Legionella-containing vacuole (LCV). Phosphoinositide (PI) lipids are crucial regulators of the identity and dynamics of host cell organelles. The PI lipid PtdIns(4)P is a hallmark of the host cell secretory pathway, and decoration of LCVs with this PI is required for pathogen vacuole maturation. The source, dynamics, and mode of accumulation of PtdIns(4)P on LCVs are largely unknown. Using Dictyostelium amoebae producing different fluorescent probes as host cells, we show here that LCVs rapidly acquire PtdIns(4)P through the continuous interaction with PtdIns(4)P-positive host vesicles derived from the Golgi apparatus. Thus, the PI lipid pattern of the secretory pathway contributes to the formation of the replication-permissive pathogen compartment.


Effects of Fragrance Ingredients on the Uptake of Legionella pneumophila into Acanthamoeba castellanii

Nomura H, Takahashi S, Tohara Y, Isshiki Y, Sakuda K, Sakuma K, Kondo S.

Department of Microbiology, School of Pharmaceutical Sciences, Josai University, Saitama, Japan.

Biocontrol Sci 2018;23(4):241-244.

Abstract: Acanthamoeba castellanii, a ubiquitous organism in water environments, is pathogenic toward humans and also is a host for bacteria of the genus Legionella, a causative agent of legionellosis. Fragrance ingredients were investigated for their antibacterial activity against planktonic Legionella pneumophila, amoebicidal activity against A. castellanii, and inhibitory effect against L. pneumophila uptake into A. castellanii. Helional® exhibited relatively high antibacterial activity [minimum inhibitory concentration (MIC), 32.0 μg/mL]. Anis aldehyde, canthoxal, helional® and vanillin exhibited amoebicidal activity (IC50 values, 58.4±2.0, 71.2±14.7, 66.8±8.3 and 49.1±2.5μg/mL, respectively). L. pneumophila pretreatment with sub-MICs (0.25×MIC) of anis aldehyde, canthoxal, cortex aldehyde® 50 percent or vanillin evidently reduced L. pneumophila uptake into A. castellanii (p<0.01). Thus, fragrance ingredients were good candidates for disinfectant against L. pneumophila and A. castellanii.


The Influence of AcanthamoebaLegionella Interaction in the Virulence of Two Different Legionella Species

Gomes TS, Gjiknuri J, Magnet A, Vaccaro L, Ollero D, Izquierdo F, Fenoy S, Hurtado C, Del Águila C.

Facultad de Farmacia, Universidad San Pablo CEU, CEU Universities, Madrid, Spain.

Front Microbiol 2018 Dec;9:2962.

Abstract: The genus Legionella comprises more than 60 species, and about half are associated with infection. Legionella pneumophila is the most commonly associated with these infections and by far the most studied, but L. non-pneumophila species, such as L. feeleiiL. anisa, etc., may also present clinical importance. Free-living amoebae are their preferred environmental host, where these bacteria not only survive but also succeed in multiplying, and this relationship can lead to an increase in bacterial virulence. The goal of this study was to evaluate the alterations of Legionella pathogenicity due to its interaction with Acanthamoeba. For this, the expression of protein effectors SdhA, LegK2, and SidK were evaluated in L. pneumophila and L. feeleii, before and after infecting Acanthamoeba. Additionally, the host response was evaluated by measuring the production of IL-6, IL-8, and IFN-γ in infected macrophages. Regarding the virulence factors, an increase in SdhA expression was observed after these bacteria infected Acanthamoeba, with a higher increase in the macrophage cultures infected with L. feeleii. Also, an increase in the expression of LegK2 was observed after infecting Acanthamoeba, but it was more intense in the cultures infected with L. pneumophila. With regard to SidK, it was increased in L. feeleii after infecting Acanthamoeba, however the same effect was not observed for L. pneumophila. In cytokine production, the effect on IL-6 and IL-8 was similar for both cytokines, increasing their concentration, but higher production was observed in the cultures infected with L. feeleii, even though it demonstrated slightly lower production with the inoculum obtained from Acanthamoeba. Concerning IFN-γ, induction was observed in both species but higher in the infection by L. pneumophila. Nevertheless, it is not known if this induction is enough to promote an efficient immune response against either L. pneumophila or L. feeleii. Altogether, these alterations seem to increase L. feeleii virulence after infecting Acanthamoeba. However, this increase does not seem to turn L. feeleii as virulent as L. pneumophila. More studies are necessary to understand the aspects influenced in these bacteria by their interaction with Acanthamoeba and, thus, identify targets to be used in future therapeutic approaches.


The Single-Domain Response Regulator LerC Functions as a Connector Protein in the Legionella pneumophila Effectors Regulatory Network

Feldheim YS, Zusman T, Kapach A, Segal G.

Department of Molecular Microbiology and Biotechnology, School of Molecular Cell Biology and Biotechnology, George S. Wise Faculty of Life Sciences, Tel-Aviv University, Tel-Aviv, Israel.

Mol Microbiol 2018 Dec;110(5):741-760.

Abstract: The intracellular pathogen Legionella pneumophila translocates more than 300 effector proteins into host cells during infection. The PmrAB two-component system (TCS) has been shown to activate the expression of a large pool of these effector-encoding genes (EEGs) and the LetAS TCS, as part of the LetAS-RsmYZ-CsrA cascade, has been shown to repress the expression of another pool of EEGs. We identified a single-domain response regulator (SDRR), named LerC, which functions as a connector protein between the PmrAB and the LetAS TCSs. The lerC gene is strongly activated by the PmrAB TCS and the LerC protein inhibits the activity of the LetAS TCS. The LerC protein specifically interacts with the HPT (histidine-phosphotransfer) domain of LetS, leading to reduced expression of the small RNAs RsmY and RsmZ, which leads to a reduced expression of the pool of EEGs regulated by the LetAS-RsmYZ-CsrA cascade. In addition, the conserved aspartic acid located in the LerC receiver domain is essential for its phosphorylation and function, suggesting that LerC functions as a phosphate-sink of LetS. Our results demonstrate a new role for SDRRs as connector proteins in regulatory networks, suggesting that members of this widespread group of proteins might function as connector proteins in other bacterial regulatory networks.


The Importance of Revisiting Legionellales Diversity

Duron O, Doublet P, Vavre F, Bouchon D.

Laboratoire Maladies Infectieuses et Vecteurs: Ecologie, Génétique, Evolution et Contrôle (MIVEGEC), Centre National de la Recherche Scientifique (CNRS) - Institut pour la Recherche et le Développement (IRD) - Université de Montpellier (UM), 911 Avenue Agropolis, F-34394 Montpellier, France.

Trends Parasitol 2018 Dec;34(12):1027-1037.

Abstract: Bacteria of the order Legionellales, such as Legionella pneumophila, the agent of Legionnaires' disease, and Coxiella burnetii, the agent of Q fever, are widely recognized as human pathogens. While our view of the Legionellales is often limited to clinical isolates, ecological surveys are continually uncovering new members of the Legionellales that do not fall into the recognized pathogenic species. Here we emphasize that most of these Legionellales are nonpathogenic forms that have evolved symbiotic lifestyles with nonvertebrate hosts. The diversity of nonpathogenic forms remains, however, largely underexplored. We conjecture that its characterization, once contrasted with the data on pathogenic species, will reveal novel highlights on the mechanisms underlying lifestyle transitions of intracellular bacteria, including the emergence of pathogenesis and mutualism, transmission routes, and host specificity.


Comparison of in Situ Sequence Type Analysis of Legionella pneumophila in Respiratory Tract Secretions and Environmental Samples of a Hospital in East Jerusalem

Jaber L, Amro M, Tair HA, Bahader SA, Alalam H, Butmeh S, Hilal DA, Brettar I, Höfle MG, Bitar DM.

Department of Microbiology and Immunology, Faculty of Medicine, Al-Quds University, Abu Dies, Palestine.

Epidemiol Infect 2018 Dec;146(16):2116-2121.

Abstract: Legionella pneumophila genotyping is important for epidemiological investigation of nosocomial and community-acquired outbreaks of legionellosis. The prevalence of legionellosis in pneumonia patients in the West Bank was monitored for the first time, and the sequence types (STs) from respiratory samples were compared with STs of environmental samples from different wards of the hospital. Sputum (n=121) and bronchoalveolar lavage (BAL) (n=74) specimens were cultured for L. pneumophila; genomic DNA was tested by 16S rRNA polymerase chain reaction (PCR) amplification. Nested PCR sequence-based typing (NPSBT) was implemented on DNA of the respiratory and environmental PCR-positive samples. Only one respiratory specimen was positive for L. pneumophila by culture. BAL gave a higher percentage of L. pneumophila-positive samples, 35% (26/74) than sputum, 15% (18/121) by PCR. NPSBT revealed the following STs: ST 1 (29%, 7/24), ST 461 (21%, 5/24), ST 1037 (4%, 1/24) from respiratory samples, STs from environmental samples: ST 1 (28.5%, 4/14), ST 187 (21.4%, 3/14) and ST 2070, ST 461, ST 1482 (7.1%, 1/14) each. This study emphasises the advantage of PCR over culture for the detection of L. pneumophila in countries where antibiotics are indiscriminately used prior to hospital admission. ST 1 was the predominant ST in both respiratory and environmental samples.


Identification of MazF Homologue in Legionella pneumophila which Cleaves RNA at the AACU Sequence

Shaku M, Park JH, Inouye M, Yamaguchi Y.

Department of Biology and Geosciences, Graduate School of Science, Osaka City University, Osaka, Japan,

J Mol Microbiol Biotechnol 2018;28(6):269-280.

Abstract: MazF is a sequence-specific endoribonuclease that is widely conserved in bacteria and archaea. Here, we found an MazF homologue (MazF-lp; LPO-p0114) in Legionella pneumophila. The mazF-lp gene overlaps 14 base pairs with the upstream gene mazE-lp (MazE-lp; LPO-p0115). The induction of mazF-lp caused cell growth arrest, while mazE-lp co-induction recovered cell growth in Escherichia coli. In vivo and in vitro primer extension experiments showed that MazF-lp is a sequence-specific endoribonuclease cleaving RNA at AACU. The endoribonuclease activity of purified MazF-lp was inhibited by purified MazE-lp. We found that MazE-lp and the MazEF-lp complex specifically bind to the palindromic sequence present in the 5'-untranslated region of the mazEF-lp operon. MazE-lp and MazEF-lp both likely function as a repressor for the mazEF-lp operon and for other genes, including icmR, whose gene product functions as a secretion chaperone for the IcmQ pore-forming protein, by specifically binding to the palindromic sequence in 5'-UTR of these genes.


Overlapping Roles for Interleukin-36 Cytokines in Protective Host Defense Against Murine Legionella pneumophila Pneumonia

Nanjo Y, Newstead MW, Aoyagi T, Zeng X, Takahashi K, Yu FS, Tateda K, Standiford TJ.

Division of Pulmonary & Critical Care Medicine, Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan, USA.

Infect Immun 2018 Dec;87(1):e00583-18.

Abstract: Legionella pneumophila causes life-threatening pneumonia culminating in acute lung injury. Innate and adaptive cytokines play an important role in host defense against L. pneumophila infection. Interleukin-36 (IL-36) cytokines are recently described members of the larger IL-1 cytokine family known to exert potent inflammatory effects. In this study, we elucidated the role for IL-36 cytokines in experimental pneumonia caused by L. pneumophila Intratracheal (i.t.) administration of L. pneumophila induced the upregulation of both IL-36α and IL-36γ mRNA and protein production in the lung. Compared to the findings for L. pneumophila-infected wild-type (WT) mice, the i.t. administration of L. pneumophila to IL-36 receptor-deficient (IL-36R-/-) mice resulted in increased mortality, a delay in lung bacterial clearance, increased L. pneumophila dissemination to extrapulmonary organs, and impaired glucose homeostasis. Impaired lung bacterial clearance in IL-36R-/- mice was associated with a significantly reduced accumulation of inflammatory cells and the decreased production of proinflammatory cytokines and chemokines. Ex vivo, reduced expression of costimulatory molecules and impaired M1 polarization were observed in alveolar macrophages isolated from infected IL-36R-/- mice compared to macrophages from WT mice. While L. pneumophila-induced mortality in IL-36α- or IL-36γ-deficient mice was not different from that in WT animals, antibody-mediated neutralization of IL-36γ in IL-36α-/- mice resulted in mortality similar to that observed in IL-36R-/- mice, indicating redundant and overlapping roles for these cytokines in experimental murine L. pneumophila pneumonia.


Peptidyl-Prolyl- cistrans-Isomerases Mip and PpiB of Legionella pneumophila

Contribute to Surface Translocation, Growth at Suboptimal Temperature, and Infection

Rasch J, Ünal CM, Klages A, Karsli Ü, Heinsohn N, Brouwer RMHJ, Richter M, Dellmann A, Steinert M.

Institut für Mikrobiologie, Technische Universität Braunschweig, Braunschweig, Germany

Infect Immun 2018 Dec;87(1):e00939-17.

Abstract: The gammaproteobacterium Legionella pneumophila is the causative agent of Legionnaires' disease, an atypical pneumonia that manifests itself with severe lung damage. L. pneumophila, a common inhabitant of freshwater environments, replicates in free-living amoebae and persists in biofilms in natural and man-made water systems. Its environmental versatility is reflected in its ability to survive and grow within a broad temperature range as well as its capability to colonize and infect a wide range of hosts, including protozoa and humans. Peptidyl-prolyl-cis/trans-isomerases (PPIases) are multifunctional proteins that are mainly involved in protein folding and secretion in bacteria. In L. pneumophila the surface associated PPIase Mip was shown to facilitate the establishment of the intracellular infection cycle in its early stages. The cytoplasmic PpiB was shown to promote cold tolerance. Here, we set out to analyze the interrelationship of these two relevant PPIases in the context of environmental fitness and infection. We demonstrate that the PPIases Mip and PpiB are important for surfactant-dependent sliding motility and adaptation to suboptimal temperatures, features that contribute to the environmental fitness of L. pneumophila Furthermore, they contribute to infection of the natural host Acanthamoeba castellanii as well as human macrophages and human explanted lung tissue. These effects were additive in the case of sliding motility or synergistic in the case of temperature tolerance and infection, as assessed by the behavior of the double mutant. Accordingly, we propose that Mip and PpiB are virulence modulators of L. pneumophila with compensatory action and pleiotropic effects.


Discovery, Structural and Biochemical Studies of a Rare Glu/Asp Specific M1 Class Aminopeptidase from Legionella pneumophila

Marapaka AK, Pillalamarri V, Gumpena R, Haque N, Bala SC, Jangam A, Addlagatta A.

Applied Biology, CSIR-Indian Institute of Chemical Technology, Hyderabad, 500 007, Telangana, India; Academy of Scientific and Innovative Research (AcSIR), Rafi Marg, New Delhi 110001,

Int J Biol Macromol 2018 Dec;120(Pt A):1111-1118.

Abstract: Aminopeptidases catalyze the hydrolysis of amino acids from the N-terminus of protein or peptide substrates. M1 family aminopeptidases are important for the pathogenicity of bacteria and play critical role in many physiological processes such as protein maturation, regulation of peptide hormone levels in humans. Most of the M1 family aminopeptidases reported till date display broad substrates specificity, mostly specific to basic and hydrophobic residues. In the current study we report the discovery of a novel M1 class aminopeptidase from Legionella pneumophila (LePepA), which cleaves only acidic residues. Biochemical and structural studies reveal that the S1 pocket is polar and positively charged. Bioinformatic analysis suggests that such active site is unique to only Legionella species and probably evolved for special needs of the microbe. Given its specific activity, LePepA could be useful in specific biotechnological applications.


Identification of Novel Legionella Genes Required for Endosymbiosis in Paramecium Based on Comparative Genome Analysis with Holospora Spp

Watanabe K, Suzuki H, Nishida T, Mishima M, Tachibana M, Fujishima M, Shimizu T, Watarai M.

The United Graduate School of Veterinary Science, Yamaguchi University, 1677-1 Yoshida, Yamaguchi 753-8515, Japan.

FEMS Microbiol Ecol 2018 Nov;94(11): fiy162.

Abstract: The relationship between Legionella and protist hosts has a huge impact when considering the infectious risk in humans because it facilitates the long-term replication and survival of Legionella in the environment. The ciliate Paramecium is considered to be a protist host for Legionella in natural environments, but the details of their endosymbiosis are largely unknown. In this study, we determined candidate Legionella pneumophila genes that are likely to be involved in the establishment of endosymbiosis in Paramecium caudatum by comparing the genomes of Legionella spp. and Holospora spp. that are obligate endosymbiotic bacteria in Paramecium spp. Among the candidate genes, each single deletion mutant for five genes (lpg0492, lpg0522, lpg0523, lpg2141 and lpg2398) failed to establish endosymbiosis in P. caudatum despite showing intracellular growth in human macrophages. The mutants exhibited no characteristic changes in terms of their morphology, multiplication rate or capacity for modulating the phagosomes in which they were contained, but their resistance to lysozyme decreased significantly. This study provides insights into novel factors required by L. pneumophila for endosymbiosis in P. caudatum and suggests that endosymbiotic organisms within conspecific hosts may have shared genes related to effective endosymbiosis establishment.


Legionella Remodels the Plasma Membrane-Derived Vacuole by Utilizing Exocyst Components as Tethers

Arasaki K, Kimura H, Tagaya M, Roy CR.

Department of Microbial Pathogenesis, Yale University School of Medicine, Boyer Center for Molecular Medicine, New Haven, CT, USA.

J Cell Biol 2018 Nov;217(11):3863-3872.

Abstract: During the initial stage of infection, Legionella pneumophila secretes effectors that promote the fusion of endoplasmic reticulum (ER)-derived vesicles with the Legionella-containing vacuole (LCV). This fusion leads to a remodeling of the plasma membrane (PM)-derived LCV into a specialized ER-like compartment that supports bacterial replication. Although the effector DrrA has been shown to activate the small GTPase Rab1, it remains unclear how DrrA promotes the tethering of host vesicles with the LCV. Here, we show that Sec5, Sec15, and perhaps Sec6, which are subunits of the exocyst that functions in the tethering of exocytic vesicles with the PM, are required for DrrA-mediated, ER-derived vesicle recruitment to the PM-derived LCV. These exocyst components were found to interact specifically with a complex containing DrrA, and the loss of Sec5 or Sec15 significantly suppressed the recruitment of ER-derived vesicles to the LCV and inhibited intracellular replication of Legionella Importantly, Sec15 is recruited to the LCV, and Rab1 activation is necessary for this recruitment.


Genomic Heterogeneity Differentiates Clinical and Environmental Subgroups of Legionella pneumophila Sequence Type 1

Mercante JW, Caravas JA, Ishaq MK, Kozak-Muiznieks NA, Raphael BH, Winchell JM.

Pneumonia Response and Surveillance Laboratory, Respiratory Diseases Branch, CDC, Atlanta, GA, USA.

PLoS One 2018 Oct;13(10):e0206110.

Abstract: Legionella spp. are the cause of a severe bacterial pneumonia known as Legionnaires' disease (LD). In some cases, current genetic subtyping methods cannot resolve LD outbreaks caused by common, potentially endemic L. pneumophila (Lp) sequence types (ST), which complicates laboratory investigations and environmental source attribution. In the United States (US), ST1 is the most prevalent clinical and environmental Lp sequence type. In order to characterize the ST1 population, we sequenced 289 outbreak and non-outbreak associated clinical and environmental ST1 and ST1-variant Lp strains from the US and, together with international isolate sequences, explored their genetic and geographic diversity. The ST1 population was highly conserved at the nucleotide level; 98% of core nucleotide positions were invariant and environmental isolates unassociated with human disease (n=99) contained ~65% more nucleotide diversity compared to clinical-sporadic (n=139) or outbreak-associated (n=28) ST1 subgroups. The accessory pangenome of environmental isolates was also ~30-60% larger than other subgroups and was enriched for transposition and conjugative transfer-associated elements. Up to ~10% of US ST1 genetic variation could be explained by geographic origin, but considerable genetic conservation existed among strains isolated from geographically distant states and from different decades. These findings provide new insight into the ST1 population structure and establish a foundation for interpreting genetic relationships among ST1 strains; these data may also inform future analyses for improved outbreak investigations.


NMR Resonance Assignments for a ProQ Homolog from Legionella pneumophila

Immer C, Hacker C, Wöhnert J.

Institute for Molecular Biosciences, Goethe University Frankfurt/M., Frankfurt, Germany.

Biomol NMR Assign 2018 Oct;12(2):319-322.

Abstract: Regulation of gene expression on a post-transcriptional level by small non-coding regulatory RNAs (sRNAs) is very common in bacteria. sRNAs base pair with sequences in their target messenger RNAs (mRNAs) and thereby regulate translation initiation or mRNA stability. Specialized RNA-binding proteins (RBPs) facilitate these regulatory sRNA/mRNA interactions by acting as RNA chaperones. A well-known example for such an RNA chaperone which is widespread in bacteria is the Hfq protein. Recently, the ProQ/FinO protein family was identified as a new class of RNA chaperones involved in sRNA based regulation. Only a few members of this protein family have been structurally characterized so far. In particular, the structural basis for RNA-binding and recognition has not yet been established for this class of proteins. Here, we report the 1H, 13C and 15N NMR resonance assignments for a ProQ homolog (Lpp 1663) from the gram-negative pathogenic bacterium Legionella pneumophila which will facilitate further structural and dynamic studies of this protein and its interaction with RNA targets.


Highlighting the Potency of Biosurfactants Produced by Pseudomonas Strains as Anti- Legionella Agents

Loiseau C, Portier E, Corre MH, Schlusselhuber M, Depayras S, Berjeaud JM, Verdon J.

Laboratoire Ecologie & Biologie des Interactions, Université de Poitiers, France.

Biomed Res Int 2018 Oct;2018:8194368.

Abstract: Legionella pneumophila, the causative agent of Legionnaires' disease, is a waterborne bacterium mainly found in man-made water systems in close association with free-living amoebae and multispecies biofilms. Pseudomonas strains, originating from various environments including freshwater systems or isolated from hospitalized patients, were tested for their antagonistic activity towards L. pneumophila. A high amount of tested strains was thus found to be active. This antibacterial activity was correlated to the presence of tensioactive agents in culture supernatants. As Pseudomonas strains were known to produce biosurfactants, these compounds were specifically extracted and purified from active strains and further characterized using reverse-phase HPLC and mass spectrometry methods. Finally, all biosurfactants tested (lipopeptides and rhamnolipids) were found active and this activity was shown to be higher towards Legionella strains compared to various other bacteria. Therefore, described biosurfactants are potent anti-Legionella agents that could be used in the water treatment industry although tests are needed to evaluate how effective they would be under field conditions.


Potential Role of Gr-1+ CD8+ T Lymphocytes as a Source of Interferon-γ and M1/M2 Polarization During the Acute Phase of Murine Legionella pneumophila Pneumonia

Kusaka Y, Kajiwara C, Shimada S, Ishii Y, Miyazaki Y, Inase N, Standiford TJ, Tateda K.

Department of Microbiology and Infectious Diseases, Toho University School of Medicine, Tokyo, Japan.

J Innate Immun 2018 Sep;10(4):328-338.

Abstract: In this study, we analyzed interferon (IFN)-γ-producing cells and M1/M2 macrophage polarization in Legionella pneumophila pneumonia following anti-Gr-1 antibody treatment. Anti-Gr-1 treatment induced an M1-to-M2 shift of macrophage subtypes in the lungs and weakly in the peripheral blood, which was associated with increased mortality in legionella-infected mice. CD8+ T lymphocytes and natural killer cells were the dominant sources of IFN-γ in the acute phase, and anti-Gr-1 treatment reduced the number of IFN-γ-producing CD8+ T lymphocytes. In the CD3-gated population, most Gr-1-positive cells were CD8+ T lymphocytes in the lungs and lymph nodes (LNs) of infected mice. Additionally, the number of IFN-γ-producing Gr-1+ CD8+ T lymphocytes in the lungs and LNs increased 2 and 4 days after L. pneumophila infection, with anti-Gr-1 treatment attenuating these populations. Antibody staining revealed that Gr-1+ CD8+ T lymphocytes were Ly6C-positive cells rather than Ly6G, a phenotype regarded as memory type cells. Furthermore, the adoptive transfer of Gr-1+ CD8+ T lymphocytes induced increases in IFN-γ, M1 shifting and reduced bacterial number in the Legionella pneumonia model. These data identified Ly6C+ CD8+ T lymphocytes as a source of IFN-γ in innate immunity and partially associated with reduced IFN-γ production, M2 polarization, and high mortality in anti-Gr-1 antibody-treated mice with L. pneumophila pneumonia.


Differential Development of Legionella Sub-Populations During Short- And Long-Term Starvation

Schrammel B, Cervero-Aragó S, Dietersdorfer E, Walochnik J, Lück C, Sommer R, Kirschner A.

Institute for Hygiene and Applied Immunology, Water Hygiene, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Austria.

Water Res 2018 Sep;141:417-427.

Abstract: Legionellae are among the most important waterborne pathogens in industrialized countries. Monitoring and surveillance of Legionella in engineered water systems is usually performed with culture-based methods. Since the advent of culture-independent techniques, it has become clear that Legionella concentrations are often several orders of magnitude higher than those measured by culture-based techniques and that a variable proportion of these non-culturable cells are viable. In engineered water systems, the formation of these viable but non-culturable (VBNC) cells can be caused by different kinds of stress, such as, and most importantly, nutrient starvation, oxidative stress and heat. In this study, the formation of VBNC cells of six Legionella strains under conditions of starvation was monitored in mono-species microcosms for up to one year using a combination of different viability indicators. Depending on the strain, complete loss of culturability was observed from 11 days to 8 weeks. During the starvation process, three distinct phases and different sub-populations of VBNC cells were identified. Until complete loss of culturability, the number of membrane-intact cells decreased rapidly to 5.5-69% of the initial cell concentration. The concentration of the sub-population with low esterase activity dropped to 0.03-55%, and the concentration of the highly esterase-active sub-population dropped to 0.01-1.2% of the initial concentration; these sub-populations remained stable for several weeks to months. Only after approximately 200 days of starvation, the number of VBNC cells started to decrease below detection limits. The most abundant VBNC sub-populations were characterized by partially damaged membranes and low esterase-activity. With this study, we showed that upon starvation, a stable VBNC Legionella community may be present over several months in a strain-dependent manner even under harsh conditions. Even after one year of starvation, a small proportion of L. pneumophila cells with high esterase-activity was detected. We speculate that this highly active VBNC subpopulation is able to infect amoebae and human macrophages.


Starved Viable but Non-Culturable (VBNC) Legionella Strains Can Infect and Replicate in Amoebae and Human Macrophages

Dietersdorfer E, Kirschner A, Schrammel B, Ohradanova-Repic A, Stockinger H, Sommer R, Walochnik J, Cervero-Aragó S.

Medical University of Vienna, Institute for Hygiene and Applied Immunology, Water Hygiene, Vienna, Austria.

Water Res 2018 Sep;141:428-438.

Abstract: Legionella infections are among the most important waterborne infections with constantly increasing numbers of cases in industrialized countries, as a result of aging populations, rising numbers of immunocompromised individuals and increased need for conditioned water due to climate change. Surveillance of water systems is based on microbiological culture-based techniques; however, it has been shown that high percentages of the Legionella populations in water systems are not culturable. In the past two decades, the relevance of such viable but non-culturable (VBNC) legionellae has been controversially discussed, and whether VBNC legionellae can directly infect human macrophages, the primary targets of Legionella infections, remains unclear. In this study, it was demonstrated for the first time that several starved VBNC Legionella strains (four L. pneumophila serogroup 1 strains, a serogroup 6 strain and a L. micdadei strain) can directly infect different types of human macrophages and amoebae even after one year of starvation in ultrapure water. However, under these conditions, the strains caused infection with reduced efficacy, as represented by the lower percentages of infected cells, prolonged time in co-culture and higher multiplicities of infection required. Interestingly, the VBNC cells remained mostly non-culturable even after multiplication within the host cells. Amoebal infection by starved VBNC Legionella, which likely occurs in oligotrophic biofilms, would result in an increase in the bacterial concentration in drinking-water systems. If cells remain in the VBNC state, the real number of active legionellae will be underestimated by the use of culture-based standard techniques. Thus, further quantitative research is needed in order to determine, whether and how many starved VBNC Legionella cells are able to cause disease in humans.


The Virulence of Legionella pneumophila Is Positively Correlated with Its Ability to Stimulate NF-κB Activation

Wang H, Lu J, Li K, Ren H, Shi Y, Qin T, Duan X, Fang M.

International College, University of Chinese Academy of Sciences, Beijing, China.

Future Microbiol 2018 Sep;13:1247-1259.

Abstract: Aim: Our work is to study the correlation between the virulence of different Legionella pneumophila in mouse model and its ability to activate NF-κB signaling pathway in vitro. Materials & methods: We measured the abilities of different strains of L. pneumophila to induce the activation of NF-κB signaling pathway in vitro. By using A/J mice, we also detected the virulence of different strains in vivo. Results & conclusion: We demonstrated that different strains of L. pneumophila induce different levels of activation to NF-κB signaling pathway in vitro. We also found that L. pneumophila strain induced higher NF-κB activation in vitro showed more severe weight losses, higher mortality, more severe lung inflammation and higher levels of serum cytokines production in mice.


The Legionella Effector Kinase LegK7 Hijacks the Host Hippo Pathway to Promote Infection

Lee PC, Machner MP.

Division of Molecular and Cellular Biology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, USA.

Cell Host Microbe 2018 Sep;24(3):429-438.

Abstract: The intracellular pathogen Legionella pneumophila encodes translocated effector proteins that modify host cell processes to support bacterial survival and growth. Here, we show that the L. pneumophila effector protein LegK7 hijacks the conserved Hippo signaling pathway by molecularly mimicking host Hippo kinase (MST1 in mammals), which is the key regulator of pathway activation. LegK7, like Hippo/MST1, phosphorylates the scaffolding protein MOB1, which triggers a signaling cascade resulting in the degradation of the transcriptional regulators TAZ and YAP1. Transcriptome analysis revealed that LegK7-mediated targeting of TAZ and YAP1 alters the transcriptional profile of mammalian macrophages, a key cellular target of L. pneumophila infection. Specifically, genes targeted by the transcription factor PPARγ, which is regulated by TAZ, displayed altered expression, and continuous interference with PPARγ activity rendered macrophages less permissive to L. pneumophila intracellular growth. Thus, a conserved L. pneumophila effector kinase exploits the Hippo pathway to promote bacterial growth and infection.


Positive Selection in F-Box Domain (lpp0233) Encoded in Legionella pneumophila Strains

Kenzaka T, Yasui M, Baba T, Nasu M, Tani K.

Faculty of Pharmacy, Osaka Ohtani University.

Biocontrol Sci 2018;23(2):53-59.

Abstract: Recent studies have shown that the genome of Legionella pneumophila is characterized by many foreign genes from a variety of eukaryotes. The eukaryotic like proteins are known to play a role in its multiplication within host cells; however, their evolutionary genetics of L. pneumophila in environments is unknown. In this study, we examined the nonsynonymous/synonymous substitution rate of eukaryotic like domain encoding genes among L. pneumophila strains. In silico analysis revealed that the nonsynonymous/synonymous substitution rate in F-box domain gene (lpp0233) was higher than those in other eukaryotic like domain and protein encoding genes and housekeeping genes. The F-box domain gene sequences in L. pneumophila strains isolated from a natural hot spring were more diversified than those of previously known strains, owing to preferential positive selection.


Targeting of RNA Polymerase II by a Nuclear Legionella pneumophila Dot/Icm Effector SnpL

Schuelein R, Spencer H, Dagley LF, Li PF, Luo L, Stow JL, Abraham G, Naderer T, Gomez-Valero L, Buchrieser C, Sugimoto C, Yamagishi J, Webb AI, Pasricha S, Hartland EL.

Department of Microbiology and Immunology, University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne, Australia.

Cell Microbiol 2018 Sep;20(9):e12852.

Abstract: The intracellular pathogen Legionella pneumophila influences numerous eukaryotic cellular processes through the Dot/Icm-dependent translocation of more than 300 effector proteins into the host cell. Although many translocated effectors localise to the Legionella replicative vacuole, other effectors can affect remote intracellular sites. Following infection, a subset of effector proteins localises to the nucleus where they subvert host cell transcriptional responses to infection. Here, we identified Lpw27461 (Lpp2587), Lpg2519 as a new nuclear-localised effector that we have termed SnpL. Upon ectopic expression or during L. pneumophila infection, SnpL showed strong nuclear localisation by immunofluorescence microscopy but was excluded from nucleoli. Using immunoprecipitation and mass spectrometry, we determined the host-binding partner of SnpL as the eukaryotic transcription elongation factor, Suppressor of Ty5 (SUPT5H)/Spt5. SUPT5H is an evolutionarily conserved component of the DRB sensitivity-inducing factor complex that regulates RNA Polymerase II dependent mRNA processing and transcription elongation. Protein interaction studies showed that SnpL bound to the central Kyprides, Ouzounis, Woese motif region of SUPT5H. Ectopic expression of SnpL led to massive upregulation of host gene expression and macrophage cell death. The activity of SnpL further highlights the ability of L. pneumophila to control fundamental eukaryotic processes such as transcription that, in the case of SnpL, leads to global upregulation of host gene expression.


Structural and Biochemical Study of the Mono-ADP-Ribosyltransferase Domain of SdeA, a Ubiquitylating/Deubiquitylating Enzyme from Legionella pneumophila

Kim L, Kwon DH, Kim BH, Kim J, Park MR, Park ZY, Song HK.

Department of Life Sciences, Korea University, 145 Anam-ro, Seongbuk-gu, Seoul 02841, Korea.

J Mol Biol 2018 Aug;430(17):2843-2856.

Abstract: Conventional ubiquitylation occurs through an ATP-dependent three-enzyme cascade (E1, E2, and E3) that mediates the covalent conjugation of the C-terminus of ubiquitin to a lysine on the substrate. SdeA, which belongs to the SidE effector family of Legionella pneumophila, can transfer ubiquitin to endoplasmic reticulum-associated Rab-family GTPases in a manner independent of E1 and E2 enzymes. The novel ubiquitin-modifying enzyme SdeA utilizes NAD+ as a cofactor to attach ubiquitin to a serine residue of the substrate. Here, to elucidate the coupled enzymatic reaction of NAD+ hydrolysis and ADP-ribosylation of ubiquitin in SdeA, we characterized the mono-ADP-ribosyltransferase domain of SdeA and show that it consists of two sub-domains termed mART-N and mART-C. The crystal structure of the mART-C domain of SdeA was also determined in free form and in complex with NAD+ at high resolution. Furthermore, the spatial orientations of the N-terminal deubiquitylase, phosphodiesterase, mono-ADP-ribosyltransferase, and C-terminal coiled-coil domains within the 180-kDa full-length SdeA were determined. These results provide insight into the unusual ubiquitylation mechanism of SdeA and expand our knowledge on the structure-function of mono-ADP-ribosyltransferases.


Biosensors for the Detection of Interaction Between Legionella pneumophila Collagen-Like Protein and Glycosaminoglycans

Su H, Li S, Terebiznik M, Guyard C, Kerman K.

Department of Physical and Environmental Sciences, University of Toronto Scarborough, 1265 Military Trail, Toronto, ON M1C 1A4, Canada.

Sensors (Basel) 2018 Aug;18(8):2668. 

Abstract: The adhesin Legionella collagen-like (Lcl) protein can bind to extracellular matrix components and mediate the binding of Legionella pneumophila to host cells. In this study, electrochemical impedance spectroscopy (EIS) and surface plasmon resonance (SPR)-based biosensors were employed to characterize these interactions between glycosaminoglycans (GAGs) and the adhesin Lcl protein. Fucoidan displayed a high affinity (KD 18 nM) for Lcl protein. Chondroitin sulfate A and dermatan sulfate differ in the position of a carboxyl group replacing D-glucuronate with D-iduronate. Our results indicated that the presence of D-iduronate in dermatan sulfate strongly hindered its interaction with Lcl. These biophysical studies provided valuable information in our understanding of adhesin-ligand interactions related to Legionella pneumophila infections.


Structural Characterization of the Hypothetical Protein Lpg2622, a New Member of the C1 Family Peptidases from Legionella pneumophila

Gong X, Zhao X, Zhang W, Wang J, Chen X, Hameed MF, Zhang N, Ge H.

School of Life Sciences, Anhui University, Hefei, China.

FEBS Lett 2018 Aug;592(16):2798-2810.

Abstract: The Legionella pneumophila type II secretion system can promote bacterial growth under a wide variety of conditions and mediates the secretion of more than 25 proteins, including the uncharacterized effector Lpg2622. Here, we determined the crystal structures of apo-Lpg2622 and Lpg2622 in complex with the cysteine protease inhibitor E64. Structural analysis suggests that Lpg2622 belongs to the C1 family peptidases. Interestingly, unlike the other structurally resolved papain-like cysteine proteases, the propeptide of Lpg2622 forms a novel super-secondary structural fold (hairpin-turn-helix) and can be categorized into a new group. In addition, the N-terminal β-sheet of the Lpg2622 propeptide plays a regulatory role on enzymatic activity. This study enhances our understanding of the classification and regulatory mechanisms of the C1 family peptidases.


Legionella pneumophila Effector Lem4 Is a Membrane-Associated Protein Tyrosine Phosphatase

Beyrakhova K, Li L, Xu C, Gagarinova A, Cygler M.

Department of Biochemistry, University of Saskatchewan, Saskatoon, Saskatchewan, Canada.

J Biol Chem 2018 Aug;293(34):13044-13058.

Abstract: Legionella pneumophila is a Gram-negative pathogenic bacterium that causes severe pneumonia in humans. It establishes a replicative niche called Legionella-containing vacuole (LCV) that allows bacteria to survive and replicate inside pulmonary macrophages. To hijack host cell defense systems, L. pneumophila injects over 300 effector proteins into the host cell cytosol. The Lem4 effector (lpg1101) consists of two domains: an N-terminal haloacid dehalogenase (HAD) domain with unknown function and a C-terminal phosphatidylinositol 4-phosphate-binding domain that anchors Lem4 to the membrane of early LCVs. Herein, we demonstrate that the HAD domain (Lem4-N) is structurally similar to mouse MDP-1 phosphatase and displays phosphotyrosine phosphatase activity. Substrate specificity of Lem4 was probed using a tyrosine phosphatase substrate set, which contained a selection of 360 phosphopeptides derived from human phosphorylation sites. This assay allowed us to identify a consensus pTyr-containing motif. Based on the localization of Lem4 to lysosomes and to some extent to plasma membrane when expressed in human cells, we hypothesize that this protein is involved in protein-protein interactions with an LCV or plasma membrane-associated tyrosine-phosphorylated host target.


The Major Outer Membrane Protein of Legionella pneumophila Lpg1974 Shows Pore-Forming Characteristics Similar to the Human Mitochondrial Outer Membrane Pore, hVDAC1

Younas F, Soltanmohammadi N, Knapp O, Benz R.

Department of Life Sciences and Chemistry, Jacobs University, Bremen, Germany.

Biochim Biophys Acta Biomembr 2018 Aug;1860(8):1544-1553.

Abstract: Legionella pneumophila is an aerobic and nonspore-forming pathogenic Gram-negative bacterium of the genus Legionella. It is the causative agent of Legionnaires' disease, also known as Legionellosis. The hosts of this organism are diverse, ranging from simple water borne protozoans such as amoebae to more complex hosts such as macrophages in humans. Genome analyses have shown the presence of genes coding for eukaryotic like proteins in several Legionella species. The presence of these proteins may assist L. pneumophila in its adaptation to the eukaryotic host. We studied the characteristics of a protein (Lpg1974) of L. pneumophila that shows remarkable homologies in length of the primary sequence and for the identity/homology of many amino acids to the voltage dependent anion channel (human VDAC1, Porin 31HL) of human mitochondria. Two different forms of Lpg1974 were overexpressed in Escherichia coli and purified to homogeneity: the one containing a putative N-terminal signal sequence and one without it. Reconstituted protein containing the signal sequence formed ion-permeable pores in lipid bilayer membranes with a conductance of approximately 5.4 nS in 1 M KCl. When the predicted N-terminal signal peptide of Lpg1974 comprising an α-helical structure similar to that at the N-terminus of hVDAC1 was removed, the channels formed in reconstitution experiments had a conductance of 7.6 nS in 1 M KCl. Both Lpg1974 proteins formed pores that were voltage-dependent and anion-selective similar to the pores formed by hVDAC1. These results suggest that Lpg1974 of L. pneumophila is indeed a structural and functional homologue to hVDAC1.


Persistent Presence of Outer Membrane Epitopes During Short- and Long-Term Starvation of Five Legionella pneumophila Strains

Schrammel B, Petzold M, Cervero-Aragó S, Sommer R, Lück C, Kirschner A.

Institute for Hygiene and Applied Immunology - Water Hygiene, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria.

BMC Microbiol 2018 Jul;18(1):75.

Abstract: Background: Legionella pneumophila, the causative agent of Legionnaire's disease, may enter a viable but non-culturable (VBNC) state triggered by environmental stress conditions. Specific outer-membrane epitopes of L. pneumophila are used in many diagnostic applications and some of them are linked to important virulence-related factors or endotoxins. However, it is not clear how the presence and status of these epitopes are influenced by environmental stress conditions. In this study, changes of outer membrane epitopes for monoclonal antibodies (mAb) from the Dresden panel and the major outer membrane protein MOMP were analysed for five L. pneumophila strains during short- and long-term starvation in ultrapure water. Results: With ELISA and single cell immuno-fluorescence analysis, we could show that for most of the investigated mAb-strain combinations the total number of mAb-stained Legionella cells stayed constant for up to 400 days. Especially the epitopes of mAb 3/1, 8/5, 26/1 and 20/1, which are specific for L. pneumophila serogroup 1 subtypes, and the mAb 9/1, specific for serogroup 6, showed long-term persistence. For most mAb-stained cells, a high percentage of viable cells was observed at least until 118 days of starvation. At the same time, we observed a reduction of the fluorescence intensity of the stained cells during starvation indicating a loss of epitopes from the cell surface. However, most of the epitopes, including the virulence-associated mAb 3/1 epitope were still present with high fluorescence intensity after 400 days of starvation in up to 50% of the starved L. pneumophila population. Conclusions: The results demonstrate the continuous presence of outer membrane epitopes of L. pneumophila during short-term and long-term starvation. Thus, culture-independent mAb-based diagnostic and detection tools, such as immuno-magnetic separation and microarray techniques are applicable for both L. pneumophila in the culturable and the VBNC state even after long-term starvation but nevertheless require careful testing before application. However, the mere presence of those epitopes is not necessarily an indication of viability or infectivity.


Legionella pneumophila Is Directly Sensitive to 2-Deoxyglucose-Phosphate via Its UhpC Transporter but Is Indifferent to Shifts in Host Cell Glycolytic Metabolism

Price JV, Jiang K, Galantowicz A, Freifeld A, Vance RE.

Department of Biology, Oberlin College, Oberlin, Ohio, USA

J Bacteriol 2018 Jul;200(16):e00176-18.

Abstract: Toll-like receptor (TLR) stimulation induces a pronounced shift to increased glycolytic metabolism in mammalian macrophages. We observed that bone marrow-derived macrophages (BMMs) increase glycolysis in response to infection with Legionella pneumophila, but the role of host macrophage glycolysis in terms of intracellular L. pneumophila replication is not currently understood. Treatment with 2-deoxyglucose (2DG) blocks L. pneumophila replication in mammalian macrophages but has no effect on bacteria grown in broth. In addition, we found that 2DG had no effect on bacteria grown in amoebae. We used a serial enrichment strategy to reveal that the effect of 2DG on L. pneumophila in macrophages requires the L. pneumophila hexose-phosphate transporter UhpC. Experiments with UhpC-deficient L. pneumophila revealed that mutant bacteria are also resistant to growth inhibition following treatment with phosphorylated 2DG in broth, suggesting that the inhibitory effect of 2DG on L. pneumophila in mammalian cells requires 2DG phosphorylation. UhpC-deficient L. pneumophila replicates without a growth defect in BMMs and protozoan host cells and also replicates without a growth defect in BMMs treated with 2DG. Our data indicate that neither TLR signaling-dependent increased macrophage glycolysis nor inhibition of macrophage glycolysis has a substantial effect on intracellular L. pneumophila replication. These results are consistent with the view that L. pneumophila can employ diverse metabolic strategies to exploit its host cells. IMPORTANCE: We explored the relationship between macrophage glycolysis and replication of an intracellular bacterial pathogen, Legionella pneumophila Previous studies demonstrated that a glycolysis inhibitor, 2-deoxyglucose (2DG), blocks replication of L. pneumophila during infection of macrophages, leading to speculation that L. pneumophila may exploit macrophage glycolysis. We isolated L. pneumophila mutants resistant to the inhibitory effect of 2DG in macrophages, identifying a L. pneumophila hexose-phosphate transporter, UhpC, that is required for bacterial sensitivity to 2DG during infection. Our results reveal how a bacterial transporter mediates the direct antimicrobial effect of a toxic metabolite. Moreover, our results indicate that neither induction nor impairment of host glycolysis inhibits intracellular replication of L. pneumophila, which is consistent with a view of L. pneumophila as a metabolic generalist.


LotA, a Legionella Deubiquitinase, Has Dual Catalytic Activity and Contributes to Intracellular Growth

Kubori T, Kitao T, Ando H, Nagai H.

Department of Microbiology, Graduate School of Medicine, Gifu University, Gifu, Japan.

Cell Microbiol 2018 Jul;20(7):e12840.

Abstract: The intracellular bacterial pathogen, Legionella pneumophila, establishes the replicative niche as a result of the actions of a large array of effector proteins delivered via the Legionella Type 4 secretion system. Many effector proteins are expected to be involved in biogenesis and regulation of the Legionella-containing vacuole (LCV) that is highly decorated with ubiquitin. Here, we identified a Legionella deubiquitinase, designated LotA, by carrying out a genome analysis to find proteins resembling the eukaryotic ovarian tumour superfamily of cysteine proteases. LotA exhibits a dual ability to cleave ubiquitin chains that is dependent on 2 distinctive catalytic cysteine residues in the eukaryotic ovarian tumour domains. One cysteine dominantly contributes to the removal of ubiquitin from the LCVs by its polyubiquitin cleavage activity. The other specifically cleaves conjugated Lys6-linked ubiquitin. After delivered by the Type 4 secretion system, LotA localises on the LCVs via its PI(3)P-binding domain. The lipid-binding ability of LotA is crucial for ubiquitin removal from the vacuoles. We further analysed the functional interaction of the protein with the recently reported noncanonical ubiquitin ligases of L. pneumophila, revealing that the effector proteins are involved in coordinated regulation that contributes to bacterial growth in the host cells.


Diverse Legionella-Like Bacteria Associated with Testate Amoebae of the Genus Arcella (Arcellinida: Amoebozoa)

Gomaa F, Gersh M, Cavanaugh CM.

Department of Organismic and Evolutionary Biology, Biological Laboratory, Harvard University, Cambridge, Massachusetts, 02138, USA.

J Eukaryot Microbiol 2018 Jul;65(5):661-668.

Abstract: Diverse species of Legionella and Legionella-like amoebal pathogens (LLAPs) have been identified as intracellular bacteria in many amoeboid protists. There are, however, other amoeboid groups such as testate amoeba for which we know little about their potential to host such bacteria. In this study, we assessed the occurrence and diversity of Legionella spp. in cultures and environmental isolates of freshwater arcellinid testate amoebae species, Arcella hemispherica, Arcella intermedia, and Arcella vulgaris, via 16S rRNA gene sequence analyses and fluorescent in situ hybridization (FISH). Analysis of the 16S rRNA gene sequences indicated that A. hemispherica, A. intermedia, and A. vulgaris host Legionella-like bacteria with 94-98% identity to other Legionella spp. based on NCBI BLAST search. Phylogenetic analysis placed Legionella-like Arcella-associated bacteria (LLAB) in three different clusters within a tree containing all other members of Legionella and LLAPs. The intracellular localization of the Legionella within Arcella hosts was confirmed using FISH with a Legionella-specific probe. This study demonstrates that the host range of Legionella and Legionella-like bacteria in the Amoebozoa extends beyond members of "naked" amoebae species, with members of the testate amoebae potentially serving an ecological role in the dispersal, protection, and replication of Legionella spp. in natural environments.


Insights into the Ubiquitin Transfer Cascade Catalyzed by the Legionella Effector SidC

Wasilko DJ, Huang Q, Mao Y.

Department of Molecular Biology and Genetics, Cornell University, Ithaca, United States.

Elife 2018 Jul;7:e36154.

Abstract: The causative agent of Legionnaires' disease, Legionella pneumophila, delivers more than 330 virulent effectors to its host to establish an intracellular membrane-bound organelle called the Legionella containing vacuole. Among the army of Legionella effectors, SidC and its paralog SdcA have been identified as novel bacterial ubiquitin (Ub) E3 ligases. To gain insight into the molecular mechanism of SidC/SdcA as Ub ligases, we determined the crystal structures of a binary complex of the N-terminal catalytic SNL domain of SdcA with its cognate E2 UbcH5C and a ternary complex consisting of the SNL domain of SidC with the Ub-linked E2 UbcH7. These two structures reveal the molecular determinants governing the Ub transfer cascade catalyzed by SidC. Together, our data support a common mechanism in the Ub transfer cascade in which the donor Ub is immobilized with its C-terminal tail locked in an extended conformation, priming the donor Ub for catalysis.


Highly Stable Single-Strand-Specific 3'-nuclease/nucleotidase from Legionella pneumophila

Trundová M, Kovaľ T, Owens RJ, Fejfarová K, Dušková J, Kolenko P, Dohnálek J.

Laboratory of Structure and Function of Biomolecules, Institute of Biotechnology of the Czech Academy of Sciences, Biocev, Průmyslová 595, 25250 Vestec, Czech Republic.

Int J Biol Macromol 2018 Jul;114:776-787.

Abstract: The Gram-negative bacterium Legionella pneumophila is one of the known opportunistic human pathogens with a gene coding for a zinc-dependent S1-P1 type nuclease. Bacterial zinc-dependent 3'-nucleases/nucleotidases are little characterized and not fully understood, including L. pneumophila nuclease 1 (Lpn1), in contrast to many eukaryotic representatives with in-depth studies available. To help explain the principle properties and role of these enzymes in intracellular prokaryotic pathogens we have designed and optimized a heterologous expression protocol utilizing E. coli together with an efficient purification procedure and performed detailed characterization of the enzyme. Replacement of Ni2+ ions by Zn2+ ions in affinity purification proved to be a crucial step in the production of pure and stable protein. The production protocol provides protein with high yield, purity, stability, and solubility for structure-function studies. We show that highly thermostable Lpn1 is active mainly towards RNA and ssDNA, with pH optima 7.0 and 6.0, respectively, with low activity towards dsDNA; the enzyme features pronounced substrate inhibition. Bioinformatic and experimental analysis, together with computer modeling and electrostatics calculations point to an unusually high positive charge on the enzyme surface under optimal conditions for catalysis. The results help explain the catalytic properties of Lpn1 and its substrate inhibition.


A Unique Cytoplasmic ATPase Complex Defines the Legionella pneumophila Type IV Secretion Channel

Chetrit D, Hu B, Christie PJ, Roy CR, Liu J.

Department of Microbial Pathogenesis, Microbial Sciences Institute and Boyer Center for Molecular Medicine, Yale School of Medicine, New Haven, CT, USA.

Nat Microbiol 2018 Jun;3(6):678-686.

Abstract: Type IV secretion systems (T4SSs) are complex machines used by bacteria to deliver protein and DNA complexes into target host cells. Conserved ATPases are essential for T4SS function, but how they coordinate their activities to promote substrate transfer remains poorly understood. Here, we show that the DotB ATPase associates with the Dot-Icm T4SS at the Legionella cell pole through interactions with the DotO ATPase. The structure of the Dot-Icm apparatus was solved in situ by cryo-electron tomography at 3.5 nm resolution and the cytoplasmic complex was solved at 3.0 nm resolution. These structures revealed a cell envelope-spanning channel that connects to the cytoplasmic complex. Further analysis revealed a hexameric assembly of DotO dimers associated with the inner membrane complex, and a DotB hexamer associated with the base of this cytoplasmic complex. The assembly of a DotB-DotO energy complex creates a cytoplasmic channel that directs the translocation of substrates through the T4SS. These data define distinct stages in Dot-Icm machine biogenesis, advance our understanding of channel activation, and identify an envelope-spanning T4SS channel.


The Legionella pneumophila Methyltransferase RomA Methylates Also Non-histone Proteins During Infection

Schuhmacher MK, Rolando M, Bröhm A, Weirich S, Kudithipudi S, Buchrieser C, Jeltsch A.

Department of Biochemistry, Institute of Biochemistry and Technical Biochemistry, University Stuttgart, Stuttgart, Germany.

J Mol Biol 2018 Jun;430(13):1912-1925.

Abstract: RomA is a SET-domain containing protein lysine methyltransferase encoded by the Gram-negative bacterium Legionella pneumophila. It is exported into human host cells during infection and has been previously shown to methylate histone H3 at lysine 14 [Rolando et al. (2013), Cell Host Microbe, 13, 395-405]. Here, we investigated the substrate specificity of RomA on peptide arrays showing that it mainly recognizes a G-K-X-(PA) sequence embedded in a basic amino acid sequence context. Based on the specificity profile, we searched for possible additional RomA substrates in the human proteome and identified 34 novel peptide substrates. For nine of these, the corresponding full-length protein or protein domains could be cloned and purified. Using radioactive and antibody-based methylation assays, we showed that seven of them are methylated by RomA, four of them strongly, one moderately, and two weakly. Mutagenesis confirmed for the seven methylated proteins that methylation occurs at target lysine residues fitting to the specificity profile. Methylation of one novel substrate (AROS) was investigated in HEK293 cells overexpressing RomA and during infection with L. pneumophila. Methylation could be detected in both conditions, confirming that RomA methylates non-histone proteins in human cells. Our data show that the bacterial methyltransferase RomA methylates also human non-histone proteins suggesting a multifaceted role in the infection process.


Acanthamoeba S13WT Relies on Its Bacterial Endosymbiont to Backpack Human Pathogenic Bacteria and Resist Legionella Infection on Solid Media

Okubo T, Matsushita M, Nakamura S, Matsuo J, Nagai H, Yamaguchi H.

Department of Medical Laboratory Science, Faculty of Health Sciences, Hokkaido University, Sapporo, Japan.

Environ Microbiol Rep 2018 Jun;10(3):344-354.

Abstract: Soil-borne amoeba Acanthamoeba S13WT has an endosymbiotic relationship with an environmental Neochlamydia bacterial strain. However, regardless of extensive experiments in liquid media, the biological advantage of the symbiosis remained elusive. We therefore explored the role of the endosymbiont in predator-prey interactions on solid media. A mixed culture of the symbiotic or aposymbiotic amoebae and GFP-expressing Escherichia coli or Salmonella Enteritidis was spotted onto the centre of a LB or B-CYE agar plate preinoculated with a ring of mCherry-expressing Legionella pneumophila (Legionella 'wall'). The spread of the amoebae on the plate was assessed using a fluorescence imaging system or scanning electron microscopy. As a result, in contrast to the aposymbiotic amoebae, the symbiotic amoebae backpacked these GFP-expressing bacteria and formed flower-like fluorescence patterns in an anticlockwise direction. Other bacteria (Pseudomonas aeruginosa and Stenotrophomonas maltophilia), but not Staphylococcus aureus, were also backpacked by the symbiotic amoebae on LB agar, although lacked the movement to anticlockwise direction. Furthermore, in contrast to the aposymbiotic amoebae, the symbiotic amoebae backpacking the E. coli broke through the Legionella 'wall' on B-CYE agar plates. Thus, we concluded that Acanthamoeba S13WT required the Neochlamydia endosymbiont to backpack human pathogenic bacteria and resist Legionella infection on solid agar.


The Extracellular Polymeric Substances of Legionella pneumophila Biofilms Contain Amyloid Structures

Peterson CP, Sauer C, Chatfield CH.

Department of Biological Sciences, SUNY Cortland, Cortland, NY, USA.

Curr Microbiol 2018 Jun;75(6):736-744.

Abstract: Human infection by bacteria of the genus Legionella most often result in the pneumonia known as Legionnaires Disease. Legionella is found as a resident of adherent biofilms in man-made water systems. Disinfection efforts to prevent Legionella infections require a better understanding of the structures that promote Legionella surface attachment and biofilm colonization. Various enzymatic treatments, including multiple carbohydrate-targeting mixtures, failed to disrupt Legionella biofilms, despite the presence of carbohydrates in the biofilms as shown by biochemical methods and concanavalin-A lectin staining. Moreover, Legionella biofilms contained amyloids as detected by three microscopic staining methods (congo red, thioflavin T, and the amyloid-specific antibody WO2). Amyloid structures were seen in biofilms of both L. pneumophila and L. longbeachae, the two Legionella species most associated with human infection. Inhibition of amyloid assembly by congo red and thioflavin T limited both self-aggregation and surface attachment of L. pneumophila, indicating that functional amyloid structures have a key role in initial biofilm formation by these pathogenic bacteria.


Spatiotemporal Changes in Bacterial Community and Microbial Activity in a Full-Scale Drinking Water Treatment Plant

Hou L, Zhou Q, Wu Q, Gu Q, Sun M, Zhang J.

Guangdong Institute of Microbiology, State Key Laboratory of Applied Microbiology Southern China, Guangdong Provincial Key Laboratory of Microbial Culture Collection and Application, Guangdong Open Laboratory of Applied Microbiology, Guangzhou, China.

Sci Total Environ 2018 Jun;625:449-459.

Abstract: To gain insight into the bacterial dynamics present in drinking water treatment (DWT) systems, the microbial community and activity in a full-scale DWT plant (DWTP) in Guangzhou, South China, were investigated using Illumina Hiseq sequencing analyses combined with cultivation-based techniques during the wet and dry seasons. Illumina sequencing analysis of 16S rRNA genes revealed a large shift in the proportion of Actinobacteria, Proteobacteria and Firmicutes during the treatment process, with the proportion of Actinobacteria decreased sharply, whereas that of Proteobacteria and Firmicutes increased and predominated in treated water. Both microbial activity and bacterial diversity during the treatment process showed obvious spatial variation, with higher levels observed during the dry season and lower levels during the wet season. Clustering analysis and principal component analysis indicated dramatic shifts in the bacterial community after chlorination, suggesting that chlorination was highly effective at influencing the bacterial community. The bacterial community structure of finished water primarily comprised Pseudomonas, Citrobacter, and Acinetobacter, and interestingly showed high similarity to biofilms on granular activated carbon. Additionally, the abundance of bacterial communities was relatively stable in finished water and did not change with the season. A large number of unique operational taxonomic units were shared during treatment steps, indicating the presence of a diverse core microbiome throughout the treatment process. Opportunistic pathogens, including Pseudomonas, Acinetobacter, Citrobacter, Mycobacterium, Salmonella, Staphylococcus, Legionella, Streptococcus and Enterococcus, were detected in water including finished water, suggesting a potential threat to drinking-water safety. We also detected bacteria isolated from each treatment step using the pure-culture method. In particular, two isolates, identified as Mycobacterium sp. and Blastococcus sp., which belong to the phylum Actinobacteria, were obtained from finished water during the dry season. Together, these results provided evidence of spatial and temporal variations in DWTPs and contributed to the beneficial manipulation of the drinking water microbiome.


Mechanism of Phosphoribosyl-Ubiquitination Mediated by a Single Legionella Effector

Akturk A, Wasilko DJ, Wu X, Liu Y, Zhang Y, Qiu J, Luo ZQ, Reiter KH, Brzovic PS, Klevit RE, Mao Y.

Weill Institute for Cell and Molecular Biology and Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY, USA.

Nature 2018 May;557(7707):729-733.

Abstract: Ubiquitination is a post-translational modification that regulates many cellular processes in eukaryotes1-4. The conventional ubiquitination cascade culminates in a covalent linkage between the C terminus of ubiquitin (Ub) and a target protein, usually on a lysine side chain1,5. Recent studies of the Legionella pneumophila SidE family of effector proteins revealed a ubiquitination method in which a phosphoribosyl ubiquitin (PR-Ub) is conjugated to a serine residue on substrates via a phosphodiester bond6-8. Here we present the crystal structure of a fragment of the SidE family member SdeA that retains ubiquitination activity and determine the mechanism of this unique post-translational modification. The structure reveals that the catalytic module contains two distinct functional units: a phosphodiesterase domain and a mono-ADP-ribosyltransferase domain. Biochemical analysis shows that the mono-ADP-ribosyltransferase domain-mediated conversion of Ub to ADP-ribosylated Ub (ADPR-Ub) and the phosphodiesterase domain-mediated ligation of PR-Ub to substrates are two independent activities of SdeA. Furthermore, we present two crystal structures of a homologous phosphodiesterase domain from the SidE family member SdeD 9 in complexes with Ub and ADPR-Ub. The structures suggest a mechanism for how SdeA processes ADPR-Ub to PR-Ub and AMP, and conjugates PR-Ub to a serine residue in substrates. Our study establishes the molecular mechanism of phosphoribosyl-linked ubiquitination and will enable future studies of this unusual type of ubiquitination in eukaryotes.


Structural Basis of Ubiquitin Modification by the Legionella Effector SdeA

Dong Y, Mu Y, Xie Y, Zhang Y, Han Y, Zhou Y, Wang W, Liu Z, Wu M, Wang H, Pan M, Xu N, Xu CQ, Yang M, Fan S, Deng H, Tan T, Liu X, Liu L, Li J, Wang J, Fang X, Feng Y.

Beijing Advanced Innovation Center for Soft Matter Science and Engineering, Beijing Key Laboratory of Bioprocess, College of Life Science and Technology, Beijing University of Chemical Technology, Beijing, China.

Nature 2018 May;557(7707):674-678.

Abstract: Protein ubiquitination is a multifaceted post-translational modification that controls almost every process in eukaryotic cells. Recently, the Legionella effector SdeA was reported to mediate a unique phosphoribosyl-linked ubiquitination through successive modifications of the Arg42 of ubiquitin (Ub) by its mono-ADP-ribosyltransferase (mART) and phosphodiesterase (PDE) domains. However, the mechanisms of SdeA-mediated Ub modification and phosphoribosyl-linked ubiquitination remain unknown. Here we report the structures of SdeA in its ligand-free, Ub-bound and Ub-NADH-bound states. The structures reveal that the mART and PDE domains of SdeA form a catalytic domain over its C-terminal region. Upon Ub binding, the canonical ADP-ribosyltransferase toxin turn-turn (ARTT) and phosphate-nicotinamide (PN) loops in the mART domain of SdeA undergo marked conformational changes. The Ub Arg72 might act as a 'probe' that interacts with the mART domain first, and then movements may occur in the side chains of Arg72 and Arg42 during the ADP-ribosylation of Ub. Our study reveals the mechanism of SdeA-mediated Ub modification and provides a framework for further investigations into the phosphoribosyl-linked ubiquitination process.


Virulence Traits of Environmental and Clinical Legionella pneumophila Multilocus Variable-Number Tandem-Repeat Analysis (MLVA) Genotypes

Sharaby Y, Rodríguez-Martínez S, Pecellin M, Sela R, Peretz A, Höfle MG, Halpern M, Brettar I.

Department of Evolutionary and Environmental Biology, Faculty of Natural Sciences, University of Haifa, Haifa, Israel.

Appl Environ Microbiol 2018 May;84(10):e00429-18. 

Abstract: Legionella pneumophila causes water-based infections resulting in severe pneumonia. Recently, we showed that different MLVA-8 (multilocus variable-number tandem-repeat analysis using 8 loci) genotypes dominated different sites of a drinking-water distribution system. Each genotype displayed a unique temperature-dependent growth behavior. Here we compared the pathogenicity potentials of different MLVA-8 genotypes of environmental and clinical strains. The virulence traits studied were hemolytic activity and cytotoxicity toward amoebae and macrophages. Clinical strains were significantly more hemolytic than environmental strains, while their cytotoxicity toward amoebae was significantly lower at 30°C. No significant differences were detected between clinical and environmental strains in cytotoxicity toward macrophages. Significant differences in virulence were observed between the environmental genotypes (Gt). Gt15 strains showed a significantly higher hemolytic activity. In contrast, Gt4 and Gt6 strains were more infective toward Acanthamoeba castellanii Moreover, Gt4 strains exhibited increased cytotoxicity toward macrophages and demonstrated a broader temperature range of amoebal lysis than Gt6 and Gt15 strains. Understanding the virulence traits of Legionella genotypes may improve the assessment of public health risks of Legionella in drinking water. IMPORTANCE: Legionella pneumophila is the causative agent of a severe form of pneumonia. Here we demonstrated that clinical strains were significantly more cytotoxic toward red blood cells than environmental strains, while their cytotoxicity toward macrophages was similar. Genotype 4 (Gt4) strains were highly cytotoxic toward amoebae and macrophages and lysed amoebae in a broader temperature range than to the other studied genotypes. The results can explain the relatively high success of Gt4 in the environment and in clinical samples; thus, Gt4 strains should be considered a main factor for the assessment of public health risks of Legionella in drinking water. Our findings shed light on the ecology, virulence, and pathogenicity potential of different L. pneumophila genotypes, which can be a valuable parameter for future modeling and quantitative microbial risk assessment of Legionella in drinking-water systems.


Small Rho GTPases and the Effector VipA Mediate the Invasion of Epithelial Cells by Filamentous Legionella pneumophila

Prashar A, Ortiz ME, Lucarelli S, Barker E, Tabatabeiyazdi Z, Shamoun F, Raju D, Antonescu C, Guyard C, Terebiznik MR.

Department of Biological Sciences, University of Toronto at Scarborough, Scarborough, ON, Canada.

Front Cell Infect Microbiol 2018 May;8:133.

Abstract: Legionella pneumophila (Lp) exhibits different morphologies with varying degrees of virulence. Despite their detection in environmental sources of outbreaks and in respiratory tract secretions and lung autopsies from patients, the filamentous morphotype of Lp remains poorly studied. We previously demonstrated that filamentous Lp invades lung epithelial cells (LECs) and replicates intracellularly in a Legionella containing vacuole. Filamentous Lp activates β1integrin and E-cadherin receptors at the surface of LECs leading to the formation of actin-rich cell membrane structures we termed hooks and membrane wraps. These structures entrap segments of a Lp filament on host cell surface and mediate bacterial internalization. Here we investigated the molecular mechanisms responsible for the actin rearrangements needed for the formation and elongation of these membrane wraps and bacterial internalization. We combined genetic and pharmacological approaches to assess the contribution of signaling downstream of β1integrin and E-cadherin receptors, and Lp Dot/Icm secretion system- translocated effectors toward the invasion process. Our studies demonstrate a multi-stage mechanism of LEC invasion by filamentous Lp. Bacterial attachment to host cells depends on signaling downstream of β1integrin and E-cadherin activation, leading to Rho GTPases-dependent activation of cellular actin nucleating proteins, Arp2/3 and mDia. This mediates the formation of primordial membrane wraps that entrap the filamentous bacteria on the cell surface. Following this, in a second phase of the invasion process the Dot/Icm translocated effector VipA mediates rapid membrane wrap elongation, leading to the engulfment of the filamentous bacteria by the LECs. Our findings provide the first description of Rho GTPases and a Dot/Icm effector VipA regulating the actin dynamics needed for the invasion of epithelial cells by Lp.


Mammalian Solute Carrier (SLC)-like Transporters of Legionella pneumophila

Best A, Jones S, Abu Kwaik Y.

Department of Microbiology and Immunology, School of Medicine, University of Louisville, Louisville, KY, USA.

Sci Rep 2018 May;8(1):8352.

Abstract: Acquisition of nutrients during intra-vacuolar growth of L. pneumophila within macrophages or amoebae is poorly understood. Since many genes of L. pneumophila are acquired by inter-kingdom horizontal gene transfer from eukaryotic hosts, we examined the presence of human solute carrier (SLC)-like transporters in the L. pneumophila genome using I-TASSER to assess structural alignments. We identified 11 SLC-like putative transporters in L. pneumophila that are structurally similar to SLCs, eight of which are amino acid transporters, and one is a tricarboxylate transporter. The two other transporters, LstA and LstB, are structurally similar to the human glucose transporter, SLC2a1/Glut1. Single mutants of lstA or lstB have decreased ability to import, while the lstA/lstB double mutant is severely defective for uptake of glucose. While lstA or lstB single mutants are not defective in intracellular proliferation within Acanthamoeba polyphaga and human monocyte-derived macrophages, the lstA/lstB double mutant is severely defective in both host cells. The two phenotypic defects of the lstA/lstB double mutant in uptake of glucose and intracellular replication are both restored upon complementation of either lstA or lstB. Our data show that the two glucose transporters, LstA and LstB, are redundant and are required for intracellular replication within human macrophages and amoebae.


Electrophoretic Mobility of Legionella pneumophila Serogroups 1 to 14

Buse HY, Hoelle JM, Muhlen C, Lytle DA.

US EPA, Office of Research and Development, National Homeland Security Research Center, Cincinnati, OH 45268, USA.

FEMS Microbiol Lett 2018 May;365(10):10.1093/femsle/fny067.

Abstract: Legionella pneumophila (Lp) is ubiquitous in the aquatic environment and can persist within drinking water distribution systems (DWDS) enabling these systems to serve as a potential source of human infections. Bacterial surface charge, deduced from electrophoretic mobility (EPM), is a well-recognized contributor to microorganism mobility, adherence and interactions with their surrounding environment. In this study, the EPM of 32 Lp strains representing serogroup (sg) 1 to 14 were measured, in 9.15 mM KH2PO4 at pH 8, to understand cell surface properties that may influence their occurrence within DWDS. EPM measurements indicated the charge of Lp varied widely between serogroups with five distinct clusters, from least to most negatively charged: (i) sg1 to 3, 5, and 12; (ii) sg6, 8, and 10; (iii) sg9 and 13; (iv) sg7, 11, and 14; and (v) sg4. The EPM of sg1 and 4 strains were pH dependent; however, values were constant between pH 6 and 9, a range typical of drinking water, suggesting that EPM differences between Lp serogroups could impact their survival within DWDS. Understanding the ecological importance of Lp surface properties (e.g. in mobility, colonization, resistance to disinfectants, etc.) within DWDS would aid in mitigation of health risks associated with this water-based pathogen.


Type II Secretion-Dependent Aminopeptidase LapA and Acyltransferase PlaC Are Redundant for Nutrient Acquisition During Legionella pneumophila Intracellular Infection of Amoebas

White RC, Gunderson FF, Tyson JY, Richardson KH, Portlock TJ, Garnett JA, Cianciotto NP.

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

mBio 2018 Apr;9(2):e00528-18.

Abstract:  Legionella pneumophila genes encoding LapA, LapB, and PlaC were identified as the most highly upregulated type II secretion (T2S) genes during infection of Acanthamoeba castellanii, although these genes had been considered dispensable on the basis of the behavior of mutants lacking either lapA and lapB or plaC A plaC mutant showed even higher levels of lapA and lapB transcripts, and a lapA lapB mutant showed heightening of plaC mRNA levels, suggesting that the role of the LapA/B aminopeptidase is compensatory with respect to that of the PlaC acyltransferase. Hence, we made double mutants and found that lapA plaC mutants have an ~50-fold defect during infection of A. castellanii These data revealed, for the first time, the importance of LapA in any sort of infection; thus, we purified LapA and defined its crystal structure, activation by another T2S-dependent protease (ProA), and broad substrate specificity. When the amoebal infection medium was supplemented with amino acids, the defect of the lapA plaC mutant was reversed, implying that LapA generates amino acids for nutrition. Since the LapA and PlaC data did not fully explain the role of T2S in infection, we identified, via proteomic analysis, a novel secreted protein (NttD) that promotes infection of A. castellanii A lapA plaC nttD mutant displayed an even greater (100-fold) defect, demonstrating that the LapA, PlaC, and NttD data explain, to a significant degree, the importance of T2S. LapA-, PlaC-, and NttD-like proteins had distinct distribution patterns within and outside the Legionella genus. LapA was notable for having as its closest homologue an A. castellanii protein.

IMPORTANCE: Transmission of L. pneumophila to humans is facilitated by its ability to grow in Acanthamoeba species. We previously documented that type II secretion (T2S) promotes L. pneumophila infection of A. castellanii Utilizing transcriptional analysis and proteomics, double and triple mutants, and crystal structures, we defined three secreted substrates/effectors that largely clarify the role of T2S during infection of A. castellanii Particularly interesting are the unique functional overlap between an acyltransferase (PlaC) and aminopeptidase (LapA), the broad substrate specificity and eukaryotic-protein-like character of LapA, and the novelty of NttD. Linking LapA to amino acid acquisition, we defined, for the first time, the importance of secreted aminopeptidases in intracellular infection. Bioinformatic investigation, not previously applied to T2S, revealed that effectors originate from diverse sources and distribute within the Legionella genus in unique ways. The results of this study represent a major advance in understanding Legionella ecology and pathogenesis, bacterial secretion, and the evolution of intracellular parasitism.


Discovery of Ubiquitin Deamidases in the Pathogenic Arsenal of Legionella pneumophila

Valleau D, Quaile AT, Cui H, Xu X, Evdokimova E, Chang C, Cuff ME, Urbanus ML, Houliston S, Arrowsmith CH, Ensminger AW, Savchenko A.

Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, ON, Canada; Department of Microbiology, Immunology and Infectious Diseases, University of Calgary, Calgary, AB, Canada.

Cell Rep 2018 Apr;23(2):568-58  

Abstract: Legionella pneumophila translocates the largest known arsenal of over 330 pathogenic factors, called "effectors," into host cells during infection, enabling L. pneumophila to establish a replicative niche inside diverse amebas and human macrophages. Here, we reveal that the L. pneumophila effectors MavC (Lpg2147) and MvcA (Lpg2148) are structural homologs of cycle inhibiting factor (Cif) effectors and that the adjacent gene, lpg2149, produces a protein that directly inhibits their activity. In contrast to canonical Cifs, both MavC and MvcA contain an insertion domain and deamidate the residue Gln40 of ubiquitin but not Gln40 of NEDD8. MavC and MvcA are functionally diverse, with only MavC interacting with the human E2-conjugating enzyme UBE2N (Ubc13). MavC deamidates the UBE2NUb conjugate, disrupting Lys63 ubiquitination and dampening NF-κB signaling. Combined, our data reveal a molecular mechanism of host manipulation by pathogenic bacteria and highlight the complex regulatory mechanisms integral to L. pneumophila's pathogenic strategy.


Crucial Role of Legionella pneumophila TolC in the Inhibition of Cellular Trafficking in the Protistan Host Paramecium tetraurelia

Nishida T, Hara N, Watanabe K, Shimizu T, Fujishima M, Watarai M.

The United Graduate School of Veterinary Science, Yamaguchi University, Yamaguchi, Japan.

Front Microbiol 2018 Apr;9:800.

Abstract: Legionella pneumophila is a facultative intracellular Gram-negative bacterium, which is a major causative agent of Legionnaires' disease. In the environment, this bacterium survives in free-living protists such as amoebae and Tetrahymena. The association of L. pneumophila and protists leads to the replication and spread of this bacterium. Thus, from a public health perspective, their association can enhance the risk of L. pneumophila infection for humans. Paramecium spp. are candidates of natural hosts of L. pneumophila, but their detailed relationships remain unclear. In the present study, we used an environmental strain, L. pneumophila Ofk308 (Ofk308) and Paramecium tetraurelia st110-1a to reveal the relationship between L. pneumophila and Paramecium spp. Ofk308 was cytotoxic to P. tetraurelia in an infection-dependent manner. We focused on TolC, a component of the type I secretion system, which is a virulence factor of L. pneumophila toward protists and found that cytotoxicity was dependent on TolC but not on other T1SS components. Further, the number of bacteria in P. tetraurelia was not associated with cytotoxicity and TolC was not involved in the mechanism of resistance against the digestion of P. tetraurelia in Ofk308. We used a LysoTracker to evaluate the maturation process of P. tetraurelia phagosomes containing Ofk308. We found that there was no difference between Ofk308 and the tolC-deletion mutant. To assess the phagocytic activity of P. tetraurelia, Texas Red-conjugated dextran-uptake assays were performed. Ofk308 inhibited phagosome formation by P. tetraurelia through a TolC-dependent mechanism. Further, we evaluated the excretion of Legionella-containing vacuoles from P. tetraurelia. We found that P. tetraurelia failed to excrete undigested Ofk308 and that Ofk308 remained within cells through a TolC-dependent mechanism. Our results suggest that TolC is essential for L. pneumophila to remain within Paramecium cells and to show cytotoxicity. Because of the high mobility and high cell division rate of Paramecium spp., living with Paramecium spp. would be beneficial for L. pneumophila to expand its habitat. To control Legionaries' disease, understanding the ecology of L. pneumophila in the environment is essential.


