Membrane Vesicles Shed by Legionella pneumophila Inhibit Fusion of Phagosomes with Lysosomes

ABSTRACT When cultured in broth to the transmissive phase, Legionella pneumophila infects macrophages by inhibiting phagosome maturation, whereas replicative-phase cells are transported to the lysosomes. Here we report that the ability of L. pneumophila to inhibit phagosome-lysosome fusion correlated with developmentally regulated modifications of the pathogen's surface, as judged by its lipopolysaccharide profile and by its binding to a sialic acid-specific lectin and to the hydrocarbon hexadecane. Likewise, the composition of membrane vesicles shed by L. pneumophila was developmentally regulated, based on binding to the lectin and to the lipopolysaccharide-specific monoclonal antibody 3/1. Membrane vesicles were sufficient to inhibit phagosome-lysosome fusion by a mechanism independent of type IV secretion, since only ∼25% of beads suspended with or coated by vesicles from transmissive phase wild type or dotA secretion mutants colocalized with lysosomal probes, whereas ∼75% of beads were lysosomal when untreated or presented with vesicles from the L. pneumophila letA regulatory mutant or E. coli. As observed previously for L. pneumophila infection of mouse macrophages, vesicles inhibited phagosome-lysosome fusion only temporarily; by 10 h after treatment with vesicles, macrophages delivered ∼72% of ingested beads to lysosomes. Accordingly, in the context of the epidemiology of the pneumonia Legionnaires' disease and virulence mechanisms of Leishmania and Mycobacteria, we discuss a model here in which L. pneumophila developmentally regulates its surface composition and releases vesicles into phagosomes that inhibit their fusion with lysosomes.

Download Full-text

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.

Download Full-text

Polar targeting and assembly of theLegionellaDot/Icm type IV secretion system (T4SS) by T6SS-related components

10.1101/315721 ◽

2018 ◽

Cited By ~ 2

Author(s):

KwangCheol C. Jeong ◽

Jacob Gyore ◽

Lin Teng ◽

Debnath Ghosal ◽

Grant J. Jensen ◽

...

Keyword(s):

Legionella Pneumophila ◽

Secretion System ◽

Type Iv Secretion ◽

Host Cells ◽

Type Iv Secretion System ◽

Membrane Complex ◽

Type Iv ◽

Legionnaires Disease ◽

Type Ivb Secretion ◽

Type Ivb Secretion System

SummaryLegionella pneumophila, the causative agent of Legionnaires’ disease, survives and replicates inside amoebae and macrophages by injecting a large number of protein effectors into the host cells’ cytoplasm via the Dot/Icm type IVB secretion system (T4BSS). Previously, we showed that the Dot/Icm T4BSS is localized to both poles of the bacterium and that polar secretion is necessary for the proper targeting of theLegionellacontaining vacuole (LCV). Here we show that polar targeting of the Dot/Icm core-transmembrane subcomplex (DotC, DotD, DotF, DotG and DotH) is mediated by two Dot/Icm proteins, DotU and IcmF, which are able to localize to the poles ofL. pneumophilaby themselves. Interestingly, DotU and IcmF are homologs of the T6SS components TssL and TssM, which are part of the T6SS membrane complex (MC). We propose thatLegionellaco-opted these T6SS components to a novel function that mediates subcellular localization and assembly of this T4SS. Finally, in depth examination of the biogenesis pathway revealed that polar targeting and assembly of theLegionellaT4BSS apparatus is mediated by an innovative “outside-inside” mechanism.

Download Full-text

Structural studies of Legionella effectors

Acta Crystallographica Section A Foundations and Advances ◽

10.1107/s2053273314094169 ◽

2014 ◽

Vol 70 (a1) ◽

pp. C583-C583

Author(s):

Kathy Wong ◽

Yinglu Zhang ◽

Guennadi Kozlov ◽

Kalle Gehring

Keyword(s):

Legionella Pneumophila ◽

Effector Proteins ◽

Type Iv Secretion ◽

Homology Search ◽

Structural Homology ◽

Nucleotide Synthesis ◽

Type Iv ◽

Conserved Domain ◽

Middle Domain ◽

Legionnaires Disease

Legionella pneumophila is a gram-negative bacterium that causes Legionnaires' disease. It uses a Dot/Icm type IV secretion system to inject effector proteins into the host cell to manipulate host processes. Currently, about 300 Icm/Dot dependent effectors of L.pneumophila have been identified. Lpg1496 is an effector protein, which contains a conserved domain from the SidE family. To date, the middle domain and the conserved SidE domain have been crystallized and the structure solved at a resolution of 1.15Å and 2.3Å, respectively. A structural homology search using the middle domain suggested a similarity to phosphoribosylaminoimidazolesuccinocarboxamide (SAICAR) synthase, an ATPase involved in purine nucleotide synthesis. We performed 1H–15N HSQC NMR titrations to show that this domain binds ATP, ADP and AMP, with the highest binding affinity for ADP. A structural homology search using the SidE domain showed a similarity to cyclic nucleotide phosphodiesterases. To further elucidate the function of lpg1496, other fragments have been cloned, