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. ekmoon@khu.ac.kr
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. cbuch@pasteur.fr
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. a.laheij@acta.nl
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; tkubori@gifu-u.ac.jp
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. mohi@umich.edu
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. machnerm@mail.nih.gov
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. L. pneumophila 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 L. pneumophila 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, L. pneumophila DUBs
are not limited to enzymes that exhibit canonical DUB activity. Some L. pneumophila 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 L. pneumophila 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 L. pneumophila 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. shivani.pasricha@unimelb.edu.au
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. marta.szysz@poczta.umcs.lublin.pl
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. ym253@cornell.edu
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. nela.malatesti@biotech.uniri.hr
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. sandrine.demaneche@amoeba-biocide.com
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. a.meir@mail.cryst.bbk.ac.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. james.garnett@kcl.ac.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. n-cianciotto@northwestern.edu
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. klevit@uw.edu
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. a.itzen@uke.de
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. chenyusy@hotmail.com
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. fengyue@mail.buct.edu.cn
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. shi2pulm@tmd.ac.jp
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. sshames@ksu.edu
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. ralph.isberg@tufts.edu
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. belyi@gamaleya.org
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. julien.verdon@univ-poitiers.fr
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. hilbi@imm.uzh.ch
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. bernd.schmeck@uni-marburg.de
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. Thierry.Soldati@unige.ch
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.
Deecker SR, Ensminger AW.
Department of Molecular Genetics, University of Toronto, Ontario, Canada. alex.ensminger@utoronto.ca
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.
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. ouyangsy@fjnu.edu.cn
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.
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. elizabeth.hartland@hudson.org.au
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.
Yan
F, Zhu Y, Zhou Y.
Institute of Microbiology, College of Life
Sciences, Zhejiang University, Hangzhou, Zhejiang, China. zhouyanlsi@zju.edu.cn
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.
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.
sandrine.demaneche@amoeba-biocide.com
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. pneumophila. Willaertia 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.
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. ghh_ahu@foxmail.com
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. hkhalil74@gmail.com
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.
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. sandy.slow@otago.ac.nz
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.
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. gils@tauex.tau.ac.il.
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.
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. apruden@vt.edu
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.
McArthur
JB, Santra A, Li W, Kooner AS, Liu Z, Yu H, Chen
X.
Department
of Chemistry, University of California, CA, USA. xiichen@ucdavis.edu
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).
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. qiujz@jlu.edu.cn
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.
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. severine.allegra@univ-st-etienne.fr
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.
Price JV, Russo D, Ji DX, Chavez RA, DiPeso L,
Lee AY, Coers J, Vance RE.
Department of Biology, Oberlin College, Oberlin, Ohio,
USA.
jprice@oberlin.edu e rvance@berkeley.edu
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.
Ge
ZH, Long QS, Yuan PB, Pan X, Shen D, Lu YJ.
School of Life Sciences, Sun Yat-sen University,
Guangzhou, China. luyj@mail.sysu.edu.cn
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.
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. ym253@cornell.edu
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.
Inaba
JI, Xu K, Kovalev N, Ramanathan H, Roy CR,
Lindenbach BD, Nagy PD.
Department
of Plant Pathology, University of Kentucky, Lexington, KY, USA. pdnagy2@uky.edu
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.
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. gabriele.meloni@utdallas.edu
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.
Fischer
JT, Hossain S, Boon EM.
Department of Chemistry and Institute of
Chemical Biology & Drug Discovery, Stony Brook University, Stony Brook, New
York, USA. elizabeth.boon@stonybrook.edu
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.
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. shaeri.mukherjee@ucsf.edu
Department of Genetics, Stanford
University School of Medicine, Stanford, CA, USA. bassik@stanford.edu
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.
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. marta.szysz@poczta.umcs.lublin.pl
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).
White
RC, Truchan HK, Zheng H, Tyson JY, Cianciotto
NP.
Department of Microbiology and Immunology,
Northwestern University Medical School, Chicago, Illinois, USA. n-cianciotto@northwestern.edu
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.
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. jdcirillo@tamu.edu
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.
Liu
X, Shin S.
Department of Microbiology, University of
Pennsylvania Perelman School of Medicine, 3610 Hamilton Walk, Johnson Pavilion
201B, Philadelphia, PA 19104, USA. sunshin@pennmedicine.upenn.edu
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.
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. xavier.charpentier@univ-lyon1.fr
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.
Gomez-Valero L, Chiner-Oms A, Comas I, Buchrieser
C.
Institut Pasteur, Departement of
Microbiology, Biologie des Bactéries Intracellulaires, Paris, France. lgomez@pasteur.fr e cbuch@pasteur.fr
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.
Weber
SS, Stoycheva D, Nimmerjahn F, Oxenius A.
Institute of Microbiology, Zürich, Switzerland. aoxenius@micro.biol.ethz.ch
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.
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.
takehara@cc.tuat.ac.jp
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.
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. abukwaik@louisville.edu
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.
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. qintian@icdc.cn
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.
Hughes
ED, Byrne BG, Swanson MS.
Department of Microbiology and Immunology, University
of Michigan Medical School, Ann Arbor, Michigan, USA. mswanson@umich.edu
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.
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. jensen@caltech.edu
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.
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. cdl1978119@sina.com
e jpchen007@163.com
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.
Gomez-Valero L,
Buchrieser C.
Institut Pasteur, Biologie des Bactéries
Intracellulaires and CNRS UMR 3525, 75724, Paris, France. cbuch@pasteur.fr
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.
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. vfuochi@unict.it
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.
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.
jdcirillo@tamu.edu
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.
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. dszamboni@fmrp.usp.br
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.
Ngwaga
T, Hydock AJ, Ganesan S, Shames SR.
Division of Biology, Kansas State
University, Manhattan, Kansas, USA. sshames@ksu.edu
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.
White
RC, Cianciotto NP.
Department of Microbiology and Immunology, Northwestern
University Medical School, Chicago, IL 60611, USA. n-cianciotto@northwestern.edu
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.
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. luyj@mail.sysu.edu.cn
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.
Nakanishi N,
Nomoto R, Tanaka S, Arikawa K, Iwamoto T.
Department of Infectious Diseases, Kobe
Institute of Health, Kobe, Japan. noriko_nakanishi@office.city.kobe.lg.jp
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.
Gomez-Valero L,
Buchrieser C.
Institut Pasteur, Biologie des Bactéries
Intracellulaires and CNRS UMR 3525, 75724, Paris, France. cbuch@pasteur.fr
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.
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. Gomez-Alvarez.Vicente@epa.gov
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.
Birteksöz-Tan
AS, Zeybek Z.
Department of Pharmaceutical Microbiology, Faculty of
Pharmacy, Istanbul University, Beyazit, Istanbul, Turkey. seherbirteksoz@hotmail.com
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.
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. horn@microbial-ecology.net
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.
Levin TC, Goldspiel BP, Malik HS.
Howard Hughes Medical Institute, Fred
Hutchinson Cancer Research Center, Seattle, USA. tlevin@fredhutch.org
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.
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. terebiznik@utsc.utoronto.ca
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.
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. mhalpern@research.haifa.ac.il
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.
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. jan.j.dubowski@usherbrooke.ca
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.
Moss
SM, Taylor IR, Ruggero D, Gestwicki JE, Shokat
KM, Mukherjee S.
Department of Microbiology and Immunology,
University of California, San Francisco, CA, USA. shaeri.mukherjee@ucsf.edu
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.
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. hilbi@imm.uzh.ch
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.
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. xingchen@pku.edu.cn
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.
Mobarez AM,
Rajabi RA, Salmanian AH, Khoramabadi N, Hosseini Doust SR.
Department of Bacteriology, Faculty of Medical Sciences,
Tarbiat Modares University, Tehran, Iran. mmmobarez@modares.ac.ir
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. lgomez@pasteur.fr
e cbuch@pasteur.fr
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.
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. g.schroeder@qub.ac.uk
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.
Gintner M, Yoneda Y, Schmölzer
C, Denner C, Kählig H, Schmid W.
AG Schmid, Institute of Organic Chemistry,
University of Vienna, Vienna, Austria. manuel.gintner@univie.ac.at
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.
Wang C, Chuai X, Liang M.
Department of Pathogenic Biology, Hebei Medical
University, Shijiazhuang, China. greencity2008@126.com
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.
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. Jason.King@sheffield.ac.uk
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.
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. ralph.isberg@tufts.edu
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.
Voth
KA, Chung IYW, van Straaten K, Li L, Boniecki MT,
Cygler M.
Department of Biochemistry, University of
Saskatchewan, Saskatoon, Canada.
miroslaw.cygler@usask.ca
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.
Bernier
AM, Bernard K.
National Microbiology Laboratory (NML)-CSCHAH
Site, Public Health Agency of Canada, Winnipeg, Canada. Kathy.bernard@canada.ca
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%.
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. ali.chakeri@ages.at
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.
Esna Ashari Z,
Brayton KA, Broschat SL.
School of Electrical Engineering and
Computer Science, Washington State University, Pullman, Washington, USA. z.esnaashariesfahan@wsu.edu
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.
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. munekazu@m.kufm.kagoshima-u.ac.jp
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.
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. luoz@purdue.edu
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.
Best AM, Abu Kwaik Y.
Center for Predictive Medicine, College
of Medicine, University of Louisville, Louisville, Kentucky. abukwaik@louisville.edu
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.
Corre
MH, Delafont V, Legrand A, Berjeaud JM, Verdon
J.
Laboratoire Ecologie et Biologie des Interactions, UMR
CNRS 7267, Université de Poitiers, Poitiers, France. julien.verdon@univ-poitiers.fr
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. Aeromonas, Bacillus, Flavobacterium,
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 Acinetobacter, Kluyvera, Rahnella, 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.
Inoue H, Agata K, Ohta H.
Tsukuba Research Laboratories, Aquas
Corporation. h_inoue0417@aquas.co.jp
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.
Rolando, Buchrieser C.
CNRS UMR 3525, Paris, France. cbuch@pasteur.fr
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.
Welin A, Weber S, Hilbi H.
Institute of Medical Microbiology, University of Zürich,
Zürich, Switzerland. awelin@imm.uzh.ch
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.
Heuner
K, Kunze M, Chen F, Eisenreich W.
Lehrstuhl für Biochemie, Technische
Universität München, Garching, Germany. wolfgang.eisenreich@mytum.de.
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.
Amaro
F, Shuman H.
Department
of Microbiology, University of Chicago, Chicago, IL, USA. hashuman@uchicago.edu
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.
Scheithauer
L, Steinert M.
Institut für Mikrobiologie, Technische
Universität Braunschweig, Braunschweig, Germany. m.steinert@tu-bs.de
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.
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. hnewton@unimelb.edu.au
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.
Maaß S, Moog G, Becher D.
Institute of Microbiology, Center for Functional
Genomics of Microbes, University of Greifswald, Greifswald, Germany. dbecher@uni-greifswald.de
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.
Pérez-Cobas
AE, Buchrieser C.
Institut Pasteur, Biologie des Bactéries
Intracellulaires, Paris, France. cbuch@pasteur.fr
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.
Ng
GZ, Solomatina A, van Driel IR, Hartland EL.
Centre for Innate Immunity and Infectious
Diseases, Hudson Institute of Medical Research, Clayton, VIC, Australia. elizabeth.hartland@hudson.org.au
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.
Brassinga AKC,
Sifri CD.
Department of Microbiology, University of
Manitoba, Winnipeg, MB, Canada. Ann.Brassinga@umanitoba.ca
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.
Frankel
G, Schroeder GN.
Wellcome-Wolfson Institute for
Experimental Medicine, Queen's University Belfast, Belfast, UK. g.schroeder@qub.ac.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.
Mascarenhas
DPA, Zamboni DS.
Department of Cell Biology, School of
Medicine of Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP,
Brazil. dszamboni@fmrp.usp.br
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.
Levanova N,
Tabakova I, Jank T, Belyi Y.
Gamaleya Research Centre, Moscow, Russia. belyi@gamaleya.org
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.
Qiu J, Luo ZQ.
Department of Biological Sciences, Purdue
Institute for Inflammation, Immunology and Infectious Disease, Purdue
University, West Lafayette, IN, USA. luoz@purdue.edu
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.
Steiner
B, Swart AL, Hilbi H.
Institute of Medical Microbiology, University of Zürich,
Zürich, Switzerland. hilbi@imm.uzh.ch
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.
Shames
SR.
Division of Biology, Kansas State University, Manhattan,
KS, USA. sshames@ksu.edu
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.
Hardy, 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, Univ Lyon, Villeurbanne, France. xavier.charpentier@univ-lyon1.fr
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.
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. christophe.gilbert.bio@univ-lyon1.fr
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.
Weber
S, Steiner B, Welin A, Hilbi H.
Institute of Medical Microbiology,
University of Zürich, Zürich, Switzerland. hilbi@imm.uzh.ch
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.
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. h-nomura@josai.ac.jp
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.
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. cagupue@ceu.es
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. feeleii, L. 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.
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. gils@tauex.tau.ac.il
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.
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. olivier.duron@ird.fr
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.
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. lina19486@hotmail.com
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.
Shaku
M, Park JH, Inouye M, Yamaguchi Y.
Department of Biology and Geosciences,
Graduate School of Science, Osaka City University, Osaka, Japan, yamaguchi@ocarina.osaka-cu.ac.jp
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.
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. ynanjyo@juntendo.ac.jp
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.
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 m.steinert@tu-bs.de
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.
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, anthony@csiriict.in
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.
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.
watarai@yamaguchi-u.ac.jp
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.
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. craig.roy@yale.edu
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.
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.
jwinchell@cdc.gov
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.
Immer C, Hacker C, Wöhnert J.
Institute for Molecular Biosciences,
Goethe University Frankfurt/M., Frankfurt, Germany. immer@bio.uni-frankfurt.de
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.
Loiseau C, Portier E, Corre MH, Schlusselhuber M, Depayras S, Berjeaud JM, Verdon
J.
Laboratoire Ecologie & Biologie des Interactions,
Université de Poitiers, France. julien.verdon@univ-poitiers.fr
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.
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. kazu@med.toho-u.ac.jp
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.
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. alexander.kirschner@meduniwien.ac.at
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.
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. alexander.kirschner@meduniwien.ac.at
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.
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.
fangm@im.ac.cn
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.
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. machnerm@nih.gov
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.
Kenzaka
T, Yasui M, Baba T, Nasu M,
Tani K.
Faculty of Pharmacy, Osaka Ohtani
University.
kenzat@osaka-ohtani.ac.jp
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.
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. elizabeth.hartland@hudson.org.au
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.
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. hksong@korea.ac.kr
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.
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. kagan.kerman@utoronto.ca
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.
Gong X, Zhao X, Zhang W, Wang J, Chen X, Hameed MF, Zhang N, Ge H.
School of Life Sciences, Anhui University, Hefei,
China.
ghh_ahu@foxmail.com
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.
Beyrakhova K,
Li L, Xu C, Gagarinova A, Cygler M.
Department of Biochemistry, University of
Saskatchewan, Saskatoon, Saskatchewan, Canada. miroslaw.cygler@usask.ca
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.
Younas F, Soltanmohammadi N, Knapp O, Benz R.
Department of Life Sciences and Chemistry,
Jacobs University, Bremen, Germany. r.benz@jacobs-university.de
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.
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. alexander.kirschner@meduniwien.ac.at
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.
Price JV, Jiang K, Galantowicz A, Freifeld A, Vance RE.
Department of Biology, Oberlin College,
Oberlin, Ohio, USA jprice@oberlin.edu
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.
Kubori T, Kitao T, Ando H, Nagai H.
Department of Microbiology, Graduate School of
Medicine, Gifu University, Gifu, Japan.
tkubori@gifu‐u.ac.jp
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.
Gomaa F, Gersh M, Cavanaugh CM.
Department of Organismic and Evolutionary
Biology, Biological Laboratory, Harvard University, Cambridge, Massachusetts,
02138, USA. fatma.gomaa@gmail.com
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.
Wasilko DJ,
Huang Q, Mao Y.
Department of Molecular Biology and Genetics, Cornell
University, Ithaca, United States. ym253@cornell.edu
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.
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. maria.trundova@ibt.cas.cz
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.
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. jliu@yale.edu
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.
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. albert.jeltsch@ibtb.uni-stuttgart.de
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.
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.
hiroyuki@med.hokudai.ac.jp
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.
Peterson CP,
Sauer C, Chatfield CH.
Department of Biological Sciences, SUNY
Cortland, Cortland, NY, USA. christa.chatfield@cortland.edu
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.
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. Zhangjm128@126.com
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.
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. ym253@cornell.edu
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.
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. fengyue@mail.buct.edu.cn
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.
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. mhalpern@research.haifa.ac.il
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.
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.
terebiznik@utsc.utoronto.ca
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.
Best A, Jones S, Abu Kwaik Y.
Department of Microbiology and Immunology,
School of Medicine, University of Louisville, Louisville, KY, USA. abukwaik@louisville.edu
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.
Buse
HY,
Hoelle JM, Muhlen C, Lytle DA.
US EPA, Office of Research and Development, National
Homeland Security Research Center, Cincinnati, OH 45268, USA.
buse.helen@epa.gov
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.
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. n-cianciotto@northwestern.edu
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.
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. alexei.savchenko@ucalgary.ca
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 UBE2N∼Ub 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.
Nishida
T, Hara N, Watanabe K, Shimizu T, Fujishima M, Watarai M.
The United Graduate School of Veterinary
Science, Yamaguchi University, Yamaguchi, Japan.
watarai@yamaguchi-u.ac.jp
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.
Mendis N, McBride P, Saoud J, Mani T, Faucher SP.
Department of Natural Resource Sciences, McGill
University, Sainte-Anne-de-Bellevue, Quebec, Canada. sebastien.faucher2@mcgill.ca
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.
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. abukwaik@louisville.edu
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.
Sousa PS, Silva IN, Moreira LM, Veríssimo A, Costa J.
Department of Life Sciences, University of Coimbra,
Coimbra, Portugal. jcosta@uc.pt
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.
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.
mostafa.mliji@pasteur.ma
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.
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. hiroyuki@med.hokudai.ac.jp
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.
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. lmcburne@asu.edu
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.
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.
hilbi@imm.uzh.ch
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.
Rolando M, Buchrieser C.
Institut Pasteur, Biologie des Bactéries
Intracellulaires, Paris, France. cbuch@pasteur.fr
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.
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.
raskin@umich.edu
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.
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. vforest@emse.fr
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.
Arslan-Aydoğdu EÖ, Kimiran A.
Istanbul University, Faculty of Science, Department of
Biology, Istanbul, Turkey. eoarslan@istanbul.edu.tr
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.
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. morteza.hadizade@gmail.com
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.
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. alexei.savchenko@ucalgary.ca
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.
Slow S, Anderson T, Biggs P,
Kennedy M, Murdoch D, Cree S.
Department of Pathology and Biomedical Science,
University of Otago, Christchurch, New Zealand. sandy.slow@otago.ac.nz
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.
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. machnerm@nih.gov
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.
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. g.waksman@mail.cryst.bbk.ac.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.
Abu Khweek A, Amer AO.
Department of Biology and Biochemistry,
Birzeit University, West Bank, Palestine.
arwakhweek@gmail.com
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.
Personnic N,
Striednig B, Hilbi H.
Institute of Medical Microbiology, University of Zürich,
Zürich, Switzerland. hilbi@imm.uzh.ch
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.
Oliva G, Sahr T, Buchrieser C.
Institut Pasteur, Biologie des Bactéries
Intracellulaires, Paris, France. cbuch@pasteur.fr
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.
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. bastian.opitz@charite.de
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.
Hiller M, Lang C, Michel W, Flieger A.
Division of Enteropathogenic Bacteria and Legionella,
Robert Koch-Institute, Wernigerode, Germany. fliegera@rki.de
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.
Mou Q, Leung PHM.
Department of Health Technology and Informatics, The
Hong Kong Polytechnic University, Kowloon, Hong Kong, China.
polly.hm.leung@polyu.edu.hk
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.
Steiner B, Weber S, Hilbi H.
Institute of Medical Microbiology, University of Zürich,
Zürich, Switzerland. hilbi@imm.uzh.ch
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.
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. neunr@udel.edu
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.
Buracco S, Peracino B, Andreini C, Bracco E, Bozzaro S.
Department of Clinical and Biological
Sciences, University of Torino, Turin, Italy.
salvatore.bozzaro@unito.it
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.
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. kazu@med.toho-u.ac.jp
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.
Schroeder GN.
Centre for Experimental Medicine, School of Medicine,
Dentistry and Biomedical Sciences, Queen's University Belfast, Belfast, United
Kingdom.
g.schroeder@qub.ac.uk
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.
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.
xavier.charpentier@univ-lyon1.fr
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.
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. bastian.opitz@charite.de
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.
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. zhuyongqun@zju.edu.cn.
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.
Dawn A, Chandra H, Ade-Browne C, Yadav J, Kumari H.
James L.
Winkle College of Pharmacy, University of Cincinnati, 231 Albert
Sabin Way, Cincinnati, OH, USA. kumariha@ucmail.uc.edu
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.
Pinotsis N,
Waksman G.
Department of Biological Sciences, Institute of
Structural and Molecular Biology, Birkbeck, London, United Kingdom.
g.waksman@ucl.ac.uk
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.
Arasaki K, Tagaya M.
School of Life Sciences, Tokyo University of Pharmacy
and Life Sciences, Hachioji, Tokyo, Japan. tagaya@toyaku.ac.jp
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.
Zhan XY, Zhu QY.
Guangzhou KingMed Center for Clinical Laboratory,
Guangzhou, China. zqy@kingmed.com.cn
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.
Lang C, Hiller M, Flieger A.
Division of Enteropathogenic Bacteria and Legionella,
Robert Koch-Institut, Wernigerode, Germany. fliegera@rki.de
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.
Bärlocher K,
Welin A, Hilbi H.
Institute of Medical Microbiology, University of Zürich,
Zurich, Switzerland. hilbi@imm.uzh.ch
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.
Ríhová J, Nováková E, Husník F, Hypša V.
Department of Parasitology, University of South Bohemia,
Ceské Budejovice, Czech Republic.
vacatko@prf.jcu.cz
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.
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. wilco@vdlugt.nl
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.
Ahmadishoar S,
Kariminik A.
Department of Microbiology, Kerman Branch,
Islamic Azad University, Kerman, Iran. a.kariminik@iauk.ac.ir
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.
Lama A, Drennan SL, Johnson RC, Rubenstein GL, Cambronne ED.
Department of Molecular Microbiology and Immunology,
Oregon Health and Science University, Portland, OR, USA.
cambronn@ohsu.edu
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.
Boamah DK, Zhou G, Ensminger AW, O'Connor TJ.
Department of Biological Chemistry, Johns
Hopkins University School of Medicine, Baltimore, MD, USA.
toconno7@jhmi.edu
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.
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. rvance@berkeley.edu
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.
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. craig.roy@yale.edu
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.
Häuslein I,
Sahr T, Escoll P, Klausner N, Eisenreich W, Buchrieser C.
Institut Pasteur, Biologie des Bactéries
Intracellulaires, Paris, France. cbuch@pasteur.fr
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.
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. christophe.ginevra@univ-lyon1.fr
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.
Pereira RPA,
Peplies J, Höfle MG, Brettar I.
Department of Vaccinology and Applied Microbiology,
Helmholtz Centre for Infection Research (HZI), Braunschweig,
Germany. manfred.hoefle@helmholtz.hzi.de
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.
Pourchez J,
Leclerc L, Girardot F, Riffard S, Prevot N, Allegra S.
University of Lyon, UJM-Saint-Etienne,
Saint-Etienne, France.
severine.allegra@univ-st-etienne.fr
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.
Proctor CR,
Dai D, Edwards MA, Pruden A.
Department of Civil and Environmental Engineering,
Virginia Tech, Blacksburg, Virginia, USA. apruden@vt.edu.
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.
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. hilbi@imm.uzh.ch
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.
Dobrowsky PH,
Khan S, Khan W.
Department of Microbiology, Faculty of Science,
Stellenbosch University, Stellenbosch, South Africa. wesaal@sun.ac.za
Environ Res 2017 Oct;158:82-93.
Abstract: Legionella and Acanthamoeba
spp. persist in harvested rainwater pasteurized at high temperatures (>72°C)
and the interaction mechanisms exhibited between these organisms need to be
elucidated. The resistance of two Legionella reference strains (Legionella
pneumophila ATCC 33152 and Legionella longbeachae ATCC 33462), three
environmental strains [Legionella longbeachae (env.), Legionella
norrlandica (env.) and Legionella rowbothamii (env.)] and Acanthamoeba
mauritaniensis ATCC 50676 to heat treatment (50-90°C) was determined by
monitoring culturability and viability [ethidium monoazide quantitative
polymerase chain reaction (EMA-qPCR)]. The expression of metabolic and virulence
genes of L. pneumophila ATCC 33152 (lolA, sidF, csrA) and L.
longbeachae (env.) (lolA) in co-culture with A. mauritaniensis ATCC
50676 during heat treatment (50-90°C) was monitored using relative qPCR. While
the culturability (CFU/mL) and viability (gene copies/mL) of the Legionella
strains reduced significantly (p<0.05) following heat treatment (60-90°C), L.
longbeachae (env.) and L. pneumophila ATCC 33152 were culturable
following heat treatment at 50-60°C. Metabolically active trophozoites and
dormant cysts of A. mauritaniensis ATCC 50676 were detected at 50°C and
60-90°C, respectively. For L. pneumophila ATCC 33152, lolA expression
remained constant, sidF expression increased and the expression of csrA
decreased during co-culture with A. mauritaniensis ATCC 50676. For L.
longbeachae (env.), while lolA was up-regulated at 50-70°C, expression was
not detected at 80-90°C and in co-culture. In conclusion, while heat treatment
may reduce the number of viable Legionella spp. in monoculture, results
indicate that the presence of A. mauritaniensis increases the virulence
of L. pneumophila during heat treatment. The virulence of Legionella
spp. in co-culture with Acanthamoeba spp. should thus be monitored in
water distribution systems where temperature (heat) is utilized for treatment.
Gradowski M,
Pawłowski K.
Department of Experimental Design and Bioinformatics,
Faculty of Agriculture and Biology, Warsaw University of Life Sciences, Warszawa,
Poland. Krzysztof_Pawlowski@sggw.pl
PeerJ 2017
Sep;5:e3849.
Abstract: Many bacterial effector proteins that
are delivered to host cells during infection are enzymes targeting host cell
signalling. Recently, Legionella pneumophila effector Lpg1137
was experimentally characterised as a serine protease that cleaves human
syntaxin 17. We present strong bioinformatic evidence that Lpg1137 is a
homologue of mitochondrial carrier proteins and is not related to known serine
proteases. We also discuss how this finding can be reconciled with the
apparently contradictory experimental results.
Allgood SC,
Romero Dueñas BP, Noll RR, Pike C, Lein S, Neunuebel MR.
Department of Biological Sciences, University of
Delaware, Newark, DE, USA. neunr@udel.edu
Front Cell
Infect Microbiol 2017 Sep;7:397.
Abstract: The facultative intracellular bacterium Legionella
pneumophila proliferates within amoebae and human alveolar macrophages,
and it is the causative agent of Legionnaires' disease, a life-threatening
pneumonia. Within host cells, L. pneumophila establishes a
replicative haven by delivering numerous effector proteins into the host cytosol,
many of which target membrane trafficking by manipulating the function of Rab
GTPases. The Legionella effector AnkX is a phosphocholine
transferase that covalently modifies host Rab1 and Rab35. However, a detailed
understanding of the biological consequence of Rab GTPase phosphocholination
remains elusive. Here, we broaden the understanding of AnkX function by
presenting three lines of evidence that it interferes with host endocytic
recycling. First, using immunogold transmission electron microscopy, we
determined that GFP-tagged AnkX ectopically produced in mammalian cells
localizes at the plasma membrane and tubular membrane compartments, sites
consistent with targeting the endocytic recycling pathway. Furthermore, the
C-terminal region of AnkX was responsible for association with the plasma
membrane, and we determined that this region was also able to bind the
phosphoinositide lipids PI(3)P and PI(4)P in vitro. Second, we
observed that mCherry-AnkX co-localized with Rab35, a regulator of recycling
endocytosis and with major histocompatibility class I protein (MHC-I), a key
immunoregulatory protein whose recycling from and back to the plasma membrane is
Rab35-dependent. Third, we report that during infection of macrophages, AnkX is
responsible for the disruption of endocytic recycling of transferrin, and AnkX's
phosphocholination activity is critical for this function. These results support
the hypothesis that AnkX targets endocytic recycling during host cell infection.
Finally, we have demonstrated that the phosphocholination activity of AnkX is
also critical for inhibiting fusion of the Legionella-containing
vacuole (LCV) with lysosomes.
Schulz C, Lai X, Bertrams W, Jung AL, Sittka-Stark A, Herkt CE, Janga H,
Zscheppang K, Stielow C, Schulte L, Hippenstiel S, Vera J, Schmeck B.
Institute for Lung Research, Universities of Giessen
and Marburg Lung Center, Philipps-University Marburg, Member of the German
Center for Lung Research (DZL), Marburg, Germany. bernd.schmeck@uni-marburg.de
Sci Rep 2017 Sep;7(1):11988.
Abstract: Immune response in the lung has to
protect the huge alveolar surface against pathogens while securing the delicate
lung structure. Macrophages and alveolar epithelial cells constitute the first
line of defense and together orchestrate the initial steps of host defense. In
this study, we analysed the influence of macrophages on type II alveolar
epithelial cells during Legionella pneumophila-infection by a systems
biology approach combining experimental work and mathematical modelling. We
found that L. pneumophila-infected THP-1-derived macrophages provoke a
pro-inflammatory activation of neighboring lung epithelial cells, but in
addition render them hypo-responsive to direct infection with the same pathogen.
We generated a kinetic mathematical model of macrophage activation and
identified a paracrine mechanism of macrophage-secreted IL-1β inducing a prolonged IRAK-1 degradation in lung
epithelial cells. This intercellular crosstalk may help to avoid an overwhelming
inflammatory response by preventing excessive local secretion of
pro-inflammatory cytokines and thereby negatively regulating the recruitment of
immune cells to the site of infection. This suggests an important but ambivalent
immunomodulatory role of macrophages in lung infection.
Mohabati Mobarez A, Ahmadrajabi R,
Khoramabadi N, Salmanian AH.
Department of Bacteriology, Faculty of Medical Sciences,
Tarbiat Modares University, Tehran, Iran. mmmobarez@modares.ac.ir
World J
Microbiol Biotechnol 2017 Sep;33(9):175.
Abstract: We report a new recombinant fusion
protein composed of full-length Legionella pneumophila flagellin A and
peptidoglycan-associated lipoprotein (PAL), rFLA-PAL, capable of inducing
protective immunity against L. pneumophila. The recombinant protein was
over expressed in Escherichia coli strain BL21 (DE3) using pET-28a (+)
expression vector (pET28a-flaA-pal) and purified by Ni2+ exchange
chromatography. Immunological properties of rFLA-PAL were assessed in a mouse
model. Female BALB/c mice, immunized with rFLA-PAL, exhibited a rapid increase
in serum antibody concentration against each of its protein portions.
Furthermore, a strong activation of both innate and adaptive cell-mediated
immunity was observed as indicated by antigen-specific splenocyte proliferation,
IFN-γ and IL-12
production, and early production of TNF-α in the serum and in
splenocyte cultures which were separately assessed against PAL and FLA. BALB/c
mice were challenged with a lethal dose of L. pneumophila intravenously.
In a 10-days follow-up after intravenous lethal challenge with L. pneumophila,
a 100% survival rate was observed for mice immunized with rFLA-PAL, same as for
those immunized with a sublethal dose of L. pneumophila. Based on the
potent immune responses observed in mice immunized with rFLA-PAL, this
recombinant fusion protein could be a potential vaccine candidate against the
intracellular pathogen L. pneumophila.
Escoll P, Song OR, Viana F, Steiner B, Lagache T, Olivo-Marin JC, Impens F,
Brodin P, Hilbi H, Buchrieser C.
Institut Pasteur, Biologie des Bactéries
Intracellulaires, Paris, France. cbuch@pasteur.fr
Cell Host Microbe 2017
Sep;22(3):302-316.
Abstract: The intracellular bacteria Legionella
pneumophila encodes a type IV secretion system (T4SS) that injects effector
proteins into macrophages in order to establish and replicate within the Legionella-containing
vacuole (LCV). Once generated, the LCV interacts with mitochondria through
unclear mechanisms. We show that Legionella uses both T4SS-independent
and T4SS-dependent mechanisms to respectively interact with mitochondria and
induce mitochondrial fragmentation that ultimately alters mitochondrial
metabolism. The T4SS effector MitF, a Ran GTPase activator, is required for
fission of the mitochondrial network. These effects of MitF occur through
accumulation of mitochondrial DNM1L, a GTPase critical for fission. Furthermore,
mitochondrial respiration is abruptly halted in a T4SS-dependent manner, while
T4SS-independent upregulation of cellular glycolysis remains elevated.
Collectively, these alterations in mitochondrial dynamics promote a Warburg-like
phenotype in macrophages that favors bacterial replication. Hence the rewiring
of cellular bioenergetics to create a replication permissive niche in host cells
is a virulence strategy of L. pneumophila.
Kubori T, Bui XT, Hubber A, Nagai
H.
Department of Infectious Disease Control, Research
Institute for Microbial Diseases, Osaka University Suita, Japan.
tkubori@gifu-u.ac.jp
Front Cell
Infect Microbiol 2017 Aug;7:384.
Abstract: Bacterial pathogens like Salmonella and Legionella establish
intracellular niches in host cells known as bacteria-containing vacuoles. In
these vacuoles, bacteria can survive and replicate. Ubiquitin-dependent
selective autophagy is a host defense mechanism to counteract infection by
invading pathogens. The Legionella effector protein RavZ
interferes with autophagy by irreversibly deconjugating LC3, an
autophagy-related ubiquitin-like protein, from a phosphoglycolipid
phosphatidylethanolamine. Using a co-infection system with Salmonella,
we show here that Legionella RavZ interferes with ubiquitin
recruitment to the Salmonella-containing vacuoles. The inhibitory
activity is dependent on the same catalytic residue of RavZ that is involved in
LC3 deconjugation. In semi-permeabilized cells infected with Salmonella,
external addition of purified RavZ protein, but not of its catalytic mutant,
induced removal of ubiquitin associated with Salmonella-containing
vacuoles. The RavZ-mediated restriction of ubiquitin recruitment to Salmonella-containing
vacuoles took place in the absence of the host system required for LC3
conjugation. These observations suggest the possibility that the targets of RavZ
deconjugation activity include not only LC3, but also ubiquitin.
Prevost MS,
Pinotsis N, Dumoux M, Hayward RD, Waksman G.
Institute of Structural and Molecular Biology,
University College London and Birkbeck, London, UK. g.waksman@mail.cryst.bbk.ac.uk
Sci Rep 2017 Aug;7(1):9450.
Abstract: Legionella pneumophila infects
human alveolar macrophages and is responsible for Legionnaire's disease, a
severe form of pneumonia. L. pneumophila encodes more than 300 putative
effectors, which are translocated into the host cell via the Dot/Icm type IV
secretion system. These effectors highjack the host's cellular processes to
allow bacterial intracellular growth and replication. Here we adopted a
multidisciplinary approach to investigate WipB, a Dot/Icm effector of unknown
function. The crystal structure of the N-terminal domain at 1.7 Å resolution
comprising residues 25 to 344 revealed that WipB harbours a Ser/Thr phosphatase
domain related to the eukaryotic phospho-protein phosphatase (PPP) family. The
C-terminal domain (residues 365-524) is sufficient to pilot the effector to
acidified LAMP1-positive lysosomal compartments, where WipB interacts with the
v-ATPase and the associated LAMTOR1 phosphoprotein, key components of the
lysosomal nutrient sensing (LYNUS) apparatus that controls the mammalian target
of rapamycin (mTORC1) kinase complex at the lysosomal surface. We propose that
WipB is a lysosome-targeted phosphatase that modulates cellular nutrient sensing
and the control of energy metabolism during Legionella infection.
de Jesús-Díaz DA,
Murphy C, Sol A, Dorer M, Isberg RR.
Graduate Program in Molecular Microbiology, Sackler
School of Graduate Biomedical Sciences, Tufts University School of Medicine,
Boston, Massachusetts, USA. ralph.isberg@tufts.edu
mBio 2017
Aug;8(4):e02345-16.
Abstract: Legionella pneumophila grows
within cells ranging from environmental amoebae to human macrophages. In spite
of this conserved strategy of pathogenesis, identification of host factors that
restrict L. pneumophila intracellular replication has not been
extended outside components of the mammalian innate immune response. We
performed a double-stranded RNA (dsRNA) screen against more than 50% of the Drosophila
melanogaster annotated open reading frames (ORFs) to identify host cell
factors that restrict L. pneumophila The majority of analyzed
dsRNAs that stimulated L. pneumophila intracellular replication
were directed against host proteins involved in protein synthesis or cell cycle
control. Consistent with disruption of the cell cycle stimulating intracellular
replication, proteins involved in translation initiation also resulted in G1 arrest.
Stimulation of replication was dependent on the stage of cell cycle arrest, as
dsRNAs causing arrest during S phase had an inhibitory effect on intracellular
replication. The inhibitory effects of S phase arrest could be recapitulated in
a human cell line, indicating that cell cycle control of L. pneumophila replication
is evolutionarily conserved. Synchronized HeLa cell populations in S phase and
challenged with L. pneumophila failed to progress through the
cell cycle and were depressed for supporting intracellular replication. Poor
bacterial replication in S phase was associated with loss of the vacuole
membrane barrier, resulting in exposure of bacteria to the cytosol and their
eventual degradation. These results are consistent with the model that S phase
is inhibitory for L. pneumophila intracellular survival as a
consequence of failure to maintain the integrity of the membrane surrounding
intracellular bacteria. IMPORTANCE: Legionella pneumophila has
the ability to replicate within human macrophages and amoebal hosts. Here, we
report that the host cell cycle influences L. pneumophila intracellular
replication. Our data demonstrate that the G1 and G2/M
phases of the host cell cycle are permissive for bacterial replication, while S
phase is toxic for the bacterium. L. pneumophila replicates
poorly within host cells present in S phase. The inability of L.
pneumophila to replicate relies on its failure to control the integrity
of its vacuole, leading to cytosolic exposure of the bacteria and eventual
degradation. The data presented here argue that growth-arrested host cells that
are encountered by L. pneumophila in either the environment or
within human hosts are ideal targets for intracellular replication because their
transit through S phase is blocked.
Pantoom S, Yang A, Wu YW.
Chemical Genomics Centre of the Max Planck Society,
Dortmund, Germany.
Autophagy 2017
Aug;13(8):1467-1469.
Abstract: RavZ, an effector protein of pathogenic Legionella
pneumophila, inhibits host macroautophagy / autophagy by deconjugation of
lipidated LC3 proteins from phosphatidylethanolamine (PE) on the autophagosome
membrane. The mechanism for how RavZ specifically recognizes and deconjugates
the lipidated LC3s is not clear. To understand the structure-function
relationship of LC3-deconjugation by RavZ, we prepared semisynthetic LC3
proteins modified with different fragments of PE or 1-hexadecanol (C16). We find
that RavZ activity is strictly dependent on the conjugated PE structure and RavZ
extracts LC3-PE from the membrane before deconjugation. Structural and
biophysical analysis of RavZ-LC3 interactions suggest that RavZ initially
recognizes LC3-PE on the membrane via its N-terminal LC3-interacting region (LIR)
motif. RavZ specifically targets to autophagosome membranes by interaction with
phosphatidylinositol 3-phosphate (PtdIns3P) via its C-terminal domain and
association with membranes via the hydrophobic α3 helix. The α3 helix is involved in extraction of the PE moiety and
docking of the fatty acid chains into the lipid-binding site of RavZ, which is
related in structure to that of the phospholipid transfer protein Sec14. The LIR
interaction and lipid binding facilitate subsequent proteolytic cleavage of
LC3-PE. The findings reveal a novel mode of host-pathogen interaction.
Ronau JA, Hochstrasser M.
Departments of Molecular Biophysics &
Biochemistry, Yale University, New Haven, CT, USA. mark.hochstrasser@yale.edu
Cell Res 2017 Jul;27(7):845-846.
Abstract:
Recently, a Legionella pneumophila effector protein was shown to have an
unprecedented ATP-independent ubiquitin ligase activity that couples
phosphoribosylated ubiquitin (PR-Ub) to serine residues of host proteins. A new
study published in Cell Research by Qiu et al. reveals that another Legionella
effector protein, SidJ, catalyzes deubiquitination of PR-Ub by cleavage of the
substrate-linked phosphodiester bond
Kwak MJ, Kim JD, Kim H, Kim C, Bowman JW, Kim S, Joo K, Lee J, Jin KS, Kim YG,
Lee NK, Jung JU, Oh BH.
Department of Biological Sciences, KAIST Institute for
the Biocentury, Korea Advanced Institute of Science and Technology, Daejeon
34141, Korea.
bhoh@kaist.ac.kr
Nat Microbiol
2017 Jul;2:17114.
Abstract: Many bacteria, including Legionella
pneumophila, rely on the type IV secretion system to translocate a
repertoire of effector proteins into the hosts for their survival and growth.
Type IV coupling protein (T4CP) is a hexameric ATPase that links translocating
substrates to the transenvelope secretion conduit. Yet, how a large number of
effector proteins are selectively recruited and processed by T4CPs remains
enigmatic. DotL, the T4CP of L. pneumophila, contains an ATPase domain
and a C-terminal extension whose function is unknown. Unlike T4CPs involved in
plasmid DNA translocation, DotL appeared to function by forming a multiprotein
complex with four other proteins. Here, we show that the C-terminal extension of
DotL interacts with DotN, IcmS, IcmW and an additionally identified subunit LvgA,
and that this pentameric assembly binds Legionella effector proteins. We
determined the crystal structure of this assembly and built an architecture of
the T4CP holocomplex by combining a homology model of the ATPase domain of DotL.
The holocomplex is a hexamer of a bipartite structure composed of a
membrane-proximal ATPase domain and a membrane-distal substrate-recognition
assembly. The presented information demonstrates the architecture and functional
dissection of the multiprotein T4CP complexes and provides important insights
into their substrate recruitment and processing.
Perpich JD,
Kalia A, Price CTD, Jones SC, Wong K, Gehring K, Kwaik YA.
Department of Microbiology and Immunology, College of
Medicine, University of Louisville, Louisville, KY, USA. abukwaik@louisville.edu
Sci Rep 2017
Jul;7(1):5123.
Abstract: Within macrophages and amoeba, the Legionella-containing
vacuole (LCV) membrane is derived from the ER. The bona fide F-box AnkB effector
protein of L. pneumophila strain AA100/130b is anchored to the cytosolic
side of the LCV membrane through host-mediated farnesylation of its C-terminal
eukaryotic "CaaX" motif. Here we show that the AnkB homologue of the
Paris strain has a frame shift mutation that led to a loss of the CaaX motif and
a concurrent generation of a unique C-terminal KNKYAP motif, which resembles the
eukaryotic di-lysine ER-retention motif (KxKxx). Our phylogenetic analyses
indicate that environmental isolates of L. pneumophila have a potential
positive selection for the ER-retention KNKYAP motif. The AnkB-Paris effector is
localized to the LCV membrane most likely through the ER-retention motif. Its
ectopic expression in HEK293T cells localizes it to the perinuclear ER region
and it trans-rescues the ankB mutant of strain AA100/130b in intra-vacuolar
replication. The di-lysine ER retention motif of AnkB-Paris is indispensable for
function; most likely as an ER retention motif that enables anchoring to the
ER-derived LCV membrane. Our findings show divergent evolution of the ankB
allele in exploiting either host farnesylation or the ER retention motif to be
anchored into the LCV membrane.
Kuhn P, Thiem S, Steinert M, Purvis D, Lugmayr V, Treutlein U, Plobner L,
Leiser RM, Hust M, Dübel S.
Biotechnology and Bioinformatics,
Institute for Biochemistry, Technische Universität Braunschweig, Braunschweig,
Germany.
s.duebel@tu-bs.de
Hum Antibodies 2017 Jul;26(1):29-38.
Abstract: Legionella are Gram-negative
bacteria that are ubiquitously present in natural and man-made water reservoirs.
When humans inhale aerosolized water contaminated with Legionella,
alveolar macrophages can be infected, which may lead to a life-threatening
pneumonia called Legionnaires' disease. Due to the universal distribution of Legionella
in water and their potential threat to human health, the Legionella
concentration in water for human use must be strictly monitored, which is
difficult since the standard detection still relies on lengthy cultivation and
analysis of bacterial morphology. In this study, an antibody against L.
pneumophila has been generated from the naïve human HAL antibody libraries
by phage-display for the first time. The panning was performed on whole
bacterial cells in order to select antibodies that bind specifically to the cell
surface of untreated Legionella. The bacterial cell wall component
lipopolysaccharide (LPS) was identified as the target structure. Specific
binding to the important pathogenic L. pneumophila strains Corby,
Philadelphia-1 and Knoxville was observed, while no binding was detected to
seven members of the families Enterobacteriaceae, Pseudomonadaceae or
Clostridiaceae. Production of this antibody in the recombinant scFv-Fc format
using either a murine or a human Fc part allowed the set-up of a sandwich-ELISA
for detection of Legionella cells. The scFv-Fc construct proved to be
very stable, even when stored for several weeks at elevated temperatures. A
sensitivity limit of 4,000 cells was achieved. The scFv-Fc antibody pair was
integrated on a biosensor, demonstrating the specific and fast detection of L.
pneumophila on a portable device. With this system, 10,000 Legionella
cells were detected within 35 min. Combined with a water filtration/concentration
system, this antibody may be developed into a promising reagent for rapid
on-site Legionella monitoring.
Buse HY, Ji P, Gomez-Alvarez V,
Pruden A, Edwards MA, Ashbolt NJ.
Pegasus Technical Services, Inc c/o US
EPA, Cincinnati, OH, USA. buse.helen@epa.gov
Microb Biotechnol 2017
Jul;10(4):773-788.
Abstract: It is unclear how the water-based
pathogen, Legionella pneumophila (Lp), and associated free-living amoeba
(FLA) hosts change or are changed by the microbial composition of drinking water
(DW) biofilm communities. Thus, this study characterized the bacterial community
structure over a 7-month period within mature (> 600-day-old) copper DW
biofilms in reactors simulating premise plumbing and assessed the impact of
temperature and introduction of Lp and its FLA host, Vermamoeba vermiformis
(Vv), co-cultures (LpVv). Sequence and quantitative PCR (qPCR) analyses
indicated a correlation between LpVv introduction and increases in Legionella
spp. levels at room temperature (RT), while at 37°C, Lp became the dominant Legionella
spp. qPCR analysis suggested Vv presence may not be directly associated with Lp
biofilm growth at RT and 37°C, but may contribute to or be associated with
non-Lp legionellae persistence at RT. Two-way PERMANOVA and PCoA revealed that
temperature was a major driver of microbiome diversity. Biofilm community
composition also changed over the seven-month period and could be associated
with significant shifts in dissolved oxygen, alkalinity and various metals in
the influent DW. Hence, temperature, biofilm age, DW quality and transient
intrusions/amplification of pathogens and FLA hosts may significantly impact
biofilm microbiomes and modulate pathogen levels over extended periods.
Xiong L, Yamasaki S, Chen H, Shi L, Mo Z.
State Key Laboratory of Respiratory
Disease, Guangzhou Institute of Respiratory Disease, The First Affiliated
Hospital of Guangzhou Medical University, Guangzhou, China. moziyao@gird.cn
Biol
Pharm Bull 2017;40(7):1035-1042.
