scholarly journals ArgR-Regulated Genes Are Derepressed in the Legionella-Containing Vacuole

2010 ◽  
Vol 192 (17) ◽  
pp. 4504-4516 ◽  
Author(s):  
Galadriel Hovel-Miner ◽  
Sebastien P. Faucher ◽  
Xavier Charpentier ◽  
Howard A. Shuman
Keyword(s):  
Legionella Pneumophila ◽  
Acanthamoeba Castellanii ◽  
Transcriptional Response ◽  
Global Gene Expression ◽  
Host Cells ◽  
Intracellular Growth ◽  
Arginine Repressor ◽  
Legionnaires Disease ◽  
Global Gene Expression Analysis ◽  
Eukaryotic Organisms

ABSTRACT Legionella pneumophila is an intracellular pathogen that infects protozoa in aquatic environments and when inhaled by susceptible human hosts replicates in alveolar macrophages and can result in the often fatal pneumonia called Legionnaires' disease. The ability of L. pneumophila to replicate within host cells requires the establishment of a specialized compartment that evades normal phagolysosome fusion called the Legionella-containing vacuole (LCV). Elucidation of the biochemical composition of the LCV and the identification of the regulatory signals sensed during intracellular replication are inherently challenging. l-Arginine is a critical nutrient in the metabolism of both prokaryotic and eukaryotic organisms. We showed that the L. pneumophila arginine repressor homolog, ArgR, is required for maximal intracellular growth in the unicellular host Acanthamoeba castellanii. In this study, we present evidence that the concentration of l-arginine in the LCV is sensed by ArgR to produce an intracellular transcriptional response. We characterized the L. pneumophila ArgR regulon by global gene expression analysis, identified genes highly affected by ArgR, showed that ArgR repression is dependent upon the presence of l-arginine, and demonstrated that ArgR-regulated genes are derepressed during intracellular growth. Additional targets of ArgR that may account for the argR mutant's intracellular multiplication defect are discussed. These results suggest that l-arginine availability functions as a regulatory signal during Legionella intracellular growth.

scholarly journals Additive Effect on Intracellular Growth by Legionella pneumophila Icm/Dot Proteins Containing a Lipobox Motif

2005 ◽  
Vol 73 (11) ◽  
pp. 7578-7587 ◽  
Author(s):  
Gal Yerushalmi ◽  
Tal Zusman ◽  
Gil Segal
Keyword(s):  
Legionella Pneumophila ◽  
Acanthamoeba Castellanii ◽  
Cysteine Residue ◽  
Secretion System ◽  
Host Cells ◽  
Intracellular Growth ◽  
Type Iv ◽  
Essential Components ◽  
Legionnaires Disease ◽  
Type Ivb Secretion

ABSTRACT Legionella pneumophila, the causative agent of Legionnaires' disease, utilizes a type IVB secretion system to subvert its host cells and grow intracellularly. This type IV secretion system is composed of 25 icm (or dot) genes that probably constitute parts of a secretion complex as well as more than 30 proteins that are translocated via this system into the host cells. Three of the Icm/Dot proteins (DotD, DotC, and IcmN) contain a lipobox motif at their N terminals and are predicted to be lipoproteins. Two of these lipoproteins (DotD and DotC) were found to be essential for intracellular growth in both HL-60-derived human macrophages and in the protozoan host Acanthamoeba castellanii, while the third lipoprotein (IcmN) was found to be partially required for intracellular growth only in A. castellanii. Mutation analysis of the lipobox cysteine residue, which was shown previously to be indispensable for the lipobox function, indicated that both DotC and DotD are partially functional without this conserved residue. Cysteine mutations in both DotC and DotD or in DotC together with an icmN deletion or in DotD together with an icmN deletion were found to be additive, indicating that each of these lipoproteins performs its function independently from the others. Analysis of the transcriptional regulation of both the dotDC operon and the icmN gene revealed that both had higher levels of expression at stationary phase which were partially dependent on the LetA regulator. Our results indicate that the lipoproteins of the L. pneumophila icm (or dot) system are essential components of the secretion system and that they perform their functions independently.