The LetA/S Two-Component System Regulates Transcriptomic Changes That Are Essential for the Culturability of Legionella pneumophila in Water

Mendis N, McBride P, Saoud J, Mani T, Faucher SP.

Department of Natural Resource Sciences, McGill University, Sainte-Anne-de-Bellevue, Quebec, Canada.

Sci Rep 2018 Apr;8(1):6764.

Abstract: Surviving the nutrient-poor aquatic environment for extended periods of time is important for the transmission of various water-borne pathogens, including Legionella pneumophila (Lp). Previous work concluded that the stringent response and the sigma factor RpoS are essential for the survival of Lp in water. In the present study, we investigated the role of the LetA/S two-component signal transduction system in the successful survival of Lp in water. In addition to cell size reduction in the post-exponential phase, LetS also contributes to cell size reduction when Lp is exposed to water. Importantly, absence of the sensor kinase results in a significantly lower survival as measured by CFUs in water at various temperatures and an increased sensitivity to heat shock. According to the transcriptomic analysis, LetA/S orchestrates a general transcriptomic downshift of major metabolic pathways upon exposure to water leading to better culturability, and likely survival, suggesting a potential link with the stringent response. However, the expression of the LetA/S regulated small regulatory RNAs, RsmY and RsmZ, is not changed in a relAspoT mutant, which indicates that the stringent response and the LetA/S response are two distinct regulatory systems contributing to the survival of Lp in water.


A Legionella pneumophila Amylase Is Essential for Intracellular Replication in Human Macrophages and Amoebae

Best A, Price C, Ozanic M, Santic M, Jones S, Abu Kwaik Y.

Department of Microbiology and Immunology, College of Medicine, University of Louisville, Louisville, KY, USA.

Sci Rep 2018 Apr;8(1):6340.

Abstract: Legionella pneumophila invades protozoa with an "accidental" ability to cause pneumonia upon transmission to humans. To support its nutrition during intracellular residence, L. pneumophila relies on host amino acids as the main source of carbon and energy to feed the TCA cycle. Despite the apparent lack of a requirement for glucose for L. pneumophila growth in vitro and intracellularly, the organism contains multiple amylases, which hydrolyze polysaccharides into glucose monomers. Here we describe one predicted putative amylase, LamB, which is uniquely present only in L. pneumophila and L. steigerwaltii among the ~60 species of Legionella. Our data show that LamB has a strong amylase activity, which is abolished upon substitutions of amino acids that are conserved in the catalytic pocket of amylases. Loss of LamB or expression of catalytically-inactive variants of LamB results in a severe growth defect of L. pneumophila in Acanthamoeba polyphaga and human monocytes-derived macrophages. Importantly, the lamB null mutant is severely attenuated in intra-pulmonary proliferation in the mouse model and is defective in dissemination to the liver and spleen. Our data show an essential role for LamB in intracellular replication of L. pneumophila in amoeba and human macrophages and in virulence in vivo.


Differences in Virulence Between Legionella pneumophila Isolates from Human and Non-human Sources Determined in Galleria mellonella Infection Model

Sousa PS, Silva IN, Moreira LM, Veríssimo A, Costa J.

Department of Life Sciences, University of Coimbra, Coimbra, Portugal.

Front Cell Infect Microbiol 2018 Apr;8:97.

Abstract: Legionella pneumophila is a ubiquitous bacterium in freshwater environments and in many man-made water systems capable of inducing pneumonia in humans. Despite its ubiquitous character most studies on L. pneumophila virulence focused on clinical strains and isolates from man-made environments, so little is known about the nature and extent of virulence variation in strains isolated from natural environments. It has been established that clinical isolates are less diverse than man-made and natural environmental strains, suggesting that only a subset of environmental isolates is specially adapted to infect humans. In this work we intended to determine if unrelated L. pneumophila strains, isolated from different environments and with distinct virulence-related genetic backgrounds, displayed differences in virulence, using the Wax Moth Galleria mellonella infection model. We found that all tested strains were pathogenic in G. mellonella, regardless of their origin. Indeed, a panoply of virulence-related phenotypes was observed sustaining the existence of significant differences on the ability of L. pneumophila strains to induce disease. Taken together our results suggest that the occurrence of human infection is not related with the increased capability of some strains to induce disease since we also found a concentration threshold above which L. pneumophila strains are equally able to cause disease. In addition, no link could be established between the sequence-type (ST) and L. pneumophila pathogenicity. We envision that in man-made water distribution systems environmental filtering selection and biotic competition acts structuring L. pneumophila populations by selecting more resilient and adapted strains that can rise to high concentration if no control measures are implemented. Therefore, public health strategies based on the sequence-based typing (STB) scheme analysis should take into account that the major disease-associated clones of L. pneumophila were not related with higher virulence in G. mellonella infection model, and that potential variability of virulence-related phenotypes was found within the same ST.


Adhesion of Legionella pneumophila on Glass and Plumbing Materials Commonly Used in Domestic Water Systems

Assaidi A, Ellouali M, Latrache H, Mabrouki M, Timinouni M, Zahir H, Tankiouine S, Barguigua A, Mliji EM.

Laboratory of Water Microbiology and Environmental Hygiene, Institut Pasteur du Maroc, Casablanca, Morocco.

Int J Environ Health Res 2018 Apr;28(2):125-133.

Abstract: We aimed to investigate the adhesion of Legionella pneumophila serogroup1 and L. pneumophila serogroup 2-15 on glass, galvanized steel, stainless steel, copper, Polyvinyl chloride (PVC), Cross-linked polyethylene (PEX-c) and Polypropylene Random Copolymer (PPR). The surface physicochemical properties of both bacterial cells and materials were estimated through contact angle measurements. The roughness and surface topography of the materials were evaluated by Atomic Force Microscopy. The two L. pneumophila serogroups and plumbing materials showed a hydrophobic character, while glass surface was hydrophilic. All strains were adhered to all materials with the exception of copper. The result showed that the adhesion of both L. pneumophila sg1 and sg2-15 was systematically expressed with high intensity on galvanized steel followed by PVC, PEX-c, PPR, stainless steel and the low intensity on glass. The extent of adhesion is in correlation with the surface roughness and acid-bases interactions, while hydrophobicity seems to have no effect in adhesion intensity.


Amoebal Endosymbiont Neochlamydia Protects Host Amoebae Against Legionella pneumophila Infection by Preventing Legionella Entry

Maita C, Matsushita M, Miyoshi M, Okubo T, Nakamura S, Matsuo J, Takemura M, Miyake M, Nagai H, Yamaguchi H.

Department of Medical Laboratory Science, Faculty of Health Sciences, Hokkaido University, Sapporo, Japan.

Microbes Infect 2018 Apr;20(4):236-244.

Abstract: Acanthamoeba isolated from environmental soil harbors the obligate intracellular symbiont Neochlamydia, which has a critical role in host amoebal defense against Legionella pneumophila infection. Here, by using morphological analysis with confocal laser scanning fluorescence microscopy and transmission electron microscopy, proteome analyses with two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) and liquid chromatography-mass spectrometry (LC/MS), and transcriptome analysis with DNA microarray, we explored the mechanism by which the Neochlamydia affected this defense. We observed that when rare uptake did occur, the symbiotic amoebae allowed Legionella to grow normally. However, the symbiotic amoebae had severely reduced uptake of Legionella when compared with the aposymbiotic amoebae. Also, in contrast to amoebae carrying the endosymbiont, the actin cytoskeleton was significantly disrupted by Legionella infection in aposymbiotic amoebae. Furthermore, despite Legionella exposure, there was little change in Neochlamydia gene expression. Taken together, we concluded that the endosymbiont Neochlamydia prevents Legionella entry to the host amoeba, resulting in the host defense against Legionella infection.


Legionella - A Threat to Groundwater: Pathogen Transport in Recharge Basin

McBurnett LR, Holt NT, Alum A, Abbaszadegan M.

School of Sustainable Engineering and the Built Environment, Ira A. Fulton Schools of Engineering, Arizona State University, USA.

Sci Total Environ 2018 Apr;621:1485-1490.

Abstract: This study elucidates the potential risk posed by Legionella during aquifer recharge practices. Experiments were conducted using pilot-scale column simulating infiltration of bacterial surrogate and pathogen, E. coli and Legionella pneumophila, under central Arizona recharge basin conditions. A column was packed with a loamy sand media collected from a recharge basin and was fitted with six sampling ports at soil depths of 15, 30, 60, 92, 122 cm and acclimated for a month with tertiary treated wastewater. Transport of Legionella appeared to be delayed compared to E. coli. The breakthrough of E. coli and Legionella at 122 cm depth occurred at 3 and 24h, respectively. Slow transport of Legionella is consistent with its pleomorphic nature and variation in size and shape under low nutrient conditions. Legionella persisted for a longer time in the column, but at lower concentrations. Given the novel results of this study, the transport of Legionella into groundwater aquifers can occur through engineering recharge basin conditions creating a potential public health risk.


Acanthamoeba and Dictyostelium as Cellular Models for Legionella Infection

Steiner B, Swart AL, Welin A, Weber S, Personnic N, Kaech A, Freyre C, Ziegler U, Klemm RW, Hilbi H.

Institute of Medical Microbiology, Medical Faculty, University of Zurich, Zurich, Switzerland.

Front Cell Infect Microbiol 2018 Mar;8:61.

Abstract: Environmental bacteria of the genus Legionella naturally parasitize free-living amoebae. Upon inhalation of bacteria-laden aerosols, the opportunistic pathogens grow intracellularly in alveolar macrophages and can cause a life-threatening pneumonia termed Legionnaires' disease. Intracellular replication in amoebae and macrophages takes place in a unique membrane-bound compartment, the Legionella-containing vacuole (LCV). LCV formation requires the bacterial Icm/Dot type IV secretion system, which translocates literally hundreds of "effector" proteins into host cells, where they modulate crucial cellular processes for the pathogen's benefit. The mechanism of LCV formation appears to be evolutionarily conserved, and therefore, amoebae are not only ecologically significant niches for Legionella spp., but also useful cellular models for eukaryotic phagocytes. In particular, Acanthamoeba castellanii and Dictyostelium discoideum emerged over the last years as versatile and powerful models. Using genetic, biochemical and cell biological approaches, molecular interactions between amoebae and Legionella pneumophila have recently been investigated in detail with a focus on the role of phosphoinositide lipids, small and large GTPases, autophagy components and the retromer complex, as well as on bacterial effectors targeting these host factors.


Legionella Effectors Explored with INSeq: New Functional Insights

Rolando M, Buchrieser C.

Institut Pasteur, Biologie des Bactéries Intracellulaires, Paris, France.

Trends Microbiol 2018 Mar;26(3):169-170.

Abstract: Legionella pneumophila secretes over 300 effector proteins that manipulate host cells. This multiplicity of effectors hampers the characterization of their individual roles. Shames et al. report a new approach to solve the enigma of Legionella effector function by using INSeq to analyse effector functions in the context of infection.


Biofilms in Full-Scale Drinking Water Ozone Contactors Contribute Viable Bacteria to Ozonated Water

Kotlarz N, Rockey N, Olson TM, Haig SJ, Sanford L, LiPuma JJ, Raskin L.

Department of Civil and Environmental Engineering, University of Michigan, Ann Arbor, Michigan, USA.

Environ Sci Technol 2018 Mar;52(5):2618-2628.

Abstract: Concentrations of viable microbial cells were monitored using culture-based and culture-independent methods across multichamber ozone contactors in a full-scale drinking water treatment plant. Membrane-intact and culturable cell concentrations in ozone contactor effluents ranged from 1200 to 3750 cells/mL and from 200 to 3850 colony forming units/mL, respectively. Viable cell concentrations decreased significantly in the first ozone contact chamber, but rose, even as ozone exposure increased, in subsequent chambers. Our results implicate microbial detachment from biofilms on contactor surfaces, and from biomass present within lime softening sediments in a hydraulic dead zone, as a possible reason for increasing cell concentrations in water samples from sequential ozone chambers. Biofilm community structures on baffle walls upstream and downstream from the dead zone were significantly different from each other (p=0.017). The biofilms downstream of the dead zone contained a significantly (p=0.036) higher relative abundance of bacteria of the genera Mycobacterium and Legionella than the upstream biofilms. These results have important implications as the effluent from ozone contactors is often treated further in biologically active filters and bacteria in ozonated water continuously seed filter microbial communities.


Deposition Pattern of Aerosolized Legionella Using an Ex Vivo Human-Porcine Respiratory Model

Perinel S, Forest V, Landraud M, Pourchez J, Girardot F, Riffard S, Stauffert M, Vergnon JM, Allegra S.

Université de Lyon, IMT Mines Saint-Etienne, Centre CIS, INSERM, SainBioSE, F-42023 Saint-Etienne, France.

Int J Hyg Environ Health 2018 Mar;221(2):252-259.

Abstract: Legionella are bacteria responsible for severe lung pathologies. However how they enter and are deposited within the respiratory tract remains poorly documented. Data using animal testing led to the establishment of mathematical models allowing the estimation of aerosol dispersion risks. But direct extrapolation to humans is questionable and experimental models more physiologically representative of the inhalation route are welcome. The aim of this study was to develop a model as close as possible to the human anatomy and physiology allowing determining the deposition pattern of aerosolized Legionella while limiting in vivo experiments. To that purpose, we adapted the chimeric respiratory tract model we previously developed. This original model consisted of a replica of the human upper respiratory airways made by additive manufacturing connected to ex vivo porcine lungs ventilated by passive expansion, as for humans in physiological conditions. These experiments didn't imply specific animal sacrifices as pigs were bred for human consumption and lungs were considered as wastes by the slaughterhouse. Fluorescent Legionella were aerosolized and visualized using Cellvizio® Lab (probe-based confocal fluorescence microscope). Legionella were found in the whole respiratory tract. Broncho-alveolar lavages were also performed and the amount of Legionella reaching the thoracic region was quantified by culture and qPCR. Legionella were found preferentially in the left upper lobe compared to the right lower lobe. To our knowledge, it is the first time that experiments mimicking so closely human exposure by inhalation are performed while limiting animal experiments and providing a model for further Legionella infectious risk assessment.


An Investigation of Virulence Factors of Legionella pneumophila Environmental Isolates

Arslan-Aydoğdu EÖ, Kimiran A.

Istanbul University, Faculty of Science, Department of Biology, Istanbul, Turkey.

Braz J Microbiol 2018 Jan-Mar;49(1):189-199.

Abstract: Nine Legionella pneumophila strains isolated from cooling towers and a standard strain (L. pneumophila serogroup 1, ATCC 33152, Philadelphia 1) were analyzed and compared in terms of motility, flagella structure, ability to form biofilms, enzymatic activities (hemolysin, nucleases, protease, phospholipase A, phospholipase C, acid phosphatase, alkaline phosphatase and lipase), hemagglutination capabilities, and pathogenicity in various host cells (Acanthamoeba castellanii ATCC 30234, mouse peritoneal macrophages and human peripheral monocytes). All the isolates of bacteria appeared to be motile and polar-flagellated and possessed the type-IV fimbria. Upon the evaluation of virulence factors, isolate 4 was found to be the most pathogenic strain, while 6 out of the 9 isolates (the isolates 1, 2, 3, 4, 5, and 7) were more virulent than the ATCC 33152 strain. The different bacterial strains exhibited differences in properties such as adhesion, penetration and reproduction in the hosts, and preferred host type. To our knowledge, this is the first study to compare the virulence of environmental L. pneumophila strains isolated in Turkey, and it provides important information relevant for understanding the epidemiology of L. pneumophila.


A Systematic in Silico Analysis of the Legionellaceae Family for Identification of Novel Drug Target Candidates

Sohrabi SM, Mohammadi M, Tabatabaiepour SN, Tabatabaiepour SZ, Hosseini-Nave H, Soltani MF, Alizadeh H, Hadizadeh M.

Physiology Research Center, Institute of Basic and Clinical Physiology Sciences, Kerman University of Medical Sciences, Kerman, Iran.

Microb Drug Resist 2019 Mar;25(2):157-166.

Abstract: The family Legionellaceae consists of Gram-negative bacteria that are widely distributed in aquatic environments around the world. This family consists of a single genus, Legionella, that is recognized as an important cause of community-acquired pneumonia and hospital-acquired pneumonia. Legionella consists of intracellular pathogens, thus cellular pharmacokinetic and pharmacodynamic properties of an antibiotic against these bacteria as well as uptake and subcellular distribution into macrophages should be considered for a successful outcome of disease. Treatment strategies for Legionella infection require a combination of multiple antibiotics. Hence, because of the possible development of resistance to the drugs during therapy, a new alternative targeted therapy is yielding promising results. In this study, a comprehensive in silico target identification pipeline was performed on members of the family Legionellaceae to identify the best targets. Using a homology-based computational pipeline method, new drug targets were identified. Of 4,358 analyzed proteins, 18 proteins, including proteins involved in metabolism (amino acid, energy, and lipid metabolisms), cellular transport, cell division, and cell motility, were selected as the final putative drug targets. These proteins play an important role in the survival and propagation of Legionella infection. In conclusion, homology-based methods could improve the identification of novel drug targets and the drug discovery process, which can potentially be effective for the prevention and treatment of Legionella infections.


The Legionella pneumophila Effector Ceg4 Is a Phosphotyrosine Phosphatase That Attenuates Activation of Eukaryotic MAPK Pathways

Quaile AT, Stogios PJ, Egorova O, Evdokimova E, Valleau D, Nocek B, Kompella PS, Peisajovich S, Yakunin AF, Ensminger AW, Savchenko A.

Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario, Canada.

J Biol Chem 2018 Mar;293(9):3307-3320. 

Abstract: Host colonization by Gram-negative pathogens often involves delivery of bacterial proteins called "effectors" into the host cell. The pneumonia-causing pathogen Legionella pneumophila delivers more than 330 effectors into the host cell via its type IVB Dot/Icm secretion system. The collective functions of these proteins are the establishment of a replicative niche from which Legionella can recruit cellular materials to grow while evading lysosomal fusion inhibiting its growth. Using a combination of structural, biochemical, and in vivo approaches, we show that one of these translocated effector proteins, Ceg4, is a phosphotyrosine phosphatase harboring a haloacid dehalogenase-hydrolase domain. Ceg4 could dephosphorylate a broad range of phosphotyrosine-containing peptides in vitro and attenuated activation of MAPK-controlled pathways in both yeast and human cells. Our findings indicate that L. pneumophila's infectious program includes manipulation of phosphorylation cascades in key host pathways. The structural and functional features of the Ceg4 effector unraveled here provide first insight into its function as a phosphotyrosine phosphatase, paving the way to further studies into L. pneumophila pathogenicity.


Complete Genome Sequence of Legionella sainthelensi Isolated from a Patient with Legionnaires' Disease

Slow S, Anderson T, Biggs P, Kennedy M, Murdoch D, Cree S.

Department of Pathology and Biomedical Science, University of Otago, Christchurch, New Zealand.

Genome Announc 2018 Feb;6(5):e01588-17.

Abstract: Legionella sainthelensi is an aquatic environmental bacterium that in humans can cause Legionnaires' disease (LD), an often-severe form of pneumonia. Here, we report the first complete genome of a L. sainthelensi clinical isolate obtained in 2001 from a patient with LD in Canterbury, New Zealand.