Abstract:
Various studies have been made to attempt to study the interaction between Legionella
pneumophila and the host cells. In this research, we successfully
constructed a L. pneumophila mutant strain that stably expressed high
levels of green fluorescent protein and used this strain to evaluate the
adherence, invasion and proliferation of L. pneumophila in association
with several cell lines, including seven cell lines [human macrophage-like cell
lines (U937, THP-1), murine macrophage-like cell lines (J774.1A, Raw264.7),
human bronchial epithelial cell lines (16HBE, Beas-2B) and human cervical cancer
cell line (HeLa)] which have been used as the host models of L. pneumophila,
and two breast carcinoma cell lines (MCF-7 and MDA-MB-231). Our results showed
that the two newly tested cell lines are able to support the intracellular
proliferation of L. pneumophila, and there were some morphological
variations during the invasion and intracellular replication of L.
pneumophila in different cell lines. These results can help us find out the
common and special patterns of invasion and proliferation of L. pneumophila
within different hosts. This is conducive to our knowledge on the relationship
and interaction between bacteria and host.
David S, Sánchez-Busó L, Harris SR, Marttinen P, Rusniok C, Buchrieser C,
Harrison TG, Parkhill J.
Pathogen Genomics, Wellcome Trust Sanger
Institute, Cambridge, United Kingdom. parkhill@sanger.ac.uk
PLoS Genet 2017 Jun;13(6):e1006855.
Abstract: Legionella pneumophila is an
environmental bacterium and the causative agent of Legionnaires' disease.
Previous genomic studies have shown that recombination accounts for a high
proportion (>96%) of diversity within several major disease-associated
sequence types (STs) of L. pneumophila. This suggests that recombination
represents a potentially important force shaping adaptation and virulence.
Despite this, little is known about the biological effects of recombination in L.
pneumophila, particularly with regards to homologous recombination (whereby
genes are replaced with alternative allelic variants). Using newly available
population genomic data, we have disentangled events arising from homologous and
non-homologous recombination in six major disease associated STs of L.
pneumophila (subsp. pneumophila), and subsequently performed a
detailed characterisation of the dynamics and impact of homologous recombination.
We identified genomic "hotspots" of homologous recombination that
include regions containing outer membrane proteins, the lipopolysaccharide (LPS)
region and Dot/Icm effectors, which provide interesting clues to the selection
pressures faced by L. pneumophila. Inference of the origin of the
recombined regions showed that isolates have most frequently imported DNA from
isolates belonging to their own clade, but also occasionally from other major
clades of the same subspecies. This supports the hypothesis that the possibility
for horizontal exchange of new adaptations between major clades of the
subspecies may have been a critical factor in the recent emergence of several
clinically important STs from diverse genomic backgrounds. However, acquisition
of recombined regions from another subspecies, L. pneumophila subsp. fraseri,
was rarely observed, suggesting the existence of a recombination barrier and/or
the possibility of ongoing speciation between the two subspecies. Finally, we
suggest that multi-fragment recombination may occur in L. pneumophila,
whereby multiple non-contiguous segments that originate from the same molecule
of donor DNA are imported into a recipient genome during a single episode of
recombination.
Pinotsis N,
Waksman G.
Department of Biological Sciences, Institute of
Structural and Molecular Biology, Birkbeck, Malet Street, London, United Kingdom.
g.waksman@ucl.ac.uk
J Biol Chem 2017
Jun;292(22):9240-9251.
Abstract: Legionnaires' disease is a severe form
of pneumonia caused by the bacterium Legionella pneumophila. L.
pneumophila pathogenicity relies on secretion of more than 300 effector
proteins by a type IVb secretion system. Among these Legionella effectors,
WipA has been primarily studied because of its dependence on a chaperone complex,
IcmSW, for translocation through the secretion system, but its role in
pathogenicity has remained unknown. In this study, we present the crystal
structure of a large fragment of WipA, WipA435. Surprisingly, this structure
revealed a serine/threonine phosphatase fold that unexpectedly targets
tyrosine-phosphorylated peptides. The structure also revealed a sequence
insertion that folds into an α-helical
hairpin, the tip of which adopts a canonical coiled-coil structure. The purified
protein was a dimer whose dimer interface involves interactions between the
coiled coil of one WipA molecule and the phosphatase domain of another. Given
the ubiquity of protein-protein interaction mediated by interactions between
coiled-coils, we hypothesize that WipA can thereby transition from a homodimeric
state to a heterodimeric state in which the coiled-coil region of WipA is
engaged in a protein-protein interaction with a tyrosine-phosphorylated host
target. In conclusion, these findings help advance our understanding of the
molecular mechanisms of an effector involved in Legionella virulence
and may inform approaches to elucidate the function of other effectors.
He J, Huang F, Chen H, Chen Q, Zhang J, Li J, Chen D, Chen J.
Department of Parasitology, West China
School of Preclinical and Forensic Medicine, Sichuan University, Chengdu, China;
Animal Disease Prevention and Food Safety Key Laboratory of Sichuan Province,
Chengdu, China. jpchen007@163.com
Immunol Lett Jun
2017;186:33-40.
Abstract: Legionella pneumophila is the
main causative agent of Legionnaires' disease, which is a severe multi-system
disease with pneumonia as the primary manifestation. We designed a recombinant
Mip-PilE-FlaA dominant epitopes vaccine against Legionella pneumophila to
prevent the disease and evaluated its immunogenicity and protective immunity.
The protein structures of Mip, PilE and FlaA were analyzed using a computer, and
the gene sequences of the dominant epitopes of the three proteins were selected
to construct and optimize the vaccine. The optimized mip, pilE, flaA and
recombinant mip-pilE-flaA gene sequences were cloned, expressed and purified.
The purified proteins were used as dominant epitopes vaccines to immunize BALB/c
mice and determine the protective immunity and immunogenicity of these purified
proteins. The identification confirmed that the recombinant mip-pilE-flaA was
successfully cloned and expressed. ELISA revealed that the Mip-PilE-FlaA group
produced the highest IgG response, and this protein may considerably improve the
production of some cytokines in BALB/c mice. Histopathology analyses of lungs
from mice immunized with Mip-PilE-FlaA revealed a certain protective effect. Our
work demonstrated that the recombinant dominant epitopes of Mip-PilE-FlaA
exhibited strong immunogenicity and immune protection, and this protein may be
an efficient epitopes vaccine candidate against Legionella pneumophila.
Li L, Faucher SP.
Department of Natural Resource Sciences,
Faculty of Agricultural and Environmental Sciences, McGill University, Montréal,
QC H9X 3V9, Canada. sebastien.faucher2@mcgill.ca
Can J
Microbiol 2017 Jun;63(6):535-545.
Abstract: The water-borne Gram-negative bacterium Legionella
pneumophila (Lp) is the causative agent of Legionnaires' disease. Lp is
typically transmitted to humans from water systems, where it grows inside
amoebae. Survival of Lp in water is central to its transmission to humans. A
transcriptomic study previously identified many genes induced by Lp in water.
One such gene, lpg2524, encodes a putative LuxR family transcriptional regulator.
It was hypothesized that this gene could be involved in the survival of Lp in
water. Deletion of lpg2524 does not affect the growth of Lp in rich medium, in
the amoeba Acanthamoeba castellanii, or in human macrophage-like THP-1
cells, showing that Lpg2524 is not required for growth in vitro and in vivo.
Nevertheless, deletion of lpg2524 results in a faster colony-forming unit (CFU)
reduction in an artificial freshwater medium, Fraquil, indicating that Lpg2524
is important for Lp to survive in water. Overexpression of Lpg2524 also results
in a survival defect, suggesting that a precise level of this transcriptional
regulator is essential for its function. However, our result shows that Lpg2524
is dispensable for survival in water when Lp is at a high cell density (109 CFU/mL),
suggesting that its regulon is regulated by another regulator activated at high
cell density.
Arh
Hig Rada Toksikol 2017 Jun;68(2):109-115.
University of
Ljubljana, Faculty of Health Sciences, Department for Sanitary Engineering,
Ljubljana, Slovenia. karmen.torkar@zf.uni-lj.si
Abstract:
Bacterial adhesion is a complex process influenced by many factors, including
hydrodynamic conditions. They affect the transfer of oxygen, nutrients, and
bacterial cells in a water supply and cooling systems. The aim of this study was
to identify hydrodynamic effects on bacterial adhesion to and detachment from
stainless steel surfaces. For this purpose, we observed the behaviour of
bacterium L. pneumophila in no-flow and laminar and turbulent flow
conditions simulated in a fluid flow chamber. The bacterial growth in no-flow
and laminar flow conditions was almost identical in the first 24 h, while at 48
and 72 h of incubation, the laminar flow stimulated bacterial growth. In the
second part of this study we found that laminar flow accelerated bacterial
adhesion in the first 48 h, but after 72 h the amount of bacterial cells exposed
to the flow dropped, probably due to detachment. In the third part we found that
the turbulent flow detached more bacterial cells than the laminar, which
indicates that the strength of shear forces determines the rate of bacterial
removal.
Truchan HK,
Christman HD, White RC, Rutledge NS, Cianciotto NP.
Department of Microbiology and Immunology, Northwestern
University Feinberg School of Medicine, Chicago, Illinois, USA. n-cianciotto@northwestern.edu
mBio 2017
Jun;8(3):e00870-17.
Abstract: Legionella pneumophila replicates
in macrophages in a host-derived phagosome, termed the Legionella-containing
vacuole (LCV). While the translocation of type IV secretion (T4S) effectors into
the macrophage cytosol is well established, the location of type II secretion
(T2S) substrates in the infected host cell is unknown. Here, we show that the
T2S substrate ProA, a metalloprotease, translocates into the cytosol of human
macrophages, where it associates with the LCV membrane (LCVM). Translocation is
detected as early as 10 h post inoculation (p.i.), which is approximately the
midpoint of the intracellular life cycle. However, it is detected as early as 6h
p.i. if ProA is hyperexpressed, indicating that translocation depends on the
timing of ProA expression and that any other factors necessary for translocation
are in place by that time point. Translocation occurs with all L.
pneumophila strains tested and in amoebae, natural hosts for L.
pneumophila It was absent in murine bone marrow-derived macrophages and
murine macrophage cell lines. The ChiA chitinase also associated with the
cytoplasmic face of the LCVM at 6h p.i. and in a T2S-dependent manner.
Galectin-3 and galectin-8, eukaryotic proteins whose localization is influenced
by damage to host membranes, appeared within the LCV of infected human but not
murine macrophages beginning at 6h p.i. Thus, we hypothesize that ProA and ChiA
are first secreted into the vacuolar lumen by the activity of the T2S and
subsequently traffic into the macrophage cytosol via a novel mechanism that
involves a semipermeable LCVM. IMPORTANCE Infection of macrophages and
amoebae plays a central role in the pathogenesis of L. pneumophila,
the agent of Legionnaires' disease. We have previously demonstrated that the T2S
system of L. pneumophila greatly contributes to intracellular
infection. However, the location of T2S substrates within the infected host cell
is unknown. This report presents the first evidence of a L. pneumophila T2S
substrate in the host cell cytosol and, therefore, the first evidence of a
non-T4S effector trafficking out of the LCV. We also provide the first
indication that the LCV is not completely intact but is instead semipermeable
and that this occurs in human but not murine macrophages. Given this
permeability, we hypothesize that other T2S substrates and LCV lumenal contents
can escape into the host cell cytosol. Thus, these substrates may represent a
battery of previously unidentified effectors that can interact with host factors
and contribute to intracellular infection by L. pneumophila.
Jwanoswki K,
Wells C, Bruce T, Rutt J, Banks T, McNealy TL.
Department of Biological Sciences,
Clemson University, Clemson, South Carolina, USA.
tmcneal@g.clemson.edu
PLoS One 2017 May;12(5):e0174245.
Abstract: Legionella pneumophila
contaminates man-made water systems and creates numerous exposure risks for
Legionnaires' Disease. Because copper/silver ionization is commonly used to
control L. pneumophila, its mechanisms of metal response and
detoxification are of significant interest. Here we describe an L.
pneumophila operon with significant similarity to the GIG operon of Cupriavidus
metallidurans. The Legionella GIG operon is present in a subset of strains
and has been acquired as part of the ICE-βox
65-kB integrative conjugative element. We assessed GIG promoter activity
following exposure of L. pneumophila to multiple concentrations of HAuCl4,
CuSO4 and AgNO3. At 37°C, control stationary phase
cultures exhibited GIG promoter activity. This activity increased significantly
in response to 20 and 50uM HAuCl4 and CuSO4 but not in
response to AgNO3. Conversely, at 26°C, cultures exhibited decreased
promoter response to copper. GIG promoter activity was also induced by HAuCl4
or CuSO4 during early biofilm establishment at both temperatures.
When an L. pneumophila GIG promoter construct was transformed into E.
coli DH5α, cultures showed
baseline expression levels that did not increase following metal addition.
Analysis of L. pneumophila transcriptional regulatory mutants suggested
that GIG up-regulation in the presence of metal ions may be influenced by the
stationary phase sigma factor, RpoS.
Bacigalupe R,
Lindsay D, Edwards G, Fitzgerald JR.
The Roslin Institute,
University of Edinburgh, Midlothian, Scotland, UK. Ross.Fitzgerald@ed.ac.uk
Emerg Infect
Dis 2017 May;23(5):750-757.
Abstract:
Legionella longbeachae is the primary cause of legionellosis in
Australasia and Southeast Asia and an emerging pathogen in Europe and the United
States; however, our understanding of the population diversity of L.
longbeachae from patient and environmental sources is limited. We analyzed
the genomes of 64 L. longbeachae isolates, of which 29 were from a
cluster of legionellosis cases linked to commercial growing media in Scotland in
2013 and 35 were non-outbreak-associated isolates from Scotland and other
countries. We identified extensive genetic diversity across the L.
longbeachae species, associated with intraspecies and interspecies gene flow,
and a wide geographic distribution of closely related genotypes. Of note, we
observed a highly diverse pool of L. longbeachae genotypes within compost
samples that precluded the genetic establishment of an infection source. These
data represent a view of the genomic diversity of L. longbeachae that
will inform strategies for investigating future outbreaks.
Ghosal D, Chang YW, Jeong KC, Vogel
JP, Jensen GJ.
California
Institute of Technology, Pasadena, CA, USA. jensen@caltech.edu
EMBO Rep 2017
May;18(5):726-732.
Abstract: Type IV secretion systems (T4SSs) are
large macromolecular machines that translocate protein and DNA and are involved
in the pathogenesis of multiple human diseases. Here, using electron
cryotomography (ECT), we report the in situ structure of the
Dot/Icm type IVB secretion system (T4BSS) utilized by the human pathogen Legionella
pneumophila This is the first structure of a type IVB secretion system,
and also the first structure of any T4SS in situ While the Dot/Icm
system shares almost no sequence similarity with type IVA secretion systems
(T4ASSs), its overall structure is seen here to be remarkably similar to
previously reported T4ASS structures (those encoded by the R388 plasmid in Escherichia
coli and the cag pathogenicity island in Helicobacter pylori).
This structural similarity suggests shared aspects of mechanism. However,
compared to the negative-stain reconstruction of the purified T4ASS from the
R388 plasmid, the L. pneumophila Dot/Icm system is
approximately twice as long and wide and exhibits several additional large
densities, reflecting type-specific elaborations and potentially better
structural preservation in situ.
Legionella Effector Lpg1137 Shuts Down ER-mitochondria Communication Through
Cleavage of Syntaxin 17
Arasaki K, Mikami Y, Shames SR,
Inoue H, Wakana Y, Tagaya M.
Department of Molecular Life Sciences, School of Life
Sciences, Tokyo University of Pharmacy and Life Sciences, Tokyo, Japan. karasaki@toyaku.ac.jp
Nat Commun 2017
May;8:15406.
Abstract: During infection of macrophages, the
pathogenic bacterium Legionella pneumophila secretes effector proteins
that induce the conversion of the plasma membrane-derived vacuole into an
endoplasmic reticulum (ER)-like replicative vacuole. These ER-like vacuoles are
ultimately fused with the ER, where the pathogen replicates. Here we show that
the L. pneumophila effector Lpg1137 is a serine protease that targets the
mitochondria and their associated membranes. Lpg1137 binds to and cleaves
syntaxin 17, a soluble N-ethylmaleimide-sensitive factor attachment protein
receptor (SNARE) protein that is known to participate in the regulation of
mitochondrial dynamics through interaction with the mitochondrial fission factor
Drp1 in fed cells and in autophagy through interaction with Atg14L and other
SNAREs in starved cells. Cleavage of syntaxin 17 inhibits not only autophagy but
also staurosporine-induced apoptosis occurring in a Bax, Drp1-dependent manner.