scholarly journals bdhA-patD Operon as a Virulence Determinant, Revealed by a Novel Large-Scale Approach for Identification of Legionella pneumophila Mutants Defective for Amoeba Infection

2009 ◽  
Vol 75 (13) ◽  
pp. 4506-4515 ◽  
Author(s):  
P. Aurass ◽  
B. Pless ◽  
K. Rydzewski ◽  
G. Holland ◽  
N. Bannert ◽  
...  
Keyword(s):  
Legionella Pneumophila ◽  
Large Scale ◽  
Human Lung ◽  
Agar Plate ◽  
Acanthamoeba Castellanii ◽  
Host Cells ◽  
Alveolar Cells ◽  
Virulence Determinant ◽  
Transposon Mutants ◽  
Legionnaires Disease

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

scholarly journals Intracellular Growth in Acanthamoeba castellanii Affects Monocyte Entry Mechanisms and Enhances Virulence of Legionella pneumophila

1999 ◽  
Vol 67 (9) ◽  
pp. 4427-4434 ◽  
Author(s):  
Jeffrey D. Cirillo ◽  
Suat L. G. Cirillo ◽  
Ling Yan ◽  
Luiz E. Bermudez ◽  
Stanley Falkow ◽  
...  
Keyword(s):  
Legionella Pneumophila ◽  
Acanthamoeba Castellanii ◽  
Host Cells ◽  
Intracellular Pathogen ◽  
Activated Macrophages ◽  
Standard Laboratory ◽  
Intracellular Growth ◽  
Growth Environment ◽  
Pathogen Entry ◽  
First Time

ABSTRACT Since Legionella pneumophila is an intracellular pathogen, entry into and replication within host cells are thought to be critical to its ability to cause disease. L. pneumophilagrown in one of its environmental hosts, Acanthamoeba castellanii, is phenotypically different from L. pneumophila grown on standard laboratory medium (BCYE agar). Although amoeba-grown L. pneumophila displays enhanced entry into monocytes compared to BCYE-grown bacteria, the mechanisms of entry used and the effects on virulence have not been examined. To explore whether amoeba-grown L. pneumophila differs from BCYE-grown L. pneumophila in these characteristics, we examined entry into monocytes, replication in activated macrophages, and virulence in mice. Entry of amoeba-grown L. pneumophilainto monocytes occurred more frequently by coiling phagocytosis, was less affected by complement opsonization, and was less sensitive to microtubule and microfilament inhibitors than was entry of BCYE-grown bacteria. In addition, amoeba-grown L. pneumophila displays increased replication in monocytes and is more virulent in A/J, C57BL/6 Beige, and C57BL/6 mice. These data demonstrate for the first time that the intra-amoebal growth environment affects the entry mechanisms and virulence of L. pneumophila.

scholarly journals Characterization of the icmH and icmF Genes Required for Legionella pneumophila Intracellular Growth, Genes That Are Present in Many Bacteria Associated with Eukaryotic Cells

2004 ◽  
Vol 72 (6) ◽  
pp. 3398-3409 ◽  
Author(s):  
Tal Zusman ◽  
Michal Feldman ◽  
Einat Halperin ◽  
Gil Segal
Keyword(s):  
Legionella Pneumophila ◽  
Rhizobium Leguminosarum ◽  
Regulatory Region ◽  
Acanthamoeba Castellanii ◽  
Regulatory Elements ◽  
Host Cells ◽  
Eukaryotic Cells ◽  
Dependent Manner ◽  
Intracellular Growth ◽  
Homologous Proteins