RavN Is a Member of a Previously Unrecognized Group of Legionella pneumophila E3 Ubiquitin Ligases

Lin YH, Lucas M, Evans TR, Abascal-Palacios G, Doms AG, Beauchene NA, Rojas AL, Hierro A, Machner MP.

Division of Molecular and Cellular Biology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, USA.

PLoS Pathog 2018 Feb;14(2):e1006897.

Abstract: The eukaryotic ubiquitylation machinery catalyzes the covalent attachment of the small protein modifier ubiquitin to cellular target proteins in order to alter their fate. Microbial pathogens exploit this post-translational modification process by encoding molecular mimics of E3 ubiquitin ligases, eukaryotic enzymes that catalyze the final step in the ubiquitylation cascade. Here, we show that the Legionella pneumophila effector protein RavN belongs to a growing class of bacterial proteins that mimic host cell E3 ligases to exploit the ubiquitylation pathway. The E3 ligase activity of RavN was located within its N-terminal region and was dependent upon interaction with a defined subset of E2 ubiquitin-conjugating enzymes. The crystal structure of the N-terminal region of RavN revealed a U-box-like motif that was only remotely similar to other U-box domains, indicating that RavN is an E3 ligase relic that has undergone significant evolutionary alteration. Substitution of residues within the predicted E2 binding interface rendered RavN inactive, indicating that, despite significant structural changes, the mode of E2 recognition has remained conserved. Using hidden Markov model-based secondary structure analyses, we identified and experimentally validated four additional L. pneumophila effectors that were not previously recognized to possess E3 ligase activity, including Lpg2452/SdcB, a new paralog of SidC. Our study provides strong evidence that L. pneumophila is dedicating a considerable fraction of its effector arsenal to the manipulation of the host ubiquitylation pathway.


Legionella DotM Structure Reveals a Role in Effector Recruiting to the Type 4B Secretion System

Meir A, Chetrit D, Liu L, Roy CR, Waksman G.

Department of Biological Sciences, Institute of Structural and Molecular Biology, Birkbeck, Malet Street, London, UK.

Nat Commun 2018 Feb;9(1):507.

Abstract: Legionella pneumophila, a causative agent of pneumonia, utilizes the Type 4B secretion (T4BS) system to translocate over 300 effectors into the host cell during infection. T4BS systems are encoded by a large gene cluster termed dot/icm, three components of which, DotL, DotM, and DotN, form the "coupling complex", which serves as a platform for recruitment of effector proteins. One class of effectors includes proteins containing Glu-rich/E-block sequences at their C terminus. However, the protein or region of the coupling complex mediating recruitment of such effectors is unknown. Here we present the crystal structure of DotM. This all alpha-helical structure exhibits patches of positively charged residues. We show that these regions form binding sites for acidic Glu-rich peptides and that mutants targeting these patches are defective in vivo in the translocation of acidic Glu-rich motif-containing effectors. We conclude that DotM forms the interacting surface for recruitment of acidic Glu-rich motif-containing Legionella effectors.


Factors Mediating Environmental Biofilm Formation by Legionella pneumophila

Abu Khweek A, Amer AO.

Department of Biology and Biochemistry, Birzeit University, West Bank, Palestine.

Front Cell Infect Microbiol 2018 Feb;8:38.

Abstract: Legionella pneumophila (L. pneumophila) is an opportunistic waterborne pathogen and the causative agent for Legionnaires' disease, which is transmitted to humans via inhalation of contaminated water droplets. The bacterium is able to colonize a variety of man-made water systems such as cooling towers, spas, and dental lines and is widely distributed in multiple niches, including several species of protozoa In addition to survival in planktonic phase, L. pneumophila is able to survive and persist within multi-species biofilms that cover surfaces within water systems. Biofilm formation by L. pneumophila is advantageous for the pathogen as it leads to persistence, spread, resistance to treatments and an increase in virulence of this bacterium. Furthermore, Legionellosis outbreaks have been associated with the presence of L. pneumophila in biofilms, even after the extensive chemical and physical treatments. In the microbial consortium-containing L. pneumophila among other organisms, several factors either positively or negatively regulate the presence and persistence of L. pneumophila in this bacterial community. Biofilm-forming L. pneumophila is of a major importance to public health and have impact on the medical and industrial sectors. Indeed, prevention and removal protocols of L. pneumophila as well as diagnosis and hospitalization of patients infected with this bacteria cost governments billions of dollars. Therefore, understanding the biological and environmental factors that contribute to persistence and physiological adaptation in biofilms can be detrimental to eradicate and prevent the transmission of L. pneumophila. In this review, we focus on various factors that contribute to persistence of L. pneumophila within the biofilm consortium, the advantages that the bacteria gain from surviving in biofilms, genes and gene regulation during biofilm formation and finally challenges related to biofilm resistance to biocides and anti-Legionella treatments.


Legionella Quorum Sensing and Its Role in Pathogen-Host Interactions

Personnic N, Striednig B, Hilbi H.

Institute of Medical Microbiology, University of Zürich, Zürich, Switzerland.

Curr Opin Microbiol 2018 Feb;41:29-35.

Abstract: Legionella pneumophila is a water-borne opportunistic pathogen causing a life-threatening pneumonia called 'Legionnaires' disease'. The Legionella quorum sensing (Lqs) system produces and responds to the α-hydroxyketone signaling molecule 3-hydroxypentadecane-4-one (Legionella autoinducer-1, LAI-1). The Lqs system controls the switch between the replicative/non-virulent and the transmissive/virulent phase of L. pneumophila, and it is a major regulator of natural competence, motility and virulence of the pathogen. Yet, beyond gene regulation, LAI-1 also directly affects pathogen-host interactions, since the signaling molecule modulates the migration of eukaryotic cells. Genes encoding Lqs homologues are present in many environmental bacteria, suggesting that α-hydroxyketone signaling is widely used for inter-bacterial as well as inter-kingdom signaling. In this review we summarize recent advances on the characterization of the Lqs system and its role in L. pneumophila-host cell interactions.


The Life Cycle of L. pneumophila: Cellular Differentiation Is Linked to Virulence and Metabolism

Oliva G, Sahr T, Buchrieser C.

Institut Pasteur, Biologie des Bactéries Intracellulaires, Paris, France.

Front Cell Infect Microbiol 2018 Jan;8:3.

Abstract: Legionella pneumophila is a gram-negative bacterium that inhabits freshwater ecosystems, where it is present in biofilm or as planktonic form. L. pneumophila is mainly found associated with protozoa, which serve as protection from hostile environments and as replication niche. If inhaled within aerosols, L. pneumophila is also able to infect and replicate in human alveolar macrophages, eventually causing the Legionnaires' disease. The transition between intracellular and extracellular environments triggers a differentiation program in which metabolic as well as morphogenetic changes occur. We here describe the current knowledge on how the different developmental states of this bacterium are regulated, with a particular emphasis on the stringent response activated during the transition from the replicative phase to the infectious phase and the metabolic features going in hand. We propose that the cellular differentiation of this intracellular pathogen is closely associated to key metabolic changes in the bacterium and the host cell, which together have a crucial role in the regulation of L. pneumophila virulence.


The Common HAQ STING Variant Impairs cGAS-dependent Antibacterial Responses and Is Associated with Susceptibility to Legionnaires' Disease in Humans

Ruiz-Moreno JS, Hamann L, Shah JA, Verbon A, Mockenhaupt FP, Puzianowska-Kuznicka M, Naujoks J, Sander LE, Witzenrath M, Cambier JC, Suttorp N, Schumann RR, Jin L, Hawn TR, Opitz B; CAPNETZ Study Group.

Department of Internal Medicine/Infectious Diseases and Pulmonary Medicine, Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany.

PLoS Pathog 2018 Jan;14(1):e1006829.

Abstract: The cyclic GMP-AMP synthase (cGAS)-STING pathway is central for innate immune sensing of various bacterial, viral and protozoal infections. Recent studies identified the common HAQ and R232H alleles of TMEM173/STING, but the functional consequences of these variants for primary infections are unknown. Here we demonstrate that cGAS- and STING-deficient murine macrophages as well as human cells of individuals carrying HAQ TMEM173/STING were severely impaired in producing type I IFNs and pro-inflammatory cytokines in response to Legionella pneumophila, bacterial DNA or cyclic dinucleotides (CDNs). In contrast, R232H attenuated cytokine production only following stimulation with bacterial CDN, but not in response to L. pneumophila or DNA. In a mouse model of Legionnaires' disease, cGAS- and STING-deficient animals exhibited higher bacterial loads as compared to wild-type mice. Moreover, the haplotype frequency of HAQ TMEM173/STING, but not of R232H TMEM173/STING, was increased in two independent cohorts of human Legionnaires' disease patients as compared to healthy controls. Our study reveals that the cGAS-STING cascade contributes to antibacterial defense against L. pneumophila in mice and men and provides important insight into how the common HAQ TMEM173/STING variant affects antimicrobial immune responses and susceptibility to infection.


Secreted Phospholipases of the Lung Pathogen Legionella pneumophila

Hiller M, Lang C, Michel W, Flieger A.

Division of Enteropathogenic Bacteria and Legionella, Robert Koch-Institute, Wernigerode, Germany.

Int J Med Microbiol 2018 Jan;308(1):168-175.

Abstract: Legionella pneumophila is an intracellular pathogen and the main causative agent of Legionnaires' disease, a potentially fatal pneumonia. The bacteria infect both mammalian cells and environmental hosts, such as amoeba. Inside host cells, the bacteria withstand the multifaceted defenses of the phagocyte and replicate within a unique membrane-bound compartment, the Legionella-containing vacuole (LCV). For establishment and maintenance of the infection, L. pneumophila secretes many proteins including effector proteins by means of different secretion systems and outer membrane vesicles. Among these are a large variety of lipolytic enzymes which possess phospholipase/lysophospholipase and/or glycerophospholipid/cholesterol acyltransferase activities. Secreted lipolytic activities may contribute to bacterial virulence, for example via modification of eukaryotic membranes, such as the LCV. In this review, we describe the secretion systems of L. pneumophila, introduce the classification of phospholipases, and summarize the state of the art on secreted L. pneumophila phospholipases. We especially highlight those enzymes secreted via the type II secretion system Lsp, via the type IVB secretion system Dot/Icm, via outer membrane vesicles, and such where the mode of secretion has not yet been defined. We also give an overview on the complexity of their activities, activation mechanisms, localization, growth-phase dependent abundance, and their role in infection.


Differential Expression of Virulence Genes in Legionella pneumophila Growing in Acanthamoeba and Human Monocytes

Mou Q, Leung PHM.

Department of Health Technology and Informatics, The Hong Kong Polytechnic University, Kowloon, Hong Kong, China.

Virulence 2018 Jan;9(1):185-196.

Abstract: Legionella pneumophila, the causative agent of Legionnaires' disease, is widely distributed throughout natural and artificial water systems and can replicate in macrophages and amoebae. Amoebae are the natural hosts of L. pneumophila, whereas macrophages are incidentally infected. The life cycle of L. pneumophila comprises a replicative phase within the Legionella-containing vacuole (LCV) and a transmissive phase during which bacterial cells become motile and are released via killing of the host. Although the host death mechanisms induced by L. pneumophila have been studied, the expression patterns of related L. pneumophila genes have not been reported. The present study compared the expression patterns of host cell death-associated genes in L. pneumophila grown in the human monocytic cell line THP-1 and Acanthamoeba castellanii. Notably, when L. pneumophila was grown in THP-1, expression of the gene flaA, which is involved in the induction of pyroptosis, was downregulated during the course of infection. In contrast, sdhA associated indirectly with host death, was upregulated. Expression of the genes vipD and sidF, which are involved in the induction and suppression of apoptosis, changed by less than 2-fold. Notably, a lower percentage of pyroptotic cells was observed among infected THP-1 cells relative to uninfected cells, and the latter exhibited stronger expression of caspase-1. A different pattern was observed when L. pneumophila was grown in A. castellanii: flaA and vipD were activated, whereas sdhA and sidF were downregulated during the later stage of replication. The percentage of non-viable (annexin-V+ PI+ or annexin-V+PI-) A. castellanii organisms increased with Legionella infection, and the expression of metacaspase-1, which is involved in encystation was up-regulated at late infection time. In summary, L. pneumophila can multiply intracellularly in both amoebae and macrophages to induce cell death and secondary infection, and this characteristic is essential for its survival in water and the lungs. The gene expression profiles observed in this study indicated the increased cytotoxicity of L. pneumophila in A. castellanii, suggesting an increased adaptation of Legionella to this host.


Formation of the Legionella-containing Vacuole: Phosphoinositide Conversion, GTPase Modulation and ER Dynamics

Steiner B, Weber S, Hilbi H.

Institute of Medical Microbiology, University of Zürich, Zürich, Switzerland.

Int J Med Microbiol 2018 Jan;308(1):49-57.

Abstract: The environmental bacterium Legionella pneumophila replicates in free-living amoeba as well as in alveolar macrophages upon inhalation of bacteria-laden aerosols. Resistance of the opportunistic pathogen to macrophages is a prerequisite to cause a severe pneumonia called Legionnaires' disease. L. pneumophila grows intracellularly in a unique, ER-associated compartment, the Legionella-containing vacuole (LCV). The bacterial Icm/Dot type IV secretion system represents an essential virulence factor, which translocates approximately 300 "effector proteins" into protozoan or mammalian host cells. Some of these effectors contribute to the formation of the LCV by targeting conserved host factors implicated in membrane dynamics, such as phosphoinositide lipids and small GTPases. Here we review recent findings on the role of phosphoinositides, small and large GTPases as well as ER dynamics for pathogen vacuole formation and intracellular replication of L. pneumophila.


Legionella Effector AnkX Interacts with Host Nuclear Protein PLEKHN1

Yu X, Noll RR, Romero Dueñas BP, Allgood SC, Barker K, Caplan JL, Machner MP, LaBaer J, Qiu J, Neunuebel MR.

Department of Biological Sciences, University of Delaware, Newark, DE, USA.

BMC Microbiol 2018 Jan;18(1):5.

Abstract: Background: The intracellular bacterial pathogen Legionella pneumophila proliferates in human alveolar macrophages, resulting in a severe pneumonia termed Legionnaires' disease. Throughout the course of infection, L. pneumophila remains enclosed in a specialized membrane compartment that evades fusion with lysosomes. The pathogen delivers over 300 effector proteins into the host cell, altering host pathways in a manner that sets the stage for efficient pathogen replication. The L. pneumophila effector protein AnkX targets host Rab GTPases and functions in preventing fusion of the Legionella-containing vacuole with lysosomes. However, the current understanding of AnkX's interaction with host proteins and the means through which it exerts its cellular function is limited. Results: Here, we investigated the protein interaction network of AnkX by using the nucleic acid programmable protein array (NAPPA), a high-density platform comprising 10,000 unique human ORFs. This approach facilitated the discovery of PLEKHN1 as a novel interaction partner of AnkX. We confirmed this interaction through multiple independent in vitro pull-down, co-immunoprecipitation, and cell-based assays. Structured illumination microscopy revealed that endogenous PLEKHN1 is found in the nucleus and on vesicular compartments, whereas ectopically produced AnkX co-localized with lipid rafts at the plasma membrane. In mammalian cells, HaloTag-AnkX co-localized with endogenous PLEKHN1 on vesicular compartments. A central fragment of AnkX (amino acids 491-809), containing eight ankyrin repeats, extensively co-localized with endogenous PLEKHN1, indicating that this region may harbor a new function. Further, we found that PLEKHN1 associated with multiple proteins involved in the inflammatory response. Conclusions: Altogether, our study provides evidence that in addition to Rab GTPases, the L. pneumophila effector AnkX targets nuclear host proteins and suggests that AnkX may have novel functions related to manipulating the inflammatory response.


Differential Effects of Iron, Zinc, and Copper on Dictyostelium discoideum Cell Growth and Resistance to Legionella pneumophila

Buracco S, Peracino B, Andreini C, Bracco E, Bozzaro S.

Department of Clinical and Biological Sciences, University of Torino, Turin, Italy.

Front Cell Infect Microbiol 2018 Jan;7:536.

Abstract: Iron, zinc, and copper play fundamental roles in eucaryotes and procaryotes, and their bioavailability regulates host-pathogen interactions. For intracellular pathogens, the source of metals is the cytoplasm of the host, which in turn manipulates intracellular metal traffic following pathogen recognition. It is established that iron is withheld from the pathogen-containing vacuole, whereas for copper and zinc the evidence is unclear. Most infection studies in mammals have concentrated on effects of metal deficiency/overloading at organismal level. Thus, zinc deficiency or supplementation correlate with high risk of respiratory tract infection or recovery from severe infection, respectively. Iron, zinc, and copper deficiency or overload affects lymphocyte proliferation/maturation, and thus the adaptive immune response. Whether they regulate innate immunity at macrophage level is open, except for iron. The early identification in a mouse mutant susceptible to mycobacterial infection of the iron transporter Nramp1 allowed dissecting Nramp1 role in phagocytes, from the social amoeba Dictyostelium to macrophages. Nramp1 regulates iron efflux from the phagosomes, thus starving pathogenic bacteria for iron. Similar studies for zinc or copper are scant, due to the large number of copper and zinc transporters. In Dictyostelium, zinc and copper transporters include 11 and 6 members, respectively. To assess the role of zinc or copper in Dictyostelium, cells were grown under conditions of metal depletion or excess and tested for resistance to Legionella pneumophila infection. Iron shortage or overload inhibited Dictyostelium cell growth within few generations. Surprisingly, zinc or copper depletion failed to affect growth. Zinc or copper overloading inhibited cell growth at, respectively, 50- or 500-fold the physiological concentration, suggesting very efficient control of their homeostasis, as confirmed by Inductively Coupled Plasma Mass Spectrometry quantification of cellular metals. Legionella infection was inhibited or enhanced in cells grown under iron shortage or overload, respectively, confirming a major role for iron in controlling resistance to pathogens. In contrast, zinc and copper depletion or excess during growth did not affect Legionella infection. Using Zinpyr-1 as fluorescent sensor, we show that zinc accumulates in endo-lysosomal vesicles, including phagosomes, and the contractile vacuole. Furthermore, we provide evidence for permeabilization of the Legionella-containing vacuole during bacterial proliferation.


Metformin Mediates Protection Against Legionella Pneumonia Through Activation of AMPK and Mitochondrial Reactive Oxygen Species

Kajiwara C, Kusaka Y, Kimura S, Yamaguchi T, Nanjo Y, Ishii Y, Udono H, Standiford TJ, Tateda K.

Department of Microbiology and Infectious Diseases, Faculty of Medicine, Toho University School of Medicine, Tokyo, Japan.

J Immunol 2018 Jan;200(2):623-631.

Abstract: In Legionella pneumophila infection, macrophages play a critical role in the host defense response. Metformin, an oral drug for type 2 diabetes, is attracting attention as a new supportive therapy against a variety of diseases, such as cancer and infectious diseases. The novel mechanisms for metformin actions include modulation of the effector functions of macrophages and other host immune cells. In this study, we have examined the effects of metformin on L. pneumophila infection in vitro and in vivo. Metformin treatment suppressed growth of L. pneumophila in a time- and concentration-dependent fashion in bone marrow-derived macrophages, RAW cells (mouse), and U937 cells (human). Metformin induced phosphorylation of AMP-activated protein kinase (AMPK) in L. pneumophila-infected bone marrow-derived macrophages, and the AMPK inhibitor Compound C negated metformin-mediated growth suppression. Also, metformin induced mitochondrial reactive oxygen species but not phagosomal NADPH oxidase-derived reactive oxygen species. Metformin-mediated growth suppression was mitigated in the presence of the reactive oxygen species scavenger glutathione. In a murine L. pneumophila pneumonia model, metformin treatment improved survival of mice, which was associated with a significant reduction in bacterial number in the lung. Similar to in vitro observations, induction of AMPK phosphorylation and mitochondrial ROS was demonstrated in the infected lungs of mice treated with metformin. Finally, glutathione treatment abolished metformin effects on lung bacterial clearance. Collectively, these data suggest that metformin promotes mitochondrial ROS production and AMPK signaling and enhances the bactericidal activity of macrophages, which may contribute to improved survival in L. pneumophila pneumonia.


The Toolbox for Uncovering the Functions of Legionella Dot/Icm Type IVb Secretion System Effectors: Current State and Future Directions

Schroeder GN.

Centre for Experimental Medicine, School of Medicine, Dentistry and Biomedical Sciences, Queen's University Belfast, Belfast, United Kingdom.

Front Cell Infect Microbiol 2018 Jan;7:528.

Abstract: The defective in organelle trafficking/intracellular multiplication (Dot/Icm) Type IVb secretion system (T4SS) is the essential virulence factor for the intracellular lifestyle and pathogenicity of Legionella species. Screens demonstrated that an individual L. pneumophila strain can use the Dot/Icm T4SS to translocate an unprecedented number of more than 300 proteins into host cells, where these, so called Icm/Dot-translocated substrates (IDTS) or effectors, manipulate host cell functions to the benefit of the bacteria. Bioinformatic analysis of the pan-genus genome predicts at least 608 orthologous groups of putative effectors. Deciphering the function of these effectors is key to understanding Legionella pathogenesis; however, the analysis is challenging. Substantial functional redundancy renders classical, phenotypic screening of single gene deletion mutants mostly ineffective. Here, I review experimental approaches that were successfully used to identify, validate and functionally characterize T4SS effectors and highlight new methods, which promise to facilitate unlocking the secrets of Legionella's extraordinary weapons arsenal.


KKL-35 Exhibits Potent Antibiotic Activity Against Legionella Species Independently of trans-Translation Inhibition

Brunel R, Descours G, Durieux I, Doublet P, Jarraud S, Charpentier X.

CIRI, Centre International de Recherche en Infectiologie, Team Horizontal Gene Transfer in Bacterial Pathogens, INSERM, U1111, Université Claude Bernard Lyon 1, CNRS, UMR5308, École Normale Supérieure de Lyon, Université Lyon, Villeurbanne, France.

Antimicrob Agents Chemother2018 Jan;62(2):e01459-17. 

Abstract: trans-Translation is a ribosome-rescue system that is ubiquitous in bacteria. Small molecules defining a new family of oxadiazole compounds that inhibit trans-translation have been found to have broad-spectrum antibiotic activity. We sought to determine the activity of KKL-35, a potent member of the oxadiazole family, against the human pathogen Legionella pneumophila and other related species that can also cause Legionnaires' disease (LD). Consistent with the essential nature of trans-translation in L. pneumophila, KKL-35 inhibited the growth of all tested strains at submicromolar concentrations. KKL-35 was also active against other LD-causing Legionella species. KKL-35 remained equally active against L. pneumophila mutants that have evolved resistance to macrolides. KKL-35 inhibited the multiplication of L. pneumophila in human macrophages at several stages of infection. No resistant mutants could be obtained, even during extended and chronic exposure. Surprisingly, KKL-35 was not synergistic with other ribosome-targeting antibiotics and did not induce the filamentation phenotype observed in cells defective for trans-translation. Importantly, KKL-35 remained active against L. pneumophila mutants expressing an alternate ribosome-rescue system and lacking transfer-messenger RNA, the essential component of trans-translation. These results indicate that the antibiotic activity of KKL-35 is not related to the specific inhibition of trans-translation and its mode of action remains to be identified. In conclusion, KKL-35 is an effective antibacterial agent against the intracellular pathogen L. pneumophila with no detectable resistance development. However, further studies are needed to better understand its mechanism of action and to assess further the potential of oxadiazoles in treatment.


Innate Sensing and Cell-Autonomous Resistance Pathways in Legionella pneumophila Infection

Naujoks J, Lippmann J, Suttorp N, Opitz B.

Charité - Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Department of Internal Medicine/Infectious Diseases and Pulmonary Medicine, Berlin, Germany.

Int J Med Microbiol 2018 Jan;308(1):161-167.

Abstract: Legionella pneumophila is a facultative intracellular bacterium which can cause a severe pneumonia called Legionnaires' disease after inhalation of contaminated water droplets and replication in alveolar macrophages. The innate immune system is generally able to sense and -in most cases- control L. pneumophila infection. Comorbidities and genetic risk factors, however, can compromise the immune system and high infection doses might overwhelm its capacity, thereby enabling L. pneumophila to grow and disseminate inside the lung. The innate immune system mediates sensing of L. pneumophila by employing e.g. NOD-like receptors (NLRs), Toll-like receptors (TLRs), as well as the cGAS/STING pathway to stimulate death of infected macrophages as well as production of proinflammatory cytokines and interferons (IFNs). Control of pulmonary L. pneumophila infection is largely mediated by inflammasome-, TNFα- and IFN-dependent macrophage-intrinsic resistance mechanisms. This article summarizes the current knowledge of innate immune responses to L. pneumophila infection in general, and of macrophage-intrinsic defense mechanisms in particular.


Structural Insights into the Roles of the IcmS-IcmW Complex in the Type IVb Secretion System of Legionella pneumophila

Xu J, Xu D, Wan M, Yin L, Wang X, Wu L, Liu Y, Liu X, Zhou Y, Zhu Y.

Life Sciences Institute and Innovation Center for Cell Signaling Network, Zhejiang University, Hangzhou, Zhejiang, China.

Proc Natl Acad Sci USA 2017 Dec;114(51):13543-13548.

Abstract: The type IVb secretion system (T4BSS) of Legionella pneumophila is a multiple-component apparatus that delivers 300 virulent effector proteins into host cells. The injected effectors modulate host cellular processes to promote bacterial infection and proliferation. IcmS and IcmW are two conserved small, acidic adaptor proteins that form a binary complex to interact with many effectors and facilitate their translocation. IcmS and IcmW can also interact with DotL, an ATPase of the type IV coupling protein complex (T4CP). However, how IcmS-IcmW recognizes effectors, and what the roles of IcmS-IcmW are in T4BSSs are unclear. In this study, we found that IcmS and IcmW form a 1:1 heterodimeric complex to bind effector substrates. Both IcmS and IcmW adopt new structural folds and have no structural similarities with known effector chaperones. IcmS has a compact global structure with an α/β fold, while IcmW adopts a fully α-folded, relatively loose architecture. IcmS stabilizes IcmW by binding to its two C-terminal α-helices. Photocrosslinking assays revealed that the IcmS-IcmW complex binds its cognate effectors via an extended hydrophobic surface, which can also interact with the C terminus of DotL. A crystal structure of the DotL-IcmS-IcmW complex reveals extensive and highly stable interactions between DotL and IcmS-IcmW. Moreover, IcmS-IcmW recruits LvgA to DotL and assembles a unique T4CP. These data suggest that IcmS-IcmW also functions as an inseparable integral component of the DotL-T4CP complex in the bacterial inner membrane. This study provides molecular insights into the dual roles of the IcmS-IcmW complex in T4BSSs.


Multifaceted Supramolecular Interactions From C-Methylresorcin[4]arene Lead to an Enhancement in In Vitro Antibacterial Activity of Gatifloxacin

Dawn A, Chandra H, Ade-Browne C, Yadav J, Kumari H.

James L. Winkle College of Pharmacy, University of Cincinnati, 231Albert Sabin Way, Cincinnati, OH, USA.

Chemistry 2017 Dec:23(72):18171-18179. 

Abstract: Mimicking the antibacterial activity of polyphenols in synthetic systems is an attractive approach for the development of new active pharmaceutical ingredients. Resorcinarenes represent a class of polyphenols, which have been exploited for decades for their attractive chemical scaffold suitable for forming host-guest complexes with hydrophobic guest molecules. However, the polyphenolic character of resorcinarenes, which could be a potential asset to the pharmaceutical industry, have been least exploited. The present work represents an unprecedented interplay of antimicrobial activity of resorcinarene together with its ability to interact chemically with an antibacterial drug gatifloxacin, improving the overall antibacterial activity. The chemistry and the clinical activities involved in this study were investigated simultaneously by spectroscopic techniques, as well as by in vitro measurement of antibacterial activity toward two human bacterial pathogens, a Gram-positive pathogen Staphylococcus aureus and a Gram-negative lung pathogen Legionella pneumophila. The initial positive result obtained from this study could revolutionize the use of synthetically modifiable resorcinarenes and their analogues in fine tuning the clinical behavior of drugs.


Crystal Structure of the Legionella pneumophila Lpg2936 in Complex with the Cofactor S-adenosyl-L-methionine Reveals Novel Insights into the Mechanism of RsmE Family Methyltransferases

Pinotsis N, Waksman G.

Department of Biological Sciences, Institute of Structural and Molecular Biology, Birkbeck, London, United Kingdom.

Protein Sci Dec 2017;26(12):2381-2391.

Abstract: The methylation of U1498 located in the 16S ribosomal RNA of Escherichia coli is an important modification affecting ribosomal activity. RsmE methyltransferases methylate specifically this position in a mechanism that requires an S-adenosyl-L-methionine (AdoMet) molecule as cofactor. Here we report the structure of Apo and AdoMet-bound Lpg2936 from Legionella pneumophila at 1.5 and 2.3 Å, respectively. The protein comprises an N-terminal PUA domain and a C-terminal SPOUT domain. The latter is responsible for protein dimerization and cofactor binding. Comparison with similar structures suggests that Lpg2936 is a RsmE-like enzyme that can target the equivalent of U1498 in the L. pneumophila ribosomal RNA, thereby potentially enhancing ribosomal activity during infection-mediated effector production. The multiple copies of the enzyme found in both structures reveal a flexible conformation of the bound AdoMet ligand. Isothermal titration calorimetry measurements suggest an asymmetric two site binding mode. Our results therefore also provide unprecedented insights into AdoMet/RsmE interaction, furthering our understanding of the RsmE catalytic mechanism.


Legionella Blocks Autophagy by Cleaving STX17 (Syntaxin 17)

Arasaki K, Tagaya M.

School of Life Sciences, Tokyo University of Pharmacy and Life Sciences, Hachioji, Tokyo, Japan.

Autophagy 2017;13(11):2008-2009.

Abstract: Pathogens subvert host defense systems including autophagy and apoptosis for their survival and proliferation. Legionella pneumophila is a Gram-negative bacterium that grows in alveolar macrophages and causes severe pneumonia. Early during infection Legionella secretes effector proteins that convert the plasma membrane-derived vacuole containing Legionella into an endoplasmic reticulum (ER)-like replicative vacuole. These vacuoles ultimately fuse with the ER, where the pathogen replicates. Recently, we showed that one of the effectors, Lpg1137, is a serine protease that targets the mitochondria-associated ER membrane (MAM) and degrades STX17 (syntaxin 17), a SNARE implicated in macroautophagy/autophagy as well as mitochondria dynamics and membrane trafficking in fed cells. Degradation of STX17 blocks autophagy and BAX-induced apoptosis.


Molecular Evolution of Virulence Genes and Non-Virulence Genes in Clinical, Natural and Artificial Environmental Legionella pneumophila Isolates

Zhan XY, Zhu QY.

Guangzhou KingMed Center for Clinical Laboratory, Guangzhou, China.

PeerJ 2017 Dec;5:e4114.

Abstract: Background: L. pneumophila is the main causative agent of Legionnaires' disease. Free-living amoeba in natural aquatic environments is the reservoir and shelter for L. pneumophila. From natural water sources, L. pneumophila can colonize artificial environments such as cooling towers and hot-water systems, and then spread in aerosols, infecting the susceptible person. Therefore, molecular phylogeny and genetic variability of L. pneumophila from different sources (natural water, artificial water, and human lung tissue) might be distinct because of the selection pressure in different environments. Several studies researched genetic differences between L. pneumophila clinical isolates and environmental isolates at the nucleotide sequence level. These reports mainly focused on the analysis of virulence genes, and rarely distinguished artificial and natural isolates. Methods: We have used 139 L. pneumophila isolates to study their genetic variability and molecular phylogeny. These isolates include 51 artificial isolates, 59 natural isolates, and 29 clinical isolates. The nucleotide sequences of two representative non-virulence (NV) genes (trpA, cca) and three representative virulence genes (icmK, lspE, lssD) were obtained using PCR and DNA sequencing and were analyzed. Results: Levels of genetic variability including haplotypes, haplotype diversity, nucleotide diversity, nucleotide difference and the total number of mutations in the virulence loci were higher in the natural isolates. In contrast, levels of genetic variability including polymorphic sites, theta from polymorphic sites and the total number of mutations in the NV loci were higher in clinical isolates. A phylogenetic analysis of each individual gene tree showed three to six main groups, but not comprising the same L. pneumophila isolates. We detected recombination events in every virulence loci of natural isolates, but only detected them in the cca locus of clinical isolates. Neutrality tests showed that variations in the virulence genes of clinical and environmental isolates were under neutral evolution. TrpA and cca loci of clinical isolates showed significantly negative values of Tajima's D, Fu and Li's D* and F*, suggesting the presence of negative selection in NV genes of clinical isolates. Discussion: Our findings reinforced the point that the natural environments were the primary training place for L. pneumophila virulence, and intragenic recombination was an important strategy in the adaptive evolution of virulence gene. Our study also suggested the selection pressure had unevenly affected these genes and contributed to the different evolutionary patterns existed between NV genes and virulence genes. This work provides clues for future work on population-level and genetics-level questions about ecology and molecular evolution of L. pneumophila, as well as genetic differences of NV genes and virulence genes between this host-range pathogen with different lifestyles.


Disulfide Loop Cleavage of Legionella pneumophila PlaA Boosts Lysophospholipase A Activity

Lang C, Hiller M, Flieger A.

Division of Enteropathogenic Bacteria and Legionella, Robert Koch-Institut, Wernigerode, Germany.

Sci Rep 2017 Nov;7(1):16313.

Abstract: L. pneumophila, an important facultative intracellular bacterium, infects the human lung and environmental protozoa. At least fifteen phospholipases A (PLA) are encoded in its genome. Three of which, namely PlaA, PlaC, and PlaD, belong to the GDSL lipase family abundant in bacteria and higher plants. PlaA is a lysophospholipase A (LPLA) that destabilizes the phagosomal membrane in absence of a protective factor. PlaC shows PLA and glycerophospholipid: cholesterol acyltransferase (GCAT) activities which are activated by zinc metalloproteinase ProA via cleavage of a disulphide loop. In this work, we compared GDSL enzyme activities, their secretion, and activation of PlaA. We found that PlaA majorly contributed to LPLA, PlaC to PLA, and both substrate-dependently to GCAT activity. Western blotting revealed that PlaA and PlaC are type II-secreted and both processed by ProA. Interestingly, ProA steeply increased LPLA but diminished GCAT activity of PlaA. Deletion of 20 amino acids within a predicted disulfide loop of PlaA had the same effect. In summary, we propose a model by which ProA processes PlaA via disulfide loop cleavage leading to a steep increase in LPLA activity. Our results help to further characterize the L. pneumophila GDSL hydrolases, particularly PlaA, an enzyme acting in the Legionella-containing phagosome.


Formation of the Legionella Replicative Compartment at the Crossroads of Retrograde Trafficking

Bärlocher K, Welin A, Hilbi H.

Institute of Medical Microbiology, University of Zürich, Zurich, Switzerland.

Front Cell Infect Microbiol 2017 Nov;7:482.

Abstract: Retrograde trafficking from the endosomal system through the Golgi apparatus back to the endoplasmic reticulum is an essential pathway in eukaryotic cells, serving to maintain organelle identity and to recycle empty cargo receptors delivered by the secretory pathway. Intracellular replication of several bacterial pathogens, including Legionella pneumophila, is restricted by the retrograde trafficking pathway. L. pneumophila employs the Icm/Dot type IV secretion system (T4SS) to form the replication-permissive Legionella-containing vacuole (LCV), which is decorated with multiple components of the retrograde trafficking machinery as well as retrograde cargo receptors. The L. pneumophila effector protein RidL is secreted by the T4SS and interferes with retrograde trafficking. Here, we review recent evidence that the LCV interacts with the retrograde trafficking pathway, discuss the possible sites of action and function of RidL in the retrograde route, and put forth the hypothesis that the LCV is an acceptor compartment of retrograde transport vesicles.


Legionella Becoming a Mutualist: Adaptive Processes Shaping the Genome of Symbiont in the Louse Polyplax Serrata

Ríhová J, Nováková E, Husník F, Hypša V.

Department of Parasitology, University of South Bohemia, Ceské Budejovice, Czech Republic.

Genome Biol Evol 2017 Nov;9(11):2946-2957.

Abstract: Legionellaceae are intracellular bacteria known as important human pathogens. In the environment, they are mainly found in biofilms associated with amoebas. In contrast to the gammaproteobacterial family Enterobacteriaceae, which established a broad spectrum of symbioses with many insect taxa, the only instance of legionella-like symbiont has been reported from lice of the genus Polyplax. Here, we sequenced the complete genome of this symbiont and compared its main characteristics to other Legionella species and insect symbionts. Based on rigorous multigene phylogenetic analyses, we confirm this bacterium as a member of the genus Legionella and propose the name Candidatus Legionella polyplacis, sp.n. We show that the genome of Ca. Legionella polyplacis underwent massive degeneration, including considerable size reduction (529.746 bp, 484 protein coding genes) and a severe decrease in GC content (23%). We identify several possible constraints underlying the evolution of this bacterium. On one hand, Ca. Legionella polyplacis and the louse symbionts Riesia and Puchtella experienced convergent evolution, perhaps due to adaptation to similar hosts. On the other hand, some metabolic differences are likely to reflect different phylogenetic positions of the symbionts and hence availability of particular metabolic function in the ancestor. This is exemplified by different arrangements of thiamine metabolism in Ca. Legionella polyplacis and Riesia. Finally, horizontal gene transfer is shown to play a significant role in the adaptive and diversification process. Particularly, we show that Ca. L. polyplacis horizontally acquired a complete biotin operon (bioADCHFB) that likely assisted this bacterium when becoming an obligate mutualist.


Growth of Legionella anisa in a Model Drinking Water System to Evaluate Different Shower Outlets and the Impact of Cast Iron Rust

van der Lugt W, Euser SM, Bruin JP, Den Boer JW, Walker JT, Crespi S.

Van der Lugt B.V., Heiligland 28A, 1821 AC, Alkmaar, The Netherlands.

Int J Hyg Environ Health 2017 Nov;220(8):1295-1308.

Abstract: Legionella continues to be a problem in water systems. This study investigated the influence of different shower mixer faucets, and the influence of the presence of cast iron rust from a drinking water system on the growth of Legionella. The research is conducted using a model of a household containing four drinking water systems. All four systems, which contained standard plumbing components including copper pipes and a water heater, were filled with unchlorinated drinking water. Furthermore, all systems had three different shower faucets: (A) a stainless-steel faucet, (B) a brass-ceramic faucet, and (C) a brass thermostatic faucet. System 1 was solely filled with drinking water. System 2 was filled with drinking water and cast iron rust. System 3 was contaminated with Legionella, and system 4 was contaminated with a Legionella, and cast iron rust. During a period of 34 months, 450 cold water samples were taken from 15 sample points of the four drinking water systems and tested for Legionella according to the Dutch Standard (NEN 6265). In system 4, with added cast iron rust, the stainless-steel mixer faucet (A) had the highest concentration of Legionella at >4.3 log10 CFU/l (>20,000 CFU/l) and was positive in 46.4% of samples. In contrast, the stainless-steel mixer faucet (A) of system 3 without cast iron rust showed 14.3% positive samples with a maximum concentration of 3.9 log10 CFU/l (7600 CFU/l) Legionella. Additionally, both contaminated systems (3 and 4), with the brass thermostatic faucet (C), tested positive for Legionella. System 3 in 85.7% of the samples, with a maximum concentration of 4.38 log10 CFU/l (24,200 CFU/l), and system 4 in 64.3% of the samples with a maximum concentration of 4.13 log10 CFU/l (13.400 CFU/l). These results suggest that both the type of faucet used in a drinking water system and the presence or absence of cast iron rust influence the growth of Legionella.


Toll-like Receptor 2 and Its Roles in Immune Responses Against Legionella pneumophila

Ahmadishoar S, Kariminik A.

Department of Microbiology, Kerman Branch, Islamic Azad University, Kerman, Iran.

Life Sci 2017 Nov;188:158-162. 

Abstract: Legionella pneumophila (L. pneumophila) is an intracellular bacterium which can be survived in the human macrophage’s phagosomes. The infectious agent is cleared in some cases and survived in others. The main mechanisms responsible for survival of L. pneumophila are yet to be clarified. It has been reported that innate immunity plays key roles in limitation and also eradication of bacterial infections. Toll like receptor 2 (TLR2) is an important cell membrane receptor which recognizes a wide range of bacterial antigens entitled pathogen associated molecular patterns (PAMPs). The aim of the current review article is to present recent data regarding the roles of TLR2 in induction of immune responses and consequently eradication of L. pneumophila. Additionally, the main mechanisms used by L. pneumophila to overcome TLR2 dependent immune responses are discussed in this review article.


Identification of Conserved ABC Importers Necessary for Intracellular Survival of Legionella pneumophila in Multiple Hosts

Lama A, Drennan SL, Johnson RC, Rubenstein GL, Cambronne ED.

Department of Molecular Microbiology and Immunology, Oregon Health and Science University, Portland, OR, USA.

Front Cell Infect Microbiol 2017 Nov;7:485. 

Abstract: It is established that the human pathogen Legionella pneumophila becomes significantly augmented for infection of macrophages after intracellular growth in amoebae when compared to like-strains cultivated in laboratory media. Based on this observation, we reasoned that the most critical virulence determinants of L.p. are expressed by responding to stimuli generated by the protozoan host specifically; a process we term "protozoan-priming." We sought to identify L.p. virulence factors that were required for replication in amoebae in order to highlight the genes necessary for production of the most infectious form of the bacterium. Using a transposon mutagenesis screen, we successfully identified 12 insertions that produced bacteria severely attenuated for growth in amoebae, while retaining a functional Dot/Icm type IVb secretion system. Seven of these insertion mutants were found dispensable for growth in macrophages, revealing attractive therapeutic targets that reside upstream of the pathogen-human interface. Two candidates identified, lpg0730 and lpg0122 were required for survival and replication in amoebae and macrophage host cells. Both genes are conserved among numerous important human pathogenic bacteria that can persist or replicate in amoebae. Each gene encodes a component of an ATP binding cassette (ABC) transport complex of unknown function. We demonstrate the lpg0730 ortholog in Francisella tularensis subsp. novicida to be essential for colonization of both protozoan and mammalian host cells, highlighting conserved survival mechanisms employed by bacteria that utilize protozoa as an environmental reservoir for replication.


From Many Hosts, One Accidental Pathogen: The Diverse Protozoan Hosts of Legionella

Boamah DK, Zhou G, Ensminger AW, O'Connor TJ.

Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD, USA.

Front Cell Infect Microbiol 2017 Nov;7:477.

Abstract: The 1976 outbreak of Legionnaires' disease led to the discovery of the intracellular bacterial pathogen Legionella pneumophila. Given their impact on human health, Legionella species and the mechanisms responsible for their replication within host cells are often studied in alveolar macrophages, the primary human cell type associated with disease. Despite the potential severity of individual cases of disease, Legionella are not spread from person-to-person. Thus, from the pathogen's perspective, interactions with human cells are accidents of time and space-evolutionary dead ends with no impact on Legionella's long-term survival or pathogenic trajectory. To understand Legionella as a pathogen is to understand its interaction with its natural hosts: the polyphyletic protozoa, a group of unicellular eukaryotes with a staggering amount of evolutionary diversity. While much remains to be understood about these enigmatic hosts, we summarize the current state of knowledge concerning Legionella's natural host range, the diversity of Legionella-protozoa interactions, the factors influencing these interactions, the importance of avoiding the generalization of protozoan-bacterial interactions based on a limited number of model hosts and the central role of protozoa to the biology, evolution, and persistence of Legionella in the environment.


Positive and Negative Regulation of the Master Metabolic Regulator mTORC1 by Two Families of Legionella pneumophila Effectors

De Leon JA, Qiu J, Nicolai CJ, Counihan JL, Barry KC, Xu L, Lawrence RE, Castellano BM, Zoncu R, Nomura DK, Luo ZQ, Vance RE.

Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA USA.

Cell Rep 2017 Nov;21(8):2031-2038.

Abstract: All pathogens must acquire nutrients from their hosts. The intracellular bacterial pathogen Legionella pneumophila, the etiological agent of Legionnaires' disease, requires host amino acids for growth within cells. The mechanistic target of rapamycin complex 1 (mTORC1) is an evolutionarily conserved master regulator of host amino acid metabolism. Here, we identify two families of translocated L. pneumophila effector proteins that exhibit opposing effects on mTORC1 activity. The Legionella glucosyltransferase (Lgt) effector family activates mTORC1, through inhibition of host translation, whereas the SidE/SdeABC (SidE) effector family acts as mTORC1 inhibitors. We demonstrate that a common activity of both effector families is to inhibit host translation. We propose that the Lgt and SidE families of effectors work in concert to liberate host amino acids for consumption by L. pneumophila.


Multiple Legionella pneumophila Effector Virulence Phenotypes Revealed Through High-Throughput Analysis of Targeted Mutant Libraries

Shames SR, Liu L, Havey JC, Schofield WB, Goodman AL, Roy CR.

Department of Microbial Pathogenesis, Yale University School of Medicine, New Haven, CT, USA.

Proc Natl Acad Sci USA 2017 Nov;114(48):E10446-E10454.

Abstract: Legionella pneumophila is the causative agent of a severe pneumonia called Legionnaires' disease. A single strain of L. pneumophila encodes a repertoire of over 300 different effector proteins that are delivered into host cells by the Dot/Icm type IV secretion system during infection. The large number of L. pneumophila effectors has been a limiting factor in assessing the importance of individual effectors for virulence. Here, a transposon insertion sequencing technology called INSeq was used to analyze replication of a pool of effector mutants in parallel both in a mouse model of infection and in cultured host cells. Loss-of-function mutations in genes encoding effector proteins resulted in host-specific or broad virulence phenotypes. Screen results were validated for several effector mutants displaying different virulence phenotypes using genetic complementation studies and infection assays. Specifically, loss-of-function mutations in the gene encoding LegC4 resulted in enhanced L. pneumophila in the lungs of infected mice but not within cultured host cells, which indicates LegC4 augments bacterial clearance by the host immune system. The effector proteins RavY and Lpg2505 were important for efficient replication within both mammalian and protozoan hosts. Further analysis of Lpg2505 revealed that this protein functions as a metaeffector that counteracts host cytotoxicity displayed by the effector protein SidI. Thus, this study identified a large cohort of effectors that contribute to L. pneumophila virulence positively or negatively and has demonstrated regulation of effector protein activities by cognate metaeffectors as being critical for host pathogenesis.


Legionella pneumophila CsrA Regulates a Metabolic Switch from Amino Acid to Glycerolipid Metabolism

Häuslein I, Sahr T, Escoll P, Klausner N, Eisenreich W, Buchrieser C.

Institut Pasteur, Biologie des Bactéries Intracellulaires, Paris, France.

Open Biol 2017 Nov;7(11):170149.

Abstract: Legionella pneumophila CsrA plays a crucial role in the life-stage-specific expression of virulence phenotypes and metabolic activity. However, its exact role is only partly known. To elucidate how CsrA impacts L. pneumophila metabolism we analysed the CsrA depended regulation of metabolic functions by comparative 13C-isotopologue profiling and oxygen consumption experiments of a L. pneumophila wild-type (wt) strain and its isogenic csrA- mutant. We show that a csrA- mutant has significantly lower respiration rates when serine, alanine, pyruvate, α-ketoglutarate or palmitate is the sole carbon source. By contrast, when grown in glucose or glycerol, no differences in respiration were detected. Isotopologue profiling uncovered that the transfer of label from [U-13C3]serine via pyruvate into the citrate cycle and gluconeogenesis was lower in the mutant as judged from the labelling patterns of protein-derived amino acids, cell-wall-derived diaminopimelate, sugars and amino sugars and 3-hydroxybutyrate derived from polyhydroxybutyrate (PHB). Similarly, the incorporation of [U-13C6]glucose via the glycolysis/Entner-Doudoroff (ED) pathway but not via the pentose phosphate pathway was repressed in the csrA- mutant. On the other hand, fluxes due to [U-13C3]glycerol utilization were increased in the csrA- mutant. In addition, we showed that exogenous [1,2,3,4-13C4]palmitic acid is efficiently used for PHB synthesis via 13C2-acetyl-CoA. Taken together, CsrA induces serine catabolism via the tricarboxylic acid cycle and glucose degradation via the ED pathway, but represses glycerol metabolism, fatty acid degradation and PHB biosynthesis, in particular during exponential growth. Thus, CsrA has a determining role in substrate usage and carbon partitioning during the L. pneumophila life cycle and regulates a switch from amino acid usage in replicative phase to glycerolipid usage during transmissive growth.


Minimum Inhibitory Concentration (MIC) Distribution Among Wild-Type Strains of Legionella pneumophila Identifies a Subpopulation with Reduced Susceptibility to Macrolides Owing to Efflux Pump Genes

Vandewalle-Capo M, Massip C, Descours G, Charavit J, Chastang J, Billy PA, Boisset S, Lina G, Gilbert C, Maurin M, Jarraud S, Ginevra C.

International Center for Infectiology Research, Université Lyon 1, Ecole normale supérieure de Lyon, Lyon, France.

Int J Antimicrob Agents Nov 2017;50(5):684-689.

Abstract: Legionnaires' disease is a severe pneumonia mainly caused by Legionella pneumophila that is treated by antibiotics. The purpose of this study was to describe the susceptibility of clinical strains of L. pneumophila to eight antibiotics used for treatment of legionellosis. The minimum inhibitory concentrations (MICs) of 109 well-characterised clinical strains of L. pneumophila serogroup 1 were determined by the broth microdilution method without charcoal and were compared with antibiotic-resistant strains selected in vitro. All strains were inhibited by low concentrations of fluoroquinolones, macrolides and rifampicin. The epidemiological cut-off values (ECOFFs) were 0.064 mg/L for ciprofloxacin, 0.064 mg/L for moxifloxacin, 0.032 mg/L for levofloxacin, 1 mg/L for erythromycin, 2 mg/L for azithromycin, 0.064 mg/L for clarithromycin, 2 mg/L for doxycycline and 0.001 mg/L for rifampicin. However, MIC distributions revealed a subpopulation of strains displaying reduced susceptibility to some macrolides (especially azithromycin), which correlated with the presence of the lpeAB genes encoding a macrolide efflux pump found specifically in sequence type (ST) ST1, ST701 and closely related STs. Thus, all isolates could be considered susceptible to the tested antibiotics, although macrolides were less active against some strains harbouring a specific efflux system.


Bacterial Community Dynamics in a Cooling Tower with Emphasis on Pathogenic Bacteria and Legionella Species Using Universal and Genus-Specific Deep Sequencing

Pereira RPA, Peplies J, Höfle MG, Brettar I.

Department of Vaccinology and Applied Microbiology, Helmholtz Centre for Infection Research (HZI), Braunschweig, Germany.

Water Res 2017 Oct;122:363-376.

Abstract: Cooling towers are the major source of outbreaks of legionellosis in Europe and worldwide. These outbreaks are mostly associated with Legionella species, primarily L. pneumophila, and its surveillance in cooling tower environments is of high relevance to public health. In this study, a combined NGS-based approach was used to study the whole bacterial community, specific waterborne and water-based bacterial pathogens, especially Legionella species, targeting the 16S rRNA gene. This approach was applied to water from a cooling tower obtained by monthly sampling for two years. The studied cooling tower was an open circuit cooling tower with lamellar cooling situated in Braunschweig, Germany. A highly diverse bacterial community was observed with 808 genera including 25 potentially pathogenic taxa using universal 16S rRNA primers. Sphingomonas and Legionella were the most abundant pathogenic genera. By applying genus-specific primers for Legionella, a diverse community with 85 phylotypes, and a representative core community with substantial temporal heterogeneity was observed. A high percentage of sequences (65%) could not be affiliated to an acknowledged species. L. pneumophila was part of the core community and the most abundant Legionella species reinforcing the importance of cooling towers as its environmental reservoir. Major temperature shifts (>10°C) were the key environmental factor triggering the reduction or dominance of the Legionella species in the Legionella community dynamics. In addition, interventions by chlorine dioxide had a strong impact on the Legionella community composition but not on the whole bacterial community. Overall, the presented results demonstrated the value of a combined NGS approach for the molecular monitoring and surveillance of health-related pathogens in man-made freshwater systems.


Experimental Human-Like Model to Assess the Part of Viable Legionella Reaching the Thoracic Region After Nebulization

Pourchez J, Leclerc L, Girardot F, Riffard S, Prevot N, Allegra S.

University of Lyon, UJM-Saint-Etienne, Saint-Etienne, France.

PLoS One 2017 Oct;12(10):e0186042.

Abstract: The incidence of Legionnaires' disease (LD) in European countries and the USA has been constantly increasing since 1998. Infection of humans occurs through aerosol inhalation. To bridge the existing gap between the concentration of Legionella in a water network and the deposition of bacteria within the thoracic region (assessment of the number of viable Legionella), we validated a model mimicking realistic exposure through the use of (i) recent technology for aerosol generation and (ii) a 3D replicate of the human upper respiratory tract. The model's sensitivity was determined by monitoring the deposition of (i) aerosolized water and Tc99m radio-aerosol as controls, and (ii) bioaerosols generated from both Escherichia coli and Legionella pneumophila sg 1 suspensions. The numbers of viable Legionella prior to and after nebulization were provided by culture, flow cytometry and qPCR. This study was designed to obtain more realistic data on aerosol inhalation (vs. animal experimentation) and deposition at the thoracic region in the context of LD. Upon nebulization, 40% and 48% of the initial Legionella inoculum was made of cultivable and non-cultivable cells, respectively; 0.7% of both populations reached the filter holder mimicking the thoracic region in this setup. These results are in agreement with experimental data based on quantitative microbial risk assessment methods and bring new methods that may be useful for preventing LD.


Interactive Effects of Temperature, Organic Carbon, and Pipe Material on Microbiota Composition and Legionella pneumophila in Hot Water Plumbing Systems

Proctor CR, Dai D, Edwards MA, Pruden A.

Department of Civil and Environmental Engineering, Virginia Tech, Blacksburg, Virginia, USA.

Microbiome 2017 Oct;5(1):130.

Abstract: Background: Several biotic and abiotic factors have been reported to influence the proliferation of microbes, including Legionella pneumophila, in hot water premise plumbing systems, but their combined effects have not been systematically evaluated. Here, we utilize simulated household water heaters to examine the effects of stepwise increases in temperature (32-53°C), pipe material (copper vs. cross-linked polyethylene (PEX)), and influent assimilable organic carbon (0-700 μg/L) on opportunistic pathogen gene copy numbers and the microbiota composition, as determined by quantitative polymerase chain reaction and 16S rRNA gene amplicon sequencing. Results: Temperature had an overarching influence on both the microbiota composition and L. pneumophila numbers. L. pneumophila peaked at 41°C in the presence of PEX (1.58×105 gene copies/mL). At 53°C, L. pneumophila was not detected. Several operational taxonomic units (OTUs) persisted across all conditions, accounting for 50% of the microbiota composition from 32 to 49°C and 20% at 53°C. Pipe material most strongly influenced microbiota composition at lower temperatures, driven by five to six OTUs enriched with each material. Copper pipes supported less L. pneumophila than PEX pipes (mean 2.5 log10 lower) at temperatures ≤41°C but showed no difference in total bacterial numbers. Differences between pipe materials diminished with elevated temperature, probably resulting from decreased release of copper ions. At temperatures ≤45°C, influent assimilable organic carbon correlated well with total bacterial numbers, but not with L. pneumophila numbers. At 53°C, PEX pipes leached organic carbon, reducing the importance of dosed organic carbon. L. pneumophila numbers correlated with a Legionella OTU and a Methylophilus OTU identified by amplicon sequencing. Conclusions: Temperature was the most effective factor for the control of L. pneumophila, while microbiota composition shifted with each stepwise temperature increase. While copper pipe may also help shape the microbiota composition and limit L. pneumophila proliferation, its benefits might be constrained at higher temperatures. Influent assimilable organic carbon affected total bacterial numbers but had minimal influence on opportunistic pathogen gene numbers or microbiota composition. These findings provide guidance among multiple control measures for the growth of opportunistic pathogens in hot water plumbing and insight into the mediating role of microbial ecological factors.


ER Remodeling by the Large GTPase Atlastin Promotes Vacuolar Growth of Legionella pneumophila

Steiner B, Swart AL, Welin A, Weber S, Personnic N, Kaech A, Freyre C, Ziegler U, Klemm RW, Hilbi H.

Institute of Medical Microbiology, University of Zürich, Zürich, Switzerland.

EMBO Rep 2017 Oct;18(10):1817-1836.

Abstract: The pathogenic bacterium Legionella pneumophila replicates in host cells within a distinct ER-associated compartment termed the Legionella-containing vacuole (LCV). How the dynamic ER network contributes to pathogen proliferation within the nascent LCV remains elusive. A proteomic analysis of purified LCVs identified the ER tubule-resident large GTPase atlastin3 (Atl3, yeast Sey1p) and the reticulon protein Rtn4 as conserved LCV host components. Here, we report that Sey1/Atl3 and Rtn4 localize to early LCVs and are critical for pathogen vacuole formation. Sey1 overproduction promotes intracellular growth of L. pneumophila, whereas a catalytically inactive, dominant-negative GTPase mutant protein, or Atl3 depletion, restricts pathogen replication and impairs LCV maturation. Sey1 is not required for initial recruitment of ER to PtdIns(4)P-positive LCVs but for subsequent pathogen vacuole expansion. GTP (but not GDP) catalyzes the Sey1-dependent aggregation of purified, ER-positive LCVs in vitro Thus, Sey1/Atl3-dependent ER remodeling contributes to LCV maturation and intracellular replication of L. pneumophila.