Lipopolysaccharide Architecture of Legionella pneumophila Grown in Broth and Host Cells

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.

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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

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.42.11.2870 ◽

1998 ◽

Vol 42 (11) ◽

pp. 2870-2876 ◽

Cited By ~ 8

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.

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

Journal of Experimental Medicine ◽

10.1084/jem.20100771 ◽

2010 ◽

Vol 207 (8) ◽

pp. 1713-1726 ◽

Cited By ~ 97

Author(s):

Christopher T.D. Price ◽

Tasneem Al-Quadan ◽

Marina Santic ◽

Snake C. Jones ◽

Yousef Abu Kwaik

Keyword(s):

Legionella Pneumophila ◽

Biological Function ◽

Host Cells ◽

Dependent Manner ◽

Protein Farnesyltransferase ◽

Type Iv ◽

Eukaryotic Proteins ◽

Bacterial Effectors

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

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Identification of Translocated Substrates of the Legionella pneumophila Dot/Icm System without the use of Eukaryotic Host Cells

Legionella ◽

10.1128/9781555815660.ch45 ◽

2014 ◽

pp. 167-176

Author(s):

Ralph R. Isberg ◽

Matthias Machner

Keyword(s):

Legionella Pneumophila ◽

Host Cells

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Molecular architecture of theLegionellaDot/Icm type IV secretion system

10.1101/312009 ◽

2018 ◽

Cited By ~ 3

Author(s):

Debnath Ghosal ◽

Yi-Wei Chang ◽

Kwang Cheol Jeong ◽

Joseph P. Vogel ◽

Grant J. Jensen

Keyword(s):

Legionella Pneumophila ◽

Cell Envelope ◽

Secretion System ◽

Host Cells ◽

Molecular Architecture ◽

Intact Cells ◽

Type Iv ◽

Type Ivb Secretion ◽

Electron Cryotomography ◽

First Time

AbstractLegionella pneumophilasurvives and replicates inside host cells by secreting ~300 effectors through the Dot/Icm type IVB secretion system (T4BSS). Understanding this machine’s structure is challenging because of its large number of components (27) and integration into all layers of the cell envelope. Previously we overcame this obstacle by imaging the Dot/Icm T4BSS in its native state within intact cells through electron cryotomography. Here we extend our observations by imaging a stabilized mutant that yielded a higher resolution map. We describe for the first time the presence of a well-ordered central channel that opens up into a windowed large (~32 nm wide) secretion chamber with an unusual 13-fold symmetry. We then dissect the complex by matching proteins to densities for many components, including all those with periplasmic domains. The placement of known and predicted structures of individual proteins into the map reveals the architecture of the T4BSS and provides a roadmap for further investigation of this amazing specialized secretion system.

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

10.1101/781849 ◽

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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Adherence of Legionella pneumophila to U-937 cells, guinea-pig alveolar macrophages, and MRC-5 cells by a novel, complement-independent binding mechanism

Canadian Journal of Microbiology ◽

10.1139/m94-137 ◽

1994 ◽

Vol 40 (10) ◽

pp. 865-872 ◽

Cited By ~ 20

Author(s):

Frank C. Gibson III ◽

Arthur O. Tzianabos ◽

Frank G. Rodgers

Keyword(s):

Monoclonal Antibodies ◽

Guinea Pig ◽

Host Cell ◽

Alveolar Macrophages ◽

Legionella Pneumophila ◽

Cell Receptor ◽

Host Cells ◽

Order Kinetic ◽

Complement Receptors ◽

Host Cell Receptor

In the absence of serum, Legionella pneumophila demonstrated wash-resistant adherence to U-937 cells, primary guinea-pig alveolar macrophages, and MRC-5 cells. Neither complement nor antibody was required for binding. The dynamics of adherence following inoculation of L. pneumophila at increasing 10-fold multiplicities of infection to each of the three host cell types resulted in a first-order kinetic relationship of binding, indicative of one bacterial adhesin molecule recognized by one host cell receptor moiety. Host cell receptor saturation studies showed that depending on the cell type, 2–8% of the bacterial inoculum adhered to cells under these nonopsonic conditions. Preliminary adhesin and receptor characterization studies were preformed to define the chemical composition of the binding structures on both the organism and the three different host cell surfaces. The adherence phenomenon was investigated using competitive binding assays in the presence of putative adhesin analogs as well as following treatments modifying the microbial and host cell surface membranes. Attachment was evaluated both by viable bacterial cell colony counts and by indirect immunofluorescent assay. With the exception of aldehyde treatments, the various membrane-modifying regimes and the presence of the adhesin analogs were shown to have no effect on organism or host cell viability. Data suggested that the L. pneumophila adhesin responsible for opsonin-independent binding to these host cells was a protein structure with lectin-like properties. Furthermore, this protein would appear to be intimately associated with carbohydrate or lipid structures located on the bacterial outer membrane. The receptor moiety present on all host cells responsible for binding L. pneumophila had properties consistent with a carbohydrate or complex saccharide structure. To evaluate the role of complement receptors as the structures necessary for L. pneumophila infection of macrophages, a battery of monoclonal antibodies were used to block the complement receptor (CR) types 1 (CD35), CR3 (CD 18, CD11b), and CR4 (CD18, CD11c). Blocking studies with CR-specific monoclonal antibodies indicated that CR1 and the integrin receptors CR3 and CR4 were not involved in the opsonin-independent binding of L. pneumophila to macrophage-like cells.Key words: Legionella, opsonin-independent attachment, bacterial adherence, complement receptors, adhesion–receptor interactions.