ABSTRACT Legionella pneumophila, the causative agent of Legionnaires' disease, replicates intracellularly within a specialized phagosome of mammalian and protozoan host cells, and the Icm/Dot type IV secretion system has been shown to be essential for this process. Unlike all the other known Icm/Dot proteins, the IcmF protein, which was described before, and the IcmH protein, which is characterized here, have homologous proteins in many bacteria (such as Yersinia pestis, Salmonella enterica, Rhizobium leguminosarum, and Vibrio cholerae), all of which associate with eukaryotic cells. Here, we have characterized the L. pneumophila icmH and icmF genes and found that both genes are present in 16 different Legionella species examined. The icmH and icmF genes were found to be absolutely required for intracellular multiplication in Acanthamoeba castellanii and partially required for intracellular growth in HL-60-derived human macrophages, for immediate cytotoxicity, and for salt sensitivity. Mutagenesis of the predicted ATP/GTP binding site of IcmF revealed that the site is partially required for intracellular growth in A. castellanii. Analysis of the regulatory region of the icmH and icmF genes, which were found to be cotranscribed, revealed that it contains at least two regulatory elements. In addition, an icmH::lacZ fusion was shown to be activated during stationary phase in a LetA- and RelA-dependent manner. Our results indicate that although the icmH and icmF genes probably have a different evolutionary origin than the rest of the icm/dot genes, they are part of the icm/dot system and are required for L. pneumophila pathogenesis.

scholarly journals Cyclic Diguanylate Signaling Proteins Control Intracellular Growth of Legionella pneumophila

mBio ◽  
2011 ◽  
Vol 2 (1) ◽  
Author(s):  
Assaf Levi ◽  
Marc Folcher ◽  
Urs Jenal ◽  
Howard A. Shuman
Keyword(s):  
Causative Agent ◽  
Legionella Pneumophila ◽  
Second Messenger ◽  
Host Cells ◽  
Diguanylate Cyclase ◽  
Intracellular Growth ◽  
Cyclic Diguanylate ◽  
Content Type ◽  
Legionnaires Disease

ABSTRACTProteins that metabolize or bind the nucleotide second messenger cyclic diguanylate regulate a wide variety of important processes in bacteria. These processes include motility, biofilm formation, cell division, differentiation, and virulence. The role of cyclic diguanylate signaling in the lifestyle ofLegionella pneumophila, the causative agent of Legionnaires’ disease, has not previously been examined. TheL. pneumophilagenome encodes 22 predicted proteins containing domains related to cyclic diguanylate synthesis, hydrolysis, and recognition. We refer to these genes ascdgS(cyclicdiguanylatesignaling) genes. Strains ofL. pneumophilacontaining deletions of all individualcdgSgenes were created and did not exhibit any observable growth defect in growth medium or inside host cells. However, when overexpressed, severalcdgSgenes strongly decreased the ability ofL. pneumophilato grow inside host cells. Expression of thesecdgSgenes did not affect the Dot/Icm type IVB secretion system, the major determinant of intracellular growth inL. pneumophila.L. pneumophilastrains overexpressing thesecdgSgenes were less cytotoxic to THP-1 macrophages than wild-typeL. pneumophilabut retained the ability to resist grazing by amoebae. In many cases, the intracellular-growth inhibition caused bycdgSgene overexpression was independent of diguanylate cyclase or phosphodiesterase activities. Expression of thecdgSgenes in aSalmonella entericaserovar Enteritidis strain that lacks all diguanylate cyclase activity indicated that severalcdgSgenes encode potential cyclases. These results indicate that components of the cyclic diguanylate signaling pathway play an important role in regulating the ability ofL. pneumophilato grow in host cells.IMPORTANCEAll bacteria must sense and respond to environmental cues. Intracellular bacterial pathogens must detect and respond to host functions that limit their ability to carry out a successful infection. Small-molecule second messengers play key roles in transmitting signals from environmental receptors to the proteins and other components that respond to signals. Cyclic diguanylate is a ubiquitous bacterial second messenger known to play an important role in many sensing and signaling systems in bacteria. The causative agent of Legionnaires’ disease,Legionella pneumophila, is an intracellular pathogen that grows inside environmental protists and human macrophages by subverting the normal processes that these cells use to capture and destroy bacteria. We show that the several cyclic diguanylate signaling components inLegionellaplay a role in the ability to grow inside both kinds of host cells. This work highlights the role of cyclic diguanylate signaling during intracellular growth.

scholarly journals Legionella pneumophila DotU and IcmF Are Required for Stability of the Dot/Icm Complex

2004 ◽  
Vol 72 (10) ◽  
pp. 5983-5992 ◽  
Author(s):  
Jessica A. Sexton ◽  
Jennifer L. Miller ◽  
Aki Yoneda ◽  
Thomas E. Kehl-Fie ◽  
Joseph P. Vogel
Keyword(s):  
Cell Surface ◽  
Legionella Pneumophila ◽  
Bacterial Species ◽  
Wide Distribution ◽  
Host Cells ◽  
Growth Defect ◽  
Intracellular Growth ◽  
Homologous Proteins ◽  
Type Iv ◽  
Cell Surface Structure

ABSTRACT Legionella pneumophila utilizes a type IV secretion system (T4SS) encoded by 26 dot/icm genes to replicate inside host cells and cause disease. In contrast to all other L. pneumophila dot/icm genes, dotU and icmF have homologs in a wide variety of gram-negative bacteria, none of which possess a T4SS. Instead, dotU and icmF orthologs are linked to a locus encoding a conserved cluster of proteins designated IcmF-associated homologous proteins, which has been proposed to constitute a novel cell surface structure. We show here that dotU is partially required for L. pneumophila intracellular growth, similar to the known requirement for icmF. In addition, we show that dotU and icmF are necessary for optimal plasmid transfer and sodium sensitivity, two additional phenotypes associated with a functional Dot/Icm complex. We found that these effects are due to the destabilization of the T4SS at the transition into the stationary phase, the point at which L. pneumophila becomes virulent. Specifically, three Dot proteins (DotH, DotG, and DotF) exhibit decreased stability in a ΔdotU ΔicmF strain. Furthermore, overexpression of just one of these proteins, DotH, is sufficient to suppress the intracellular growth defect of the ΔdotU ΔicmF mutant. This suggests a model where the DotU and IcmF proteins serve to prevent DotH degradation and therefore function to stabilize the L. pneumophila T4SS. Due to their wide distribution among bacterial species and their genetic linkage to known or predicted cell surface structures, we propose that this function in complex stabilization may be broadly conserved.

scholarly journals Subinhibitory Concentrations of Antimicrobial Agents Reduce the Uptake of Legionella pneumophila into Acanthamoeba castellanii and U937 Cells by Altering the Expression of Virulence-Associated Antigens

1998 ◽  
Vol 42 (11) ◽  
pp. 2870-2876 ◽  
Author(s):  
P. Christian Lück ◽  
Jürgen W. Schmitt ◽  
Arne Hengerer ◽  
Jürgen H. Helbig
Keyword(s):  
Monoclonal Antibodies ◽  
Membrane Protein ◽  
Outer Membrane ◽  
Outer Membrane Protein ◽  
Legionella Pneumophila ◽  
Antimicrobial Agents ◽  
Acanthamoeba Castellanii ◽  
Host Cells ◽  
Uncharacterized Protein ◽  
Subinhibitory Concentrations

ABSTRACT We determined the MICs of ampicillin, ciprofloxacin, erythromycin, imipenem, and rifampin for two clinical isolates of Legionella pneumophila serogroup 1 by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay and by quantitative culture. To test the influence of subinhibitory concentrations (sub-MICs) of antimicrobial agents on Legionella uptake into Acanthamoeba castellanii and U937 macrophage-like cells, both strains were pretreated with 0.25 MICs of the antibiotics for 24 h. In comparison to that for the untreated control, subinhibitory concentrations of antibiotics significantly reducedLegionella uptake into the host cells. Measurement of the binding of monoclonal antibodies against several Legionellaantigens by enzyme-linked immunoassays indicated that sub-MIC antibiotic treatment reduced the expression of the macrophage infectivity potentiator protein (Mip), the Hsp 60 protein, the outer membrane protein (OmpM), an as-yet-uncharacterized protein of 55 kDa, and a few lipopolysaccharide (LPS) epitopes. In contrast, the expression of some LPS epitopes recognized by monoclonal antibodies 8/5 and 30/4 as well as a 45-kDa protein, a 58-kDa protein, and the major outer membrane protein (OmpS) remained unaffected.

scholarly journals Human Lung Tissue Explants Reveal Novel Interactions during Legionella pneumophila Infections

2013 ◽  
Vol 82 (1) ◽  
pp. 275-285 ◽  
Author(s):  
Jens Jäger ◽  
Sebastian Marwitz ◽  
Jana Tiefenau ◽  
Janine Rasch ◽  
Olga Shevchuk ◽  
...  
Keyword(s):  
Lung Tissue ◽  
Legionella Pneumophila ◽  
Human Lung ◽  
Transcriptional Response ◽  
Human Infection ◽  
Bacterial Invasion ◽  
Human Lung Tissue ◽  
Content Type ◽  
Tissue Explants ◽  
Legionnaires Disease

ABSTRACTHistological and clinical investigations describe late stages of Legionnaires' disease but cannot characterize early events of human infection. Cellular or rodent infection models lack the complexity of tissue or have nonhuman backgrounds. Therefore, we developed and applied a novel model forLegionella pneumophilainfection comprising living human lung tissue. We stimulated lung explants withL. pneumophilastrains and outer membrane vesicles (OMVs) to analyze tissue damage, bacterial replication, and localization as well as the transcriptional response of infected tissue. Interestingly, we found that extracellular adhesion ofL. pneumophilato the entire alveolar lining precedes bacterial invasion and replication in recruited macrophages. In contrast, OMVs predominantly bound to alveolar macrophages. Specific damage to septa and epithelia increased over 48 h and was stronger in wild-type-infected and OMV-treated samples than in samples infected with the replication-deficient, type IVB secretion-deficient DotA−strain. Transcriptome analysis of lung tissue explants revealed a differential regulation of 2,499 genes after infection. The transcriptional response included the upregulation of uteroglobin and the downregulation of the macrophage receptor with collagenous structure (MARCO). Immunohistochemistry confirmed the downregulation of MARCO at sites of pathogen-induced tissue destruction. Neither host factor has ever been described in the context ofL. pneumophilainfections. This work demonstrates that the tissue explant model reproduces realistic features of Legionnaires' disease and reveals new functions for bacterial OMVs during infection. Our model allows us to characterize early steps of human infection which otherwise are not feasible for investigations.

scholarly journals Peptidyl-Prolyl-cis/trans-Isomerases Mip and PpiB ofLegionella pneumophilaContribute to Surface Translocation, Growth at Suboptimal Temperature, and Infection

2018 ◽  
Vol 87 (1) ◽  
Author(s):  
J. Rasch ◽  
C. M. Ünal ◽  
A. Klages ◽  
Ü. Karsli ◽  
N. Heinsohn ◽  
...  
Keyword(s):  
Legionella Pneumophila ◽  
Acanthamoeba Castellanii ◽  
Temperature Tolerance ◽  
Lung Damage ◽  
Atypical Pneumonia ◽  
Content Type ◽  
Wide Range ◽  
Legionnaires Disease ◽  
Environmental Fitness ◽  
Sliding Motility

ABSTRACTThe gammaproteobacteriumLegionella pneumophilais 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. InL. pneumophilathe 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 ofL. pneumophila. Furthermore, they contribute to infection of the natural hostAcanthamoeba castellaniias 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 ofL. pneumophilawith compensatory action and pleiotropic effects.

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

2019 ◽  
Author(s):  
Ila S. Anand ◽  
Won Young Choi ◽  
Ralph R. Isberg
Keyword(s):  
Membrane Trafficking ◽  
Legionella Pneumophila ◽  
Host Cells ◽  
Rab Gtpases ◽  
Intracellular Growth ◽  
Mutant Proteins ◽  
Host Cytoplasm ◽  
Downstream Effectors ◽  
Cell Death Response ◽  
Host Membrane

SummaryLegionella 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 antagonize 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 destabilize the replication vacuole, EEA1 and Rab11FIP1 showed enhanced colocalization with the vacuole surrounding the sdhA mutant compared with the WT vacuole. Depletion of Rab5 isoforms or Rab11b reduced this aberrant colocalization. These findings are consistent with SdhA interfering with both endocytic and recycling membrane trafficking events that act to destabilize vacuole integrity during infection.

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