Thus, L. pneumophila can shut down ER-mitochondria communication through
cleavage of syntaxin 17.
Speiser Y, Zusman T, Pasechnek A, Segal G.
Department of Molecular Microbiology and Biotechnology,
George S. Wise Faculty of Life Sciences, Tel-Aviv University, Tel-Aviv, Israel. gils@tauex.tau.ac.il
Infect Immun 2017
May;85(6):e00121-17.
Abstract:
The nitrogen phosphotransferase system (PTSNtr) is a regulatory
cascade present in many bacteria, where it controls different functions. This
system is usually composed of three basic components: enzyme INtr (EINtr),
NPr, and EIIANtr (encoded by the ptsP, ptsO,
and ptsN genes, respectively). In Legionella
pneumophila, as well as in many other Legionella species,
the EIIANtr component is missing. However, we found that
deletion mutations in both ptsP and ptsO are
partially attenuated for intracellular growth. Furthermore, these two PTSNtr components
were found to be required for maximal expression of effector-encoding genes
regulated by the transcriptional activator PmrA. Genetic analyses which include
the construction of single and double deletion mutants and overexpression of
wild-type and mutated forms of EINtr, NPr, and PmrA indicated that
the PTSNtr components affect the expression of PmrA-regulated
genes via PmrA and independently from PmrB and that EINtr and
NPr are part of the same cascade and require their conserved histidine residues
in order to function. Furthermore, expression of the Legionella micdadei EIINtr component
in L. pneumophila resulted in a reduction in the levels of
expression of PmrA-regulated genes which was completely dependent on the L.
pneumophila PTS components and the L. micdadei EIINtr conserved
histidine residue. Moreover, reconstruction of the L. pneumophila PTS in
vitro indicated that EINtr is phosphorylated by
phosphoenolpyruvate (PEP) and transfers its phosphate to NPr. Our results
demonstrate that the L. pneumophila incomplete PTSNtr is
functional and involved in the expression of effector-encoding genes regulated
by PmrA.
Tlapák H, Rydzewski K, Schulz T, Weschka D, Schunder E, Heuner K.
Cellular Interactions of Bacterial
Pathogens, ZBS 2, Robert Koch Institute, Berlin, Germany heunerk@rki.de
J Bacteriol 2017
May;199(11):e00018-17.
Abstract: Legionella oakridgensis causes
Legionnaires' disease but is known to be less virulent than Legionella
pneumophilaL. oakridgensis is one of the Legionella species
that is nonflagellated. The genes of the flagellar regulon are absent, except
those encoding the alternative sigma-28 factor (FliA) and its anti-sigma-28
factor (FlgM). Similar to L. oakridgensis, Legionella
adelaidensis and Legionella londiniensis, located in the
same phylogenetic clade, have no flagellar regulon, although both are positive
for fliA and flgM Here, we investigated the
role and function of both genes to better understand the role of FliA, the
positive regulator of flagellin expression, in nonflagellated strains. We
demonstrated that the FliA gene of L. oakridgensis encodes a
functional sigma-28 factor that enables the transcription start from the
sigma-28-dependent promoter site. The investigations have shown that FliA is
necessary for full fitness of L. oakridgensis Interestingly,
expression of FliA-dependent genes depends on the growth phase and temperature,
as already shown for L. pneumophila strains that are
flagellated. In addition, we demonstrated that FlgM is a negative regulator of
FliA-dependent gene expression. FlgM seems to be degraded in a growth-phase- and
temperature-dependent manner, instead of being exported into the medium as
reported for most bacteria. The degradation of FlgM leads to an increase of FliA
activity. IMPORTANCE: A less virulent Legionella species, L.
oakridgensis, causes Legionnaires' disease and is known to not have
flagella, even though L. oakridgensis has the regulator of
flagellin expression (FliA). This protein has been shown to be involved in the
expression of virulence factors. Thus, the strain was chosen for use in this
investigation to search for FliA target genes and to identify putative virulence
factors of L. oakridgensis One of the five major target genes
of FliA identified here encodes the anti-FliA sigma factor FlgM. Interestingly,
in contrast to most homologs in other bacteria, FlgM in L. oakridgensis seems
not to be transported from the cell so that FliA gets activated. In L.
oakridgensis, FlgM seems to be degraded by protease activities.
Massip C, Descours G, Ginevra C, Doublet P, Jarraud S, Gilbert C.
CIRI, Centre International de Recherche en
Infectiologie, "Legionella pathogenesis" team, Inserm, U1111,
Université Lyon 1, CNRS, UMR5308, École Normale Supérieure de Lyon, Univ Lyon,
France. christophe.gilbert.bio@univ-lyon1.fr
Antimicrob Chemother 2017 May;72(5):1327-1333.
Abstract:
Objectives: A
previous study on 12 in vitro -selected azithromycin-resistant Legionella
pneumophila lineages showed that ribosomal mutations were major macrolide
resistance determinants. In addition to these mechanisms that have been well
described in many species, mutations upstream of lpeAB operon, homologous to
acrAB in Escherichia coli, were identified in two lineages. In this study,
we investigated the role of LpeAB and of these mutations in macrolide resistance
of L. pneumophila. Methods: The
role of LpeAB was studied by testing the antibiotic susceptibility of WT,
deleted and complemented L. pneumophila Paris strains. Translational
fusion experiments using GFP as a reporter were conducted to investigate the
consequences of the mutations observed in the upstream sequence of lpeAB operon.
Results: We
demonstrated the involvement of LpeAB in an efflux pump responsible for a
macrolide-specific reduced susceptibility of L. pneumophila Paris strain.
Mutations in the upstream sequence of lpeAB operon were associated with an
increased protein expression. Increased expression was also observed under
sub-inhibitory macrolide concentrations in strains with both mutated and WT
promoting regions. Conclusions: LpeAB
are components of an efflux pump, which is a macrolide resistance determinant in
L. pneumophila Paris strain. Mutations observed in the upstream sequence
of lpeAB operon in resistant lineages led to an overexpression of this efflux
pump. Sub-inhibitory concentrations of macrolides themselves participated in
upregulating this efflux and could constitute a first step in the acquisition of
a high macrolide resistance level.
Jentho E, Bodden M, Schulz C, Jung AL, Seidel K, Schmeck B, Bertrams W.
Institute for Lung Research/iLung, German
Center for Lung Research, Universities of Giessen and Marburg Lung Center,
Philipps-University Marburg, Marburg, Germany.
bernd.schmeck@uni-marburg.de
PLoS One 2017
Apr;12(4):e0176204.
Abstract: Background: Legionella
pneumophila (L. pneumophila) is a causative agent of severe pneumonia.
It is highly adapted to intracellular replication and manipulates host cell
functions like vesicle trafficking and mRNA translation to its own advantage.
However, it is still unknown to what extent microRNAs (miRNAs) are involved in
the Legionella-host cell interaction. Methods: WT
and MyD88-/-murine bone marrow-derived macrophages (BMM) were infected with L.
pneumophila, the transcriptome was analyzed by high throughput qPCR array (microRNAs)
and conventional qPCR (mRNAs), and mRNA-miRNA interaction was validated by
luciferase assays with 3´-UTR mutations and western blot. Results: L.
pneumophila infection caused a pro-inflammatory reaction and significant
miRNA changes in murine macrophages. In MyD88-/-cells, induction of inflammatory
markers, such as Ccxl1/Kc, Il6 and miR-146a-5p was reduced. Induction of
miR-125a-3p was completely abrogated in MyD88-/-cells. Target prediction
analyses revealed N-terminal asparagine amidase 1 (NTAN1), a factor from the
n-end rule pathway, to be a putative target of miR-125a-3p. This interaction
could be confirmed by luciferase assay and western blot. Conclusion: Taken
together, we characterized the miRNA regulation in L. pneumophila
infection with regard to MyD88 signaling and identified NTAN1 as a target of
miR-125a-3p. This finding unravels a yet unknown feature of Legionella-host
cell interaction, potentially relevant for new treatment options.
Yang A, Pantoom S, Wu YW.
Institute of Chemical Biology and Precision Therapy,
Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou, China.
yaowen.wu@mpi-dortmund.mpg.de
Elife 2017
Apr;6:e23905.
Abstract:
Autophagy is a conserved cellular process involved in the elimination of
proteins and organelles. It is also used to combat infection with pathogenic
microbes. The intracellular pathogen Legionella pneumophila manipulates
autophagy by delivering the effector protein RavZ to deconjugate Atg8/LC3
proteins coupled to phosphatidylethanolamine (PE) on autophagosomal membranes.
To understand how RavZ recognizes and deconjugates LC3-PE, we prepared
semisynthetic LC3 proteins and elucidated the structures of the RavZ:LC3
interaction. Semisynthetic LC3 proteins allowed the analysis of
structure-function relationships. RavZ extracts LC3-PE from the membrane before
deconjugation. RavZ initially recognizes the LC3 molecule on membranes via its
N-terminal LC3-interacting region (LIR) motif. The RavZ α3 helix is involved in extraction of the PE moiety and
docking of the acyl chains into the lipid-binding site of RavZ that is related
in structure to that of the phospholipid transfer protein Sec14. Thus, Legionella has
evolved a novel mechanism to specifically evade host autophagy.
Casson CN, Doerner JL, Copenhaver AM, Ramirez J, Holmgren AM, Boyer MA, Siddarthan
IJ, Rouhanifard SH, Raj A, Shin S.
Department of Microbiology, Perelman School of
Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
sunshin@mail.med.upenn.edu
PLoS Pathog 2017
Apr;13(4):e1006309.
Abstract: Early responses mounted by both
tissue-resident and recruited innate immune cells are essential for host defense
against bacterial pathogens. In particular, both neutrophils and Ly6Chi
monocytes are rapidly recruited to sites of infection. While neutrophils and
monocytes produce bactericidal molecules, such as reactive nitrogen and oxygen
species, both cell types are also capable of synthesizing overlapping sets of
cytokines important for host defense. Whether neutrophils and monocytes perform
redundant or non-redundant functions in the generation of anti-microbial
cytokine responses remains elusive. Here, we sought to define the contributions
of neutrophils and Ly6Chi monocytes to cytokine production and host defense
during pulmonary infection with Legionella pneumophila, responsible for
the severe pneumonia Legionnaires' disease. We found that both neutrophils and
monocytes are critical for host defense against L. pneumophila. Both
monocytes and neutrophils contribute to maximal IL-12 and IFNγ responses, and monocytes are also required for TNF
production. Moreover, natural killer (NK) cells, NKT cells, and γδ T cells are sources of IFNγ, and monocytes direct IFNγ production by these cell types. Thus, neutrophils and
monocytes cooperate in eliciting an optimal cytokine response that promotes
effective control of bacterial infection.
Schmölders
J, Manske C,
Otto A, Hoffmann C, Steiner B, Welin A, Becher D, Hilbi H.
Max von Pettenkofer Institute,
Ludwig-Maximilians University, Munich, Germany. hilbi@imm.uzh.ch
Mol Cell
Proteomics 2017 Apr;16(4):622-641.
Abstract: Legionella pneumophila is an
opportunistic bacterial pathogen that causes a severe lung infection termed
"Legionnaires' disease." The pathogen replicates in environmental
protozoa as well as in macrophages within a unique membrane-bound compartment,
the Legionella-containing-vacuole (LCV). LCV formation requires the
bacterial Icm/Dot type IV secretion system, which translocates ca. 300 "effector
proteins" into host cells, where they target distinct host factors. The L.
pneumophila "pentuple" mutant (Δpentuple) lacks 5 gene clusters (31% of the effector
proteins) and replicates in macrophages but not in Dictyostelium
discoideum amoeba. To elucidate the host factors defining a
replication-permissive compartment, we compare here the proteomes of intact LCVs
isolated from D. discoideum or macrophages infected with Δpentuple or the parental strain Lp02. This analysis
revealed that the majority of host proteins are shared in D. discoideum or
macrophage LCVs containing the mutant or the parental strain, respectively,
whereas some proteins preferentially localize to distinct LCVs. The small GTPase
Rap1 was identified on D. discoideum LCVs containing strain
Lp02 but not the Δpentuple mutant and
on macrophage LCVs containing either strain. The localization pattern of active
Rap1 on D. discoideum or macrophage LCVs was confirmed by
fluorescence microscopy and imaging flow cytometry, and the depletion of Rap1 by
RNA interference significantly reduced the intracellular growth of L.
pneumophila Thus, comparative proteomics identified Rap1 as a novel LCV
host component implicated in intracellular replication of L. pneumophila.
Brown AS, Yang C, Hartland EL, van Driel IR.
Department of Biochemistry and Molecular Biology, Bio21
Molecular Science and Biotechnology Institute, University of Melbourne,
Melbourne, Victoria, Australia. i.vandriel@unimelb.edu.au
J Leukoc Biol
2017 Apr;101(4):875-886.
Abstract: Legionella pneumophila causes
Legionnaires' disease, a severe and potentially fatal bacterial pneumonia in
immunocompromised individuals. Despite the understanding that a robust
inflammatory response is important for control of L. pneumophila infection,
our understanding of the network of molecular and cellular events within the
lung that function to clear the bacterium is not clearly understood. This review
compiles our understanding of the various molecular and cellular pathways
stimulated upon infection with L. pneumophila and considers
recently published advances that focus on the immune response to L.
pneumophila in the lungs of mice. This includes a cooperative network
of tissue-resident and inflammatory phagocytes, including alveolar macrophages
(AM)s, neutrophils, and inflammatory monocytes/monocyte-derived cells (MC) that
contribute to the acute inflammatory response and restrict the bacteria via
distinct intracellular pathways. The understanding of this difference in
cellular activity in response to infection provides insight into the innate
immune responses within the tissues in general and may prompt novel means of
clinical management of bacterial infections in an era of increasing emergence of
antibiotic resistance.
Mascarenhas DP,
Zamboni DS.
Department of Cell Biology, School of Medicine of
Ribeirão Preto, University of São Paulo, São Paulo, Brazil. dszamboni@fmrp.usp.br
eukoc Biol 2017
Apr;101(4):841-849.
Abstract: Inflammasomes are multimeric protein
complexes that assemble in the cytosol of many types of cells, including innate
immune cells. The inflammasomes can be activated in response to infection or in
response to stress signals that induce damage in the host cell membranes. These
platforms trigger inflammatory processes, cell death, and the control of
microbial replication. Many inflammasomes have been described so far, including
NLRP3, NAIP/NLRC4, caspase-11, and AIM2. The ligand for NLRP3 is still
unidentified, but the efflux of K+ is essential for NLRP3
activation. By contrast, inflammasomes, such as those composed of NAIP/NLRC4,
caspase-11, and AIM2, can be activated by bacterial flagellin, LPS, and dsDNA.
The knowledge of inflammasome biology has advanced tremendously in the last
decade, fostered by the use of model organisms, such as Legionella
pneumophila This bacterium evolved, infecting unicellular protozoa in
freshwater environments, and the human infection is accidental. Thus, L.
pneumophila did not evolve sophisticated mechanisms to inhibit
mammalian innate immunity. For this reason, it has emerged as a very appropriate
model of a pathogenic microbe for the investigation of inflammasome biology. In
this review, we highlight the current information regarding the biology of
inflammasomes and emphasize the advances achieved using L. pneumophila We
also describe the inflammasomes activated in response to L. pneumophila infection
and discuss the effector mechanisms that operate to clear the infection.
Mentasti M,
Cassier P, David S, Ginevra C, Gomez-Valero L, Underwood A, Afshar B, Etienne J,
Parkhill J, Chalker V, Buchrieser C, Harrison TG, Jarraud S; ESCMID Study
Group for Legionella Infections (ESGLI).
French National Reference Center of Legionella,
Hospices Civils de Lyon, France; International Center for Infectiology Research,
Inserm U1111, CNRS, UMR5308, University Lyon1, Ecole Normale Supérieure de Lyon,
Lyon, F-69008, France. Sophie.jarraud@univ-lyon1.fr
Clin Microbiol Infect 2017
Apr;23(4):264.e1-264.e9.
Abstract: Objectives: Legionella
pneumophila serogroup 1 (Lp1) sequence type 47 is the leading cause of
legionellosis in north-western Europe, but, surprisingly, it is rarely isolated
from environmental samples. Comparative genomics was applied to develop a PCR
assay and to better understand the evolution of this strain. Methods: Comparative
analysis of 36 genomes representative of the Lp species was used to identify
specific PCR targets, which were then evaluated in silico on 545 sequenced
genomes and in vitro on 436 Legionella strains, 106 respiratory samples,
and three environmental samples from proven ST47 sources. Phylogenetic analyses
were performed to understand the evolution of ST47. Results: The
gene LPO_1073 was characterized as being 100% conserved in all 129 ST47 genomes
analysed. A real-time PCR designed to detect LPO_1073 was positive for all 110
ST47 strains tested and agreed with culture and typing results previously
obtained for 106 respiratory samples. The three environmental samples were also
positive. Surprisingly, 26 of the 44 ST109 strains tested among 342 non-ST47
strains scored positive for LPO_1073. SNP-based phylogenetic analysis was
undertaken to understand this result: the PCR-positive ST109 genomes were almost
identical to ST47 genomes, with the exception of a recombined region probably
acquired by ST47 from a ST62(-like) strain. Conclusion: The
genomic analysis allowed the design of a highly specific PCR assay for rapid
detection of ST47 strains. Furthermore, it allowed us to uncover the evolution
of ST47 strains from ST109 by homologous recombination with ST62. We hypothesize
that this recombination generated the leading cause of legionellosis in
north-western Europe.
Kang YS, Kirby JE.
Department of Pathology, Beth Israel Deaconess Medical
Center and Harvard Medical School, Boston, Massachusetts, USA. jekirby@bidmc.harvard.edu
Infect Immun 2017
Apr;85(5):e00991-16.
Abstract: We established a new Brucella
neotomae in vitro model system for study of type IV secretion
system-dependent (T4SS) pathogenesis in the Brucella genus.
Importantly, B. neotomae is a rodent pathogen, and unlike B.
abortus, B. melitensis, and B. suis, B.
neotomae has not been observed to infect humans. It therefore can be
handled more facilely using biosafety level 2 practices. More particularly,
using a series of novel fluorescent protein and lux operon
reporter systems to differentially label pathogens and track intracellular
replication, we confirmed T4SS-dependent intracellular growth of B.
neotomae in macrophage cell lines. Furthermore, B. neotomae exhibited
early endosomal (LAMP-1) and late endoplasmic reticulum (calreticulin)-associated
phagosome maturation. These findings recapitulate prior observations for
human-pathogenic Brucella spp. In addition, during coinfection
experiments with Legionella pneumophila, we found that defective
intracellular replication of a B. neotomae T4SS virB4 mutant
was rescued and baseline levels of intracellular replication of wild-type B.
neotomae were significantly stimulated by coinfection with wild-type
but not T4SS mutant L. pneumophila. Using confocal microscopy, it
was determined that intracellular colocalization of B. neotomae and L.
pneumophila was required for rescue and that colocalization came at a
cost to L. pneumophila fitness. These findings were not
completely expected based on known temporal and qualitative differences in the
intracellular life cycles of these two pathogens. Taken together, we have
developed a new system for studying in vitro Brucella pathogenesis
and found a remarkable T4SS-dependent interplay between Brucella and Legionella during
macrophage coinfection.
Qin T, Zhou H, Ren H, Liu W.
State Key Laboratory for Infectious
Disease Prevention and Control, National Institute for Communicable Disease
Control and Prevention, Chinese Center for Disease Control and Prevention,
Beijing, China. qintian@icdc.cn
Front Microbiol 2017 Mar;8:388.
Abstract: The genus Legionella comprises
over 60 species, which are important human pathogens. Secretion systems in Legionella
pneumophila have been studied extensively because of the essential role
of protein secretion in bacterial infection. However, there are few reports
describing the secretion systems in non-L. pneumophila species. In
this study, we analyzed the distribution of secretion systems in L.
pneumophila and 18 species of non-L. pneumophila based on
whole genome sequences. A total of 74 whole genome sequences from 19 species of Legionella were
analyzed. Type II and IVB secretion systems were detected in all Legionella strains,
but the type I secretion systems was restricted to L. pneumophila. The
type IVA secretion system was randomly distributed among different species.
Furthermore, we found the type VI secretion system in three non-L.
pneumophila strains (Legionella cherrii DSM 19213, Legionella
dumoffii Tex-KL, and Legionella gormanii ATCC 33297).
In population structure analysis, L. pneumophila formed a
conservative cluster and was located at the terminal of the evolutionary tree.
At the same time, L. pneumophila, especially eight clone groups (named
MCGG1-MCGG8), showed higher intracellular growth ability than non-L.
pneumophila species. These results suggest that L. pneumophila has
acquired additional secretion systems during evolution, resulting in increased
pathogenicity.
Sharaby Y, Rodríguez-Martínez S, Oks O, Pecellin M, Mizrahi H, Peretz A, Brettar
I, Höfle MG, Halpern M.
Department of Evolutionary and Environmental Biology,
Faculty of Natural Sciences, University of Haifa, Haifa, Israel. mhalpern@research.haifa.ac.il
Appl Environ
Microbiol 2017 Mar;83(8):e03295-16.
Abstract:
Legionella pneumophila causes waterborne infections resulting in
severe pneumonia. High-resolution genotyping of L. pneumophila isolates
can be achieved by multiple-locus variable-number tandem-repeat analysis (MLVA).
Recently, we found that different MLVA genotypes of L. pneumophila dominated
different sites in a small drinking-water network, with a genotype-related
temperature and abundance regime. The present study focuses on understanding the
temperature-dependent growth kinetics of the genotypes that dominated the water
network. Our aim was to model mathematically the influence of temperature on the
growth kinetics of different environmental and clinical L. pneumophila genotypes
and to compare it with the influence of their ecological niches. Environmental
strains showed a distinct temperature preference, with significant differences
among the growth kinetics of the three studied genotypes (Gt4, Gt6, and Gt15).
Gt4 strains exhibited superior growth at lower temperatures (25 and 30°C),
while Gt15 strains appeared to be best adapted to relatively higher temperatures
(42 and 45°C). The temperature-dependent growth traits of the environmental
genotypes were consistent with their distribution and temperature preferences in
the water network. Clinical isolates exhibited significantly higher growth rates
and reached higher maximal cell densities at 37°C and 42°C than the
environmental strains. Further research on the growth preferences of L.
pneumophila clinical and environmental genotypes will result in a
better understanding of their ecological niches in drinking-water systems as
well as in the human body. IMPORTANCE: Legionella pneumophila is a
waterborne pathogen that threatens humans in developed countries. The bacteria
inhabit natural and man-made freshwater environments. Here we demonstrate that
different environmental L. pneumophila genotypes have different
temperature-dependent growth kinetics. Moreover, Legionella strains
that belong to the same species but were isolated from environmental and
clinical sources possess adaptations for growth at different temperatures. These
growth preferences may influence the bacterial colonization at specific
ecological niches within the drinking-water network. Adaptations for growth at
human body temperatures may facilitate the abilities of some L.
pneumophila strains to infect and cause illness in humans. Our findings
may be used as a tool to improve Legionella monitoring in
drinking-water networks. Risk assessment models for predicting the risk of
legionellosis should take into account not only Legionella concentrations
but also the temperature-dependent growth kinetics of the isolates.
Quero S, García-Núñez M, Párraga-Niño N, Pedro-Botet ML, Mateu L,
Sabrià M.
Unitat de Malalties Infeccioses, Fundació Institut
Investigació en Ciències de la Salut Germans Trias i Pujol,
Badalona, Barcelona, Spain. mgarcia@igtp.cat
Curr Microbiol 2017
Mar;74(3):344-355.
Abstract: Molecular epidemiologic studies of Legionella
have shown different molecular types coexisting in the same environment, with
only one having the ability to trigger an outbreak. We therefore studied the
proteome of isolates of these different molecular types in search of the
proteins responsible for infection. In this study, we performed a differential
proteomic analysis between patient-related and non-patient-related environmental
isolates using two-dimensional difference gel electrophoresis (2D-DIGE) combined
with mass spectrometry. Sixty-three spots were observed as being different
between the two groups; 31 spots were identified corresponding to 23 different
proteins. Patient-related isolates overexpressed proteins associated with
metabolism, with enzymes of the tricarboxylic acid cycle and the degradation
pathways being the most abundant proteins identified. However, the largest group
of non-patient-related proteins was associated with stress response. Furthermore,
the MOMP protein was located in different spots depending on their
patient-related or non-patient-related origin, suggesting different
post-translational modifications. According to these results, different
bacterial adaptation pathways are activated in stress conditions which influence
their ability to produce infection.
Mallama CA,
McCoy-Simandle K, Cianciotto NP.
Department of Microbiology and Immunology, Northwestern
University Medical School, Chicago, Illinois, USA. n-cianciotto@northwestern.edu
Infect Immun 2017
Mar;85(4):e00897-16.
Abstract: Previously,
we reported that mutants of Legionella
pneumophila lacking a type II secretion (T2S) system elicit higher
levels of cytokines (e.g., interleukin-6 [IL-6]) following infection of U937
cells, a human macrophage-like cell line. We now show that this effect of T2S is
also manifest upon infection of human THP-1 macrophages and peripheral blood
monocytes but does not occur during infection of murine macrophages. Supporting
the hypothesis that T2S acts to dampen the triggering of an innate immune
response, we observed that the mitogen-activated protein kinase (MAPK) and
nuclear transcription factor kappa B (NF-κB)
pathways are more highly stimulated upon infection with the T2S mutant than upon
infection with the wild type. By using short hairpin RNA to deplete proteins
involved in specific pathogen-associated molecular pattern (PAMP) recognition
pathways, we determined that the dampening effect of the T2S system was not
dependent on nucleotide binding oligomerization domain (NOD)-like receptors (NLRs),
retinoic acid-inducible protein I (RIG-I)-like receptors (RLRs), double-stranded
RNA (dsRNA)-dependent protein kinase receptor (PKR), or TIR domain-containing
adaptor inducing interferon beta (TRIF) signaling or an apoptosis-associated
speck-like protein containing a CARD (ASC)- or caspase-4-dependent inflammasome.
However, the dampening effect of T2S on IL-6 production was significantly
reduced upon gene knockdown of myeloid differentiation primary response 88
(MyD88), TANK binding kinase 1 (TBK1), or Toll-like receptor 2 (TLR2). These
data indicate that the L. pneumophila T2S system dampens the
signaling of the TLR2 pathway in infected human macrophages. We also document
the importance of PKR, TRIF, and TBK1 in cytokine secretion during L.
pneumophila infection of macrophages.
Hoppe
J, Ünal CM, Thiem S, Grimpe L, Goldmann T, Gaßler N, Richter M, Shevchuk O,
Steinert M.
Institut für Mikrobiologie, Technische
Universität Braunschweig Braunschweig, Germany.
m.steinert@tu-bs.de
Front Cell
Infect Microbiol 2017 Mar;7:63.
Abstract: Legionnaires' disease is an acute
fibrinopurulent pneumonia. During infection Legionella pneumophila adheres
to the alveolar lining and replicates intracellularly within recruited
macrophages. Here we provide a sequence and domain composition analysis of the L.
pneumophila PilY1 protein, which has a high homology to PilY1 of Pseudomonas
aeruginosa. PilY1 proteins of both pathogens contain a von Willebrand factor
A (vWFa) and a C-terminal PilY domain. Using cellular fractionation, we assigned
the L. pneumophila PilY1 as an outer membrane protein that is
only expressed during the transmissive stationary growth phase. PilY1
contributes to infection of human lung tissue explants (HLTEs). A detailed
analysis using THP-1 macrophages and A549 lung epithelial cells revealed that
this contribution is due to multiple effects depending on host cell type.
Deletion of PilY1 resulted in a lower replication rate in THP-1 macrophages but
not in A549 cells. Further on, adhesion to THP-1 macrophages and A549 epithelial
cells was decreased. Additionally, the invasion into non-phagocytic A549
epithelial cells was drastically reduced when PilY1 was absent. Complementation
variants of a PilY1-negative mutant revealed that the C-terminal PilY domain is
essential for restoring the wild type phenotype in adhesion, while the
putatively mechanosensitive vWFa domain facilitates invasion into non-phagocytic
cells. Since PilY1 also promotes twitching motility of L. pneumophila,
we discuss the putative contribution of this newly described virulence factor
for bacterial dissemination within infected lung tissue.
Price C, Merchant M, Jones S, Best A, Von Dwingelo J, Lawrenz MB, Alam N,
Schueler-Furman O, Kwaik YA.
Department of Microbiology and Immunology, College of
Medicine, University of Louisville Louisville, KY, USA.
abukwaik@louisville.edu
Front Cell
Infect Microbiol 2017 Mar;7:54.
Abstract: FIH-mediated post-translational
modification through asparaginyl hydroxylation of eukaryotic proteins impacts
regulation of protein-protein interaction. We have identified the FIH
recognition motif in 11 Legionella pneumophila translocated
effectors, YopM of Yersinia, IpaH4.5 of Shigella and
an ankyrin protein of Rickettsia. Mass spectrometry analyses of the
AnkB and AnkH effectors of L. pneumophila confirm their
asparaginyl hydroxylation. Consistent with localization of the AnkB effector to
the Legionella-containing vacuole (LCV) membrane and its
modification by FIH, our data show that FIH and its two interacting proteins,
Mint3 and MT1-MMP are acquired by the LCV in a Dot/Icm type IV
secretion-dependent manner. Chemical inhibition or RNAi-mediated knockdown of
FIH promotes LCV-lysosomes fusion, diminishes decoration of the LCV with
polyubiquitinated proteins, and abolishes intra-vacuolar replication of L.
pneumophila. These data show acquisition of the host FIH by a
pathogen-containing vacuole and that asparaginyl-hydroxylation of translocated
effectors is indispensable for their function.
Hubber A, Kubori T, Coban C, Matsuzawa T, Ogawa M, Kawabata T, Yoshimori T, Nagai
H.
Research Institute for Microbial Diseases, Osaka
University, Osaka, Japan.
Sci Rep 2017
Mar;7:44795.
Abstract: The evolutionarily
conserved processes of endosome-lysosome maturation and macroautophagy are
established mechanisms that limit survival of intracellular bacteria. Similarly,
another emerging mechanism is LC3-associated phagocytosis (LAP). Here we report
that an intracellular vacuolar pathogen, Legionella dumoffii, is
specifically targeted by LAP over classical endocytic maturation and
macroautophagy pathways. Upon infection, the majority of L. dumoffii
resides in ER-like vacuoles and replicate within this niche, which involves
inhibition of classical endosomal maturation. The establishment of the
replicative niche requires the bacterial Dot/Icm type IV secretion system
(T4SS). Intriguingly, the remaining subset of L. dumoffii transiently
acquires LC3 to L. dumoffii-containing vacuoles in a Dot/Icm
T4SS-dependent manner. The LC3-decorated vacuoles are bound by an apparently
undamaged single membrane and fail to associate with the molecules implicated in
selective autophagy, such as ubiquitin or adaptors. The process requires
toll-like receptor 2, Rubicon, diacylglycerol signaling and downstream NADPH
oxidases, whereas ULK1 kinase is dispensable. Together, we have discovered an
intracellular pathogen, the survival of which in infected cells is limited
predominantly by LAP. The results suggest that L. dumoffii is a valuable
model organism for examining the mechanistic details of LAP, particularly
induced by bacterial infection.
Nishida T, Watanabe K, Tachibana M,
Shimizu T, Watarai M.
The United Graduate School of Veterinary Science,
Yamaguchi University, Yamaguchi, Japan. watarai@yamaguchi-u.ac.jp
Plasmid 2017
Mar;90:30-37.