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Proteomic Characterization of the Whole Secretome of Legionella pneumophila and Functional Analysis of Outer Membrane Vesicles

Infection and Immunity ◽

10.1128/iai.01396-07 ◽

2008 ◽

Vol 76 (5) ◽

pp. 1825-1836 ◽

Cited By ~ 116

Author(s):

Frank Galka ◽

Sun Nyunt Wai ◽

Harald Kusch ◽

Susanne Engelmann ◽

Michael Hecker ◽

...

Keyword(s):

Outer Membrane ◽

Legionella Pneumophila ◽

Protein Identification ◽

Membrane Vesicles ◽

Outer Membrane Vesicles ◽

Laser Scanning Microscopy ◽

Host Cells ◽

Confocal Laser ◽

Lipolytic Enzyme ◽

New Findings

ABSTRACT Secretion of effector molecules is one of the major mechanisms by which the intracellular human pathogen Legionella pneumophila interacts with host cells during infection. Specific secretion machineries which are responsible for the subfraction of secreted proteins (soluble supernatant proteins [SSPs]) and the production of bacterial outer membrane vesicles (OMVs) both contribute to the protein composition of the extracellular milieu of this lung pathogen. Here we present comprehensive proteome reference maps for both SSPs and OMVs. Protein identification and assignment analyses revealed a total of 181 supernatant proteins, 107 of which were specific to the SSP fraction and 33 of which were specific to OMVs. A functional classification showed that a large proportion of the identified OMV proteins are involved in the pathogenesis of Legionnaires' disease. Zymography and enzyme assays demonstrated that the SSP and OMV fractions possess proteolytic and lipolytic enzyme activities which may contribute to the destruction of the alveolar lining during infection. Furthermore, it was shown that OMVs do not kill host cells but specifically modulate their cytokine response. Binding of immunofluorescently stained OMVs to alveolar epithelial cells, as visualized by confocal laser scanning microscopy, suggested that there is delivery of a large and complex group of proteins and lipids in the infected tissue in association with OMVs. On the basis of these new findings, we discuss the relevance of protein sorting and compartmentalization of virulence factors, as well as environmental aspects of the vesicle-mediated secretion.

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The Legionella pneumophila effector RavY contributes to a replication-permissive vacuolar environment during infection

Infection and Immunity ◽

10.1128/iai.00261-21 ◽

2021 ◽

Author(s):

Luying Liu ◽

Craig R. Roy

Keyword(s):

Legionella Pneumophila ◽

Effector Proteins ◽

Type Iv Secretion ◽

Host Cells ◽

Effector Protein ◽

Intracellular Replication ◽

Phagocytic Cells ◽

Host Molecules ◽

Type Iv ◽

Legionnaires Disease

Legionella pneumophila is the causative agent of Legionnaires’ Disease and is capable replicating inside phagocytic cells such as mammalian macrophages. The Dot/Icm type IV secretion system is a L. pneumophila virulence factor that is essential for successful intracellular replication. During infection, L. pneumophila builds a replication permissive vacuole by recruiting multiple host molecules and hijacking host cellular signaling pathways, a process mediated by the coordinated functions of multiple Dot/Icm effector proteins. RavY is a predicted Dot/Icm effector protein found to be important for optimal L. pneumophila replication inside host cells. Here, we demonstrate that RavY is a Dot/Icm-translocated effector protein that is dispensable for axenic replication of L. pneumophila , but critical for optimal intracellular replication of the bacteria. RavY is not required for avoidance of endosomal maturation, nor does RavY contribute to the recruitment of host molecules found on replication-permissive vacuoles, such as ubiquitin, RAB1a, and RTN4. Vacuoles containing L. pneumophila ravY mutants promote intracellular survival but limit replication. The replication defect of the L. pneumophila ravY mutant was complemented when the mutant was in the same vacuole as wild type L. pneumophila . Thus, RavY is an effector that is essential for promoting intracellular replication of L. pneumophila once the specialized vacuole has been established.

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

mBio ◽

10.1128/mbio.00405-20 ◽

2020 ◽

Vol 11 (2) ◽

Cited By ~ 3

Author(s):

A. Leoni Swart ◽

Bernhard Steiner ◽

Laura Gomez-Valero ◽

Sabina Schütz ◽

Mandy Hannemann ◽

...

Keyword(s):

Host Cell ◽

Legionella Pneumophila ◽

Small Gtpase ◽

Effector Proteins ◽

Host Cells ◽

Divergent Evolution ◽

Ran Gtpase ◽

Content Type ◽

Gtpase Cycle ◽

Gtpase Ran

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.

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