Abstract: In this study, a cryptic plasmid pOfk55
from Legionella pneumophila was isolated and characterized. pOfk55
comprised 2584bp with a GC content of 37.3% and contained three putative open
reading frames (ORFs). orf1 encoded a protein of 195 amino acids and the
putative protein shared 39% sequence identity with a putative plasmid
replication protein RepL. ORF1 was needed for replication in L. pneumophila
but pOfk55 did not replicate in Escherichia coli. orf2 and orf3 encoded
putative hypothetical proteins of 114 amino acids and 78 amino acids,
respectively, but the functions of the putative proteins ORF2 and OFR3 are not
clear. The transfer mechanism for pOfk55 was independent on the type IVB
secretion system in the original host. A L. pneumophila-E. coli
shuttle vector, pNT562 (5058bp, KmR), was constructed by In-Fusion
Cloning of pOfk55 with a kanamycin-resistance gene from pUTmini-Tn5Km and the
origin of replication from pBluescript SK(+) (pNT561). Multiple cloning sites
from pBluescript SK(+) as well as the tac promoter region and lacI gene from
pAM239-GFP were inserted into pNT561 to construct pNT562. The transformation
efficiency of pNT562 in L. pneumophila strains ranged from 1.6×101 to
1.0×105CFU/ng. The relative number of pNT562 was estimated at 5.7±1.0
copies and 73.6% of cells maintained the plasmid after 1week in liquid culture
without kanamycin. A green fluorescent protein (GFP) expression vector, pNT563,
was constructed by ligating pNT562 with the gfpmut3 gene from pAM239-GFP. pNT563
was introduced into L. pneumophila Lp02 and E. coli DH5α, and both strains
expressed GFP successfully. These results suggest that the shuttle vector is
useful for genetic studies in L. pneumophila.
Speir M, Vogrin A, Seidi A, Abraham G, Hunot S, Han Q, Dorn GW 2nd, Masters SL,
Flavell RA, Vince JE, Naderer T.
Department of Biochemistry and Molecular Biology,
Biomedicine Discovery Institute, Monash University Clayton, VIC, Australia.
thomas.naderer@monash.edu
Front Cell
Infect Microbiol 2017 Feb;7:35.
Abstract: The human pathogen Legionella
pneumophila must evade host cell death signaling to enable replication
in lung macrophages and to cause disease. After bacterial growth, however, L.
pneumophila is thought to induce apoptosis during egress from
macrophages. The bacterial effector protein, SidF, has been shown to control
host cell survival and death by inhibiting pro-apoptotic BNIP3 and BCL-RAMBO
signaling. Using live-cell imaging to follow the L. pneumophila-macrophage
interaction, we now demonstrate that L. pneumophila evades host
cell apoptosis independent of SidF. In the absence of SidF, L.
pneumophila was able to replicate, cause loss of mitochondria membrane
potential, kill macrophages, and establish infections in lungs of mice.
Consistent with this, deletion of BNIP3 and BCL-RAMBO did not affect
intracellular L. pneumophila replication, macrophage death
rates, and in vivo bacterial virulence. Abrogating
mitochondrial cell death by genetic deletion of the effectors of intrinsic
apoptosis, BAX, and BAK, or the regulator of mitochondrial permeability
transition pore formation, cyclophilin-D, did not affect bacterial growth or the
initial killing of macrophages. Loss of BAX and BAK only marginally limited the
ability of L. pneumophila to efficiently kill all macrophages
over extended periods. L. pneumophila induced killing of
macrophages was delayed in the absence of capsase-11 mediated pyroptosis.
Together, our data demonstrate that L. pneumophila evades host
cell death responses independently of SidF during replication and can induce
pyroptosis to kill macrophages in a timely manner.
Zhang L, Li Y, Wang X, Shangguan
Z, Zhou H, Wu Y, Wang L, Ren H, Hu Y, Lin M, Qin T.
Wenzhou Center for Disease Control and Prevention,
Wenzhou, China. zhleyi@126.com
Int J Environ Res Public Health 2017
Feb;14(3):222.
Abstract:
Natural
and engineered water systems are the main sources of Legionnaires' disease. It
is essential from a public health perspective to survey water environments for
the existence of Legionella. To analyze the main serogroups,
genotypes and pathogenicity of the pathogen, a stratified sampling method was
adopted to collect water samples randomly from shower water, cooling tower
water, and local public hot springs in Wenzhou, China. Suspected strains were
isolated from concentrated water samples. Serum agglutination assay and
real-time PCR (Polymerase chain reaction) were used to identify L.
pneumophila. Sequence-based typing (SBT) and pulsed-field gel
electrophoresis (PFGE) were used to elucidate the genetic polymorphisms in the
collected isolates. The intracellular growth ability of the isolates was
determined through their interaction with J774 cells and plating them onto BCYE
(Buffered Charcoal Yeast Extract) agar plates. Overall, 25.56% (46/180) of water
samples were Legionella-positive; fifty-two strains were isolated,
and two kinds of serogroups were co-detected from six water samples from 2015 to
2016. Bacterial concentrations ranged from 20 CFU/100 mL to 10,720 CFU/100 mL.
In detail, the Legionella-positive rates of shower water, cooling
tower water and hot springs water were 15.45%, 13.33%, and 62.5%, respectively.
The main serogroups were LP1 (30.69%) and LP3 (28.85%) and all strains carried
the dot gene. Among them, 52 isolates and another 10 former isolates were
analyzed by PFGE. Nineteen distinct patterns were observed in 52 strains
isolated from 2015 to 2016 with three patterns being observed in 10 strains
isolated from 2009 to 2014. Seventy-three strains containing 52 from this study
and 21 former isolates were selected for SBT analysis and divided into 25
different sequence types in 4 main clonal groups belonging to 4 homomorphic
types. Ten strains were chosen to show their abilities to grow and multiply in
J744 cells. Taken together, our results demonstrate a high prevalence and
genetic polymorphism of Legionella in Wenzhou's environmental
water system. The investigated environmental water sources pose a potential
threat to the public where intervention could help to prevent the occurrence of
Legionnaires' disease.
Sahr T, Rusniok C, Impens F, Oliva G, Sismeiro O, Coppée JY, Buchrieser C.
Institut Pasteur, Biologie des Bactéries
Intracellulaires, Paris France. cbuch@pasteur.fr
PLoS Genet 2017
Feb;13(2):e1006629.
Abstract: The carbon storage regulator protein
CsrA regulates cellular processes post-transcriptionally by binding to
target-RNAs altering translation efficiency and/or their stability. Here we
identified and analyzed the direct targets of CsrA in the human pathogen Legionella
pneumophila. Genome wide transcriptome, proteome and RNA
co-immunoprecipitation followed by deep sequencing of a wild type and a csrA
mutant strain identified 479 RNAs with potential CsrA interaction sites located
in the untranslated and/or coding regions of mRNAs or of known non-coding sRNAs.
Further analyses revealed that CsrA exhibits a dual regulatory role in virulence
as it affects the expression of the regulators FleQ, LqsR, LetE and RpoS but it
also directly regulates the timely expression of over 40 Dot/Icm substrates.
CsrA controls its own expression and the stringent response through a regulatory
feedback loop as evidenced by its binding to RelA-mRNA and links it to quorum
sensing and motility. CsrA is a central player in the carbon, amino acid, fatty
acid metabolism and energy transfer and directly affects the biosynthesis of
cofactors, vitamins and secondary metabolites. We describe the first L.
pneumophila riboswitch, a thiamine pyrophosphate riboswitch whose regulatory
impact is fine-tuned by CsrA, and identified a unique regulatory mode of CsrA,
the active stabilization of RNA anti-terminator conformations inside a coding
sequence preventing Rho-dependent termination of the gap operon through
transcriptional polarity effects. This allows L. pneumophila to regulate
the pentose phosphate pathway and the glycolysis combined or individually
although they share genes in a single operon. Thus the L. pneumophila
genome has evolved to acclimate at least five different modes of regulation by
CsrA giving it a truly unique position in its life cycle.
Park B, Park G,
Kim J, Lim SA, Lee KM.
Global Research Laboratory, Department of Biochemistry
and Molecular Biology, Korea University College of Medicine, Seoul, Korea. kyunglee@korea.ac.kr
Arch Pharm Res 2017 Feb;40(2):131-145.
Abstract: Legionella pneumophila is an
etiological agent of the severe pneumonia known as Legionnaires' disease (LD).
This gram-negative bacterium is thought to replicate naturally in various
freshwater amoebae, but also replicates in human alveolar macrophages. Inside
host cells, legionella induce the production of non-endosomal replicative
phagosomes by injecting effector proteins into the cytosol. Innate immune
responses are first line defenses against legionella during early phases of
infection and distinguish between legionella and host cells using
germline-encoded pattern recognition receptors such as Toll-like receptors,
NOD-like receptors, and RIG-I-like receptors, which sense pathogen-associated
molecular patterns that are absent in host cells. During pulmonary legionella
infections, various inflammatory cells such as macrophages, neutrophils, natural
killer (NK) cells, large mononuclear cells, B cells, and CD4+ and CD8+ T cells
are recruited into infected lungs, and predominantly occupy interstitial areas
to control legionella. During pulmonary legionella infections, the interplay
between distinct cytokines and chemokines also modulates innate host responses
to clear legionella from the lungs. Recognition by NK cell receptors triggers
effector functions including secretion of cytokines and chemokines and leads to
lysis of target cells. Crosstalk between NK cells and dendritic cells, monocytes,
and macrophages provides a major first-line defense against legionella infection,
whereas activation of T and B cells resolves the infection and mounts
legionella-specific memory in the host.
Kotewicz
KM, Ramabhadran V,
Sjoblom N, Vogel JP, Haenssler E, Zhang M, Behringer J, Scheck RA, Isberg RR.
Department of Molecular Biology and Microbiology, Tufts
University School of Medicine, Boston, MA, USA. ralph.isberg@tufts.edu
Cell Host Microbe 2017 Feb;21(2):169-181.
Abstract: Intracellular pathogens manipulate host
organelles to support replication within cells. For Legionella pneumophila,
the bacterium translocates proteins that establish an endoplasmic reticulum (ER)-associated
replication compartment. We show here that the bacterial Sde proteins target
host reticulon 4 (Rtn4) to control tubular ER dynamics, resulting in tubule
rearrangements as well as alterations in Rtn4 associated with the replication
compartment. These rearrangements are triggered via Sde-promoted ubiquitin
transfer to Rtn4, occurring almost immediately after bacterial uptake. Ubiquitin
transfer requires two sequential enzymatic activities from a single Sde
polypeptide: an ADP-ribosyltransferase and a nucleotidase/phosphohydrolase. The
ADP-ribosylated moiety of ubiquitin is a substrate for the nucleotidase/phosphohydrolase,
resulting in either transfer of ubiquitin to Rtn4 or phosphoribosylation of
ubiquitin in the absence of a ubiquitination target. Therefore, a single
bacterial protein drives a multistep biochemical pathway to control
ubiquitination and tubular ER function independently of the host ubiquitin
machinery.
Tanner JR, Patel PG, Hellinga JR, Donald LJ, Jimenez C, LeBlanc JJ, Brassinga
AKC.
Department of Microbiology, Faculty of Science,
University of Manitoba, Winnipeg, Manitoba, Canada. Ann.Brassinga@umanitoba.ca
J Bacteriol 2017
Feb;199(5):e00690-16.
Abstract:
Nominally an environmental organism, Legionella pneumophila is
an intracellular parasite of protozoa but is also the causative agent of the
pneumonia termed Legionnaires' disease, which results from inhalation of
aerosolized bacteria by susceptible humans. Coordination of gene expression by a
number of identified regulatory factors, including OxyR, assists L.
pneumophila in adapting to the stresses of changing environments. L.
pneumophila OxyR (OxyRLp) is an ortholog of Escherichia
coli OxyR; however, OxyRLp was shown elsewhere to be
functionally divergent, such that it acts as a transcription regulator
independently of the oxidative stress response. In this study, the use of
improved gene deletion methods has enabled us to generate an unmarked in-frame
deletion of oxyR in L. pneumophila Lack of
OxyRLp did not affect in vitro growth or
intracellular growth in Acanthamoeba castellanii protozoa and
U937-derived macrophages. The expression of OxyRLp does not
appear to be regulated by CpxR, even though purified recombinant CpxR bound a
DNA sequence similar to that reported for CpxR elsewhere. Surprisingly, a lack
of OxyRLp resulted in elevated activity of the promoters located
upstream of icmR and the lpg1441-cpxA operon,
and OxyRLp directly bound to these promoter regions, suggesting
that OxyRLp is a direct repressor. Interestingly, a strain
overexpressing OxyRLp demonstrated reduced intracellular growth
in A. castellanii but not in U937-derived macrophages,
suggesting that balanced expression control of the two-component CpxRA system is
necessary for survival in protozoa. Taken together, this study suggests that
OxyRLp is a functionally redundant transcriptional regulator in L.
pneumophila under the conditions evaluated herein. IMPORTANCE: Legionella
pneumophila is an environmental pathogen, with its transmission to the
human host dependent upon its ability to replicate in protozoa and survive
within its aquatic niche. Understanding the genetic factors that contribute to L.
pneumophila survival within each of these unique environments will be
key to limiting future point-source outbreaks of Legionnaires' disease. The
transcriptional regulator L. pneumophila OxyR (OxyRLp)
has been previously identified as a potential regulator of virulence traits
warranting further investigation. This study demonstrated that oxyR is
nonessential for L. pneumophila survival in vitro and in
vivo via mutational analysis. While the mechanisms of how OxyRLp expression
is regulated remain elusive, this study shows that OxyRLp negatively
regulates the expression of the cpxRA two-component system
necessary for intracellular survival in protozoa.
Massis LM, Assis-Marques MA, Castanheira FV, Capobianco YJ, Balestra AC, Escoll P,
Wood RE, Manin GZ, Correa VM, Alves-Filho JC, Cunha FQ, Buchrieser C, Borges MC,
Newton HJ, Zamboni DS.
Department of Medical Clinic, School of Medicine of
Ribeirão Preto, University of São Paulo, FMRP/USP, Ribeirão Preto, Brazil.
dszamboni@fmrp.usp.br
J Infect Dis 2017
Feb;215(3):440-451.
Abstract:
Background: Legionella
longbeachae (Llo) and Legionella pneumophila (Lpn) are the most
common pneumonia-causing agents of the genus. Although both species can be
lethal to humans and are highly prevalent, little is known about the molecular
pathogenesis of Llo infections. In murine models of infection, Lpn infection is
self-limited, whereas Llo infection is lethal. Methods: We
used mouse macrophages, human macrophages, human epithelial cells, and mouse
infections in vivo to evaluate multiple parameters of the infection. Results: We
determined that the Llo Dot/Icm secretion system is critical for virulence.
Different than Lpn, Llo disseminates and the animals develop a severe pulmonary
failure, as demonstrated by lung mechanics and blood oxygenation assays. As
compared to Lpn, Llo is immunologically silent and fails to trigger the
production of cytokines in human pulmonary epithelial cells and in mouse and
human macrophages. Infections in Tnfr1-/-, Ifng-/-, and Il12p40-/- mice
supported the participation of cytokines for the resistance phenotype. Conclusions: Both
Lpn and Llo require the Dot/Icm system for pathogenesis, but the infection
outcome is strikingly different. Llo is immunologically silent, highly virulent,
and lethal. The differences reported herein may reflect unappreciated clinical
differences in patients infected with Lpn or Llo.
Jung AL, Hoffmann K, Herkt CE, Schulz C, Bertrams W, Schmeck B.
Department of Medicine, Pulmonary and
Critical Care Medicine, University Medical Center Giessen and Marburg, German. bernd.schmeck@uni-marburg.de
J Vis Exp 2017 Feb;(120):55146.
Abstract: Bacteria are able to secrete a variety
of molecules via various secretory systems. Besides the secretion of molecules
into the extracellular space or directly into another cell, Gram-negative
bacteria can also form outer membrane vesicles (OMVs). These membrane vesicles
can deliver their cargo over long distances, and the cargo is protected from
degradation by proteases and nucleases. Legionella pneumophila (L.
pneumophila) is an intracellular, Gram-negative pathogen that causes a
severe form of pneumonia. In humans, it infects alveolar macrophages, where it
blocks lysosomal degradation and forms a specialized replication vacuole.
Moreover, L. pneumophila produces OMVs under various growth conditions.
To understand the role of OMVs in the infection process of human macrophages, we
set up a protocol to purify bacterial membrane vesicles from liquid culture. The
method is based on differential ultracentrifugation. The enriched OMVs were
subsequently analyzed with regard to their protein and lipopolysaccharide (LPS)
amount and were then used for the treatment of a human monocytic cell line or
murine bone marrow-derived macrophages. The pro-inflammatory responses of those
cells were analyzed by enzyme-linked immunosorbent assay. Furthermore,
alterations in a subsequent infection were analyzed. To this end, the bacterial
replication of L. pneumophila in macrophages was studied by
colony-forming unit assays. Here, we describe a detailed protocol for the
purification of L. pneumophila OMVs from liquid culture by
ultracentrifugation and for the downstream analysis of their pro-inflammatory
potential on macrophages.
Hochstrasser R,
Hilbi H.
Department of Medicine, Institute of
Medical Microbiology, University of Zürich Zürich, Switzerland.
hilbi@imm.uzh.ch
Front Microbiol 2017 Feb;8:79.
Abstract: The ubiquitous Gram-negative bacterium Legionella
pneumophila parasitizes environ mental amoebae and, upon inhalation,
replicates in alveolar macrophages, thus causing a life-threatening pneumonia
called "Legionnaires' disease." The opportunistic pathogen employs a
bi-phasic life cycle, alternating between a replicative, non-virulent phase and
a stationary, transmissive/virulent phase. L. pneumophila employs
the Lqs (Legionella quorum sensing) system as a major regulator of
the growth phase switch. The Lqs system comprises the autoinducer synthase LqsA,
the homologous sensor kinases LqsS and LqsT, as well as a prototypic response
regulator termed LqsR. These components produce, detect, and respond to the α-hydroxyketone
signaling molecule LAI-1 (Legionella autoinducer-1,
3-hydroxypentadecane-4-one). LAI-1-mediated signal transduction through the
sensor kinases converges on LqsR, which dimerizes upon phosphorylation. The Lqs
system regulates the bacterial growth phase switch, pathogen-host cell
interactions, motility, natural competence, filament production, and expression
of a chromosomal "fitness island." Yet, LAI-1 not only mediates
bacterial intra-species signaling, but also modulates the motility of eukaryotic
cells through the small GTPase Cdc42 and thus promotes inter-kingdom signaling.
Taken together, the low molecular weight compound LAI-1 produced by L.
pneumophila and sensed by the bacteria as well as by eukaryotic cells
plays a major role in pathogen-host cell interactions.
Shen Y, Huang C, Lin J, Wu W,
Ashbolt NJ, Liu WT, Nguyen TH.
Department of Civil and Environmental Engineering,
University of Illinois at Urbana-Champaign, Urbana, Illinois, USA.
senyun11@gmail.com
Environ Sci
Technol 2017 Feb;51(4):2087-2095.
Abstract: Legionella pneumophila, the most
commonly identified causative agent in drinking water associated with disease
outbreaks, can be harbored by and released from drinking water biofilms. In this
study, the release of biofilm-associated L. pneumophila under simulated
drinking water flow containing a disinfectant residual was examined. Meanwhile,
the inactivation and infectivity (to amoebae) of the released L. pneumophila
were studied. To simulate drinking water system conditions, biofilms were
prepared under either disinfectant exposure (predisinfected biofilms) or
disinfectant-free (untreated biofilms) conditions, respectively. For experiments
with water flow containing a disinfectant to release the biofilm-associated L.
pneumophila from these two types of biofilms, the L. pneumophila
release kinetics values from predisinfected and untreated biofilms under flow
condition were not statistically different (one-way ANOVA, p>0.05). However,
inactivation of the L. pneumophila released from predisinfected biofilms
was 1-2 times higher and amoeba infectivity was 2-29 times lower than that from
untreated biofilms. The higher disinfectant resistance of L. pneumophila released
from untreated biofilms was presumably influenced by the detachment of a larger
amount of biofilm material (determined by 16S rRNA qPCR) surrounding the
released L. pneumophila. This study highlights the interaction among
disinfectant residual, biofilms, and L. pneumophila, which provides
guidelines to assess and control pathogen risk.
Descours G, Ginevra C, Jacotin N,
Forey F, Chastang J, Kay E, Etienne J, Lina G, Doublet P, Jarraud S.
CIRI,
Centre International de Recherche en Infectiologie, Equipe Pathogénèse des Légionelles,
Lyon, France. ghislaine.descours@univ-lyon1.fr
Antimicrob Agents Chemother 2017
Feb;61(3):e02188-16.
Abstract:
Monitoring the emergence of antibiotic resistance is a recent issue in the
treatment of Legionnaires' disease. Macrolides are recommended as first-line
therapy, but resistance mechanisms have not been studied in Legionella species.
Our aim was to determine the molecular basis of macrolide resistance in L.
pneumophila Twelve independent lineages from a common susceptible L.
pneumophila ancestral strain were propagated under conditions of
erythromycin or azithromycin pressure to produce high-level macrolide resistance.
Whole-genome sequencing was performed on 12 selected clones, and we investigated
mutations common to all lineages. We reconstructed the dynamics of mutation for
each lineage and demonstrated their involvement in decreased susceptibility to
macrolides. The resistant mutants were produced in a limited number of passages
to obtain a 4,096-fold increase in erythromycin MICs. Mutations affected highly
conserved 5-amino-acid regions of L4 and L22 ribosomal proteins and of domain V
of 23S rRNA (G2057, A2058, A2059, and C2611 nucleotides). The early mechanisms
mainly affected L4 and L22 proteins and induced a 32-fold increase in the MICs
of the selector drug. Additional mutations related to 23S rRNA mostly occurred
later and were responsible for a major increase of macrolide MICs, depending on
the mutated nucleotide, the substitution, and the number of mutated genes among
the three rrl copies. The major mechanisms of the decreased
susceptibility to macrolides in L. pneumophila and their
dynamics were determined. The results showed that macrolide resistance could be
easily selected in L. pneumophila and warrant further
investigations in both clinical and environmental settings.
van der Kooij D, Bakker GL, Italiaander R,
Veenendaal HR, Wullings BA.
KWR Watercycle Research Institute,
Nieuwegein, the Netherlands. Dick.van.der.Kooij@kwrwater.nl
Appl Environ
Microbiol 2017 Feb;83(5):e02737-16.
Abstract: Legionella pneumophila in
potable water installations poses a potential health risk, but quantitative
information about its replication in biofilms in relation to water quality is
scarce. Therefore, biofilm formation on the surfaces of glass and chlorinated
polyvinyl chloride (CPVC) in contact with tap water at 34 to 39°C was
investigated under controlled hydraulic conditions in a model system inoculated
with biofilm-grown L. pneumophila The biofilm on glass (average
steady-state concentration, 23±9 pg ATP cm-2) exposed to treated
aerobic groundwater (0.3 mg C liter-1; 1 μg assimilable organic carbon [AOC] liter-1)
did not support growth of the organism, which also disappeared from the biofilm
on CPVC (49±9 pg ATP cm-2) after initial growth. L.
pneumophila attained a level of 4.3 log CFU cm-2 in the
biofilms on glass (1,055±225 pg ATP cm-2) and CPVC (2,755±460 pg
ATP cm-2) exposed to treated anaerobic groundwater (7.9 mg C liter-1;
10 μg AOC liter-1).
An elevated biofilm concentration and growth of L. pneumophila were
also observed with tap water from the laboratory. The Betaproteobacteria
Piscinibacter and Methyloversatilis and amoeba
resisting Alphaproteobacteria predominated in the clones and
isolates retrieved from the biofilms. In the biofilms, the Legionella colony
count correlated significantly with the total cell count (TCC), heterotrophic
plate count, ATP concentration, and presence of Vermamoeba vermiformis.
This amoeba was rarely detected at biofilm concentrations of <100 pg ATP cm-2.
A threshold concentration of approximately 50 pg ATP cm-2 (TCC=1×106 to
2×106 cells cm-2) was derived for growth of L.
pneumophila in biofilms. IMPORTANCE: Legionella pneumophila is
the etiologic agent in more than 10,000 cases of Legionnaires' disease that are
reported annually worldwide and in most of the drinking water-associated disease
outbreaks reported in the United States. The organism proliferates in biofilms
on surfaces exposed to warm water in engineered freshwater installations. An
investigation with a test system supplied with different types of warm drinking
water without disinfectant under controlled hydraulic conditions showed that
treated aerobic groundwater (0.3 mg liter-1 of organic carbon)
induced a low biofilm concentration that supported no or very limited growth of L.
pneumophila. Elevated biofilm concentrations and L. pneumophila colony
counts were observed on surfaces exposed to two types of extensively treated
groundwater, containing 1.8 and 7.9 mg C liter-1 and complying
with the microbial water quality criteria during distribution. Control measures
in warm tap water installations are therefore essential for preventing growth of L.
pneumophila.
Liu Y, Zhu W, Tan Y, Nakayasu ES, Staiger CJ, Luo ZQ.
Purdue Institute for Inflammation, Immunology and
Infectious Diseases and Department of Biological Sciences, Purdue University,
West Lafayette, IN, USA. luoz@purdue.edu
PLoS Pathog 2017
Jan;13(1):e1006186.
Abstract:
Legionella pneumophila, the etiological agent of
Legionnaires' disease, replicates intracellularly in protozoan and human hosts.
Successful colonization and replication of this pathogen in host cells requires
the Dot/Icm type IVB secretion system, which translocates approximately 300
effector proteins into the host cell to modulate various cellular processes. In
this study, we identified RavK as a Dot/Icm substrate that targets the host
cytoskeleton and reduces actin filament abundance in mammalian cells upon
ectopic expression. RavK harbors an H95EXXH99 motif associated with diverse
metalloproteases, which is essential for the inhibition of yeast growth and for
the induction of cell rounding in HEK293T cells. We demonstrate that the actin
protein itself is the cellular target of RavK and that this effector cleaves
actin at a site between residues Thr351 and Phe352. Importantly, RavK-mediated
actin cleavage also occurs during L. pneumophila infection. Cleavage by
RavK abolishes the ability of actin to form polymers. Furthermore, an F352A
mutation renders actin resistant to RavK-mediated cleavage; expression of the
mutant in mammalian cells suppresses the cell rounding phenotype caused by RavK,
further establishing that actin is the physiological substrate of RavK. Thus, L.
pneumophila exploits components of the host cytoskeleton by multiple
effectors with distinct mechanisms, highlighting the importance of modulating
cellular processes governed by the actin cytoskeleton in the intracellular life
cycle of this pathogen.
Oliva G, Sahr T, Rolando M, Knoth M, Buchrieser C.
Institut Pasteur, Biologie des Bactéries
Intracellulaires, Paris, France. cbuch@pasteur.fr
mBio 2017
Jan;8(1):e02182-16.
Abstract: Legionella
pneumophila is an environmental bacterium that parasitizes protozoa,
but it may also infect humans, thereby causing a severe pneumonia called
Legionnaires' disease. To cycle between the environment and a eukaryotic host, L.
pneumophila is regulating the expression of virulence factors in a life
cycle-dependent manner: replicating bacteria do not express virulence factors,
whereas transmissive bacteria are highly motile and infective. Here we show that
Hfq is an important regulator in this network. Hfq is highly expressed in
transmissive bacteria but is expressed at very low levels in replicating
bacteria. A L. pneumophila hfq deletion mutant exhibits reduced abilities
to infect and multiply in Acanthamoeba castellanii at environmental
temperatures. The life cycle-dependent regulation of Hfq expression depends on a
unique cis-encoded small RNA named Anti-hfq that is transcribed antisense of the
hfq transcript and overlaps its 5' untranslated region. The Anti-hfq sRNA is
highly expressed only in replicating L. pneumophila where it regulates
hfq expression through binding to the complementary regions of the hfq
transcripts. This results in reduced Hfq protein levels in exponentially growing
cells. Both the small noncoding RNA (sRNA) and hfq mRNA are bound and stabilized
by the Hfq protein, likely leading to the cleavage of the RNA duplex by the
endoribonuclease RNase III. In contrast, after the switch to transmissive
bacteria, the sRNA is not expressed, allowing now an efficient expression of the
hfq gene and consequently Hfq. Our results place Hfq and its newly identified
sRNA anti-hfq in the center of the regulatory network governing L.
pneumophila differentiation from nonvirulent to virulent bacteria. Importance: The
abilities of L. pneumophila to replicate intracellularly and to cause
disease depend on its capacity to adapt to different extra- and intracellular
environmental conditions. Therefore, a timely and fine-tuned expression of
virulence factors and adaptation traits is crucial. Yet, the regulatory circuits
governing the life cycle of L. pneumophila from replicating to virulent
bacteria are only partly uncovered. Here we show that the life cycle-dependent
regulation of the RNA chaperone Hfq relies on a small regulatory RNA encoded
antisense to the hfq-encoding gene through a base pairing mechanism. Furthermore,
Hfq regulates its own expression in an autoregulatory loop. The discovery of
this RNA regulatory mechanism in L. pneumophila is an important step
forward in the understanding of how the switch from inoffensive, replicating to
highly virulent, transmissive L. pneumophila is regulated.
Kawamoto Y,
Morinaga Y, Kimura Y, Kaku N, Kosai K, Uno N, Hasegawa H, Yanagihara K.
Department of Laboratory Medicine, Nagasaki University
Graduate School of Biomedical Sciences, Nagasaki, Japan. y-morina@nagasaki-u.ac.jp
J Infect
Chemother 2017 Jan;23(1):51-55.
Abstract: Background: TNF-α plays an important role in the pathogenesis of Legionella
pneumophila (Lp)-induced pneumonia. Patients undergoing anti-TNF-α therapy are at an increased risk of Lp infection. Lp
infects both phagocytic and non-phagocytic cells such as airway epithelial cells;
however, the role of TNF-α
in airway epithelial cells is unknown. Methods: Human
airway epithelial cell line NCI-H292 was infected with Lp NUL1 strain. After
infection, both intracellular growth of Lp and cell death were evaluated after
treating the cells with or without TNF-α.
Apoptosis was examined by performing activated caspase-3/7 staining and by using
a pan-caspase inhibitor. Results: Lp
infected and replicated in NCI-H292 cells in a time-dependent manner, and TNF-α treatment of Lp-infected NCI-H292 cells inhibited Lp
replication. Inhibitory effects of TNF-α
on Lp replication were suppressed after treatment with a TNF-α-neutralizing antibody. Lp infection increased
extracellular lactate dehydrogenase levels and decreased the number of living
cells. Increased number of Lp-infected NCI-H292 cells showed caspase-3/7
activation, indicating they underwent apoptosis. TNF-α
treatment inhibited Lp replication by increasing the apoptosis of NCI-H292 cells.
Conclusions: Thus,
our results suggested that airway epithelial cells were involved in the
pathogenesis of Lp infection and that TNF-α
played a protective role by inhibiting the intracellular replication of Lp and
by increasing the apoptosis of Lp-infected airway epithelial cells. However, Lp
infection should be investigated further in patients undergoing anti-TNF-α therapy who develop pneumonia.
Hilbi H, Nagai H, Kubori T, Roy CR.
Department of Microbial Pathogenesis,
Yale University, New Haven, CT, USA. craig.roy@yale.edu
Curr Top Microbiol Immunol 2017;413:221-242.
Abstract: Legionella species are Gram-negative ubiquitous environmental bacteria, which thrive in biofilms and parasitize protozoa. Employing an evolutionarily conserved mechanism, the opportunistic pathogens also replicate intracellularly in mammalian macrophages. This feature is a prerequisite for the pathogenicity of Legionella pneumophila, which causes the vast majority of clinical cases of a severe pneumonia, termed "Legionnaires' disease." In macrophages as well as in amoeba, L. pneumophila grows in a distinct membrane-bound compartment, the Legionella-containing vacuole (LCV). Formation of this replication-permissive pathogen compartment requires the bacterial Dot/Icm type IV secretion system (T4SS). Through the T4SS as many as 300 different "effector" proteins are injected into host cells, where they presumably subvert pivotal processes. Less than 40 Dot/Icm substrates have been characterized in detail to date, a number of which show unprecedented biological activities. Some of these effector proteins target host cell small GTPases, phosphoinositide lipids, the chelator phytate, the ubiquitination machinery, the retromer complex, the actin cytoskeleton, or the autophagy pathway. A recently discovered class of L. pneumophila effectors modulates the activity of other effectors and is termed "metaeffectors." Here, we summarize recent insight into the cellular functions and biochemical activities of L. pneumophila effectors and metaeffectors targeting the host's endocytic, retrograde, or autophagic pathways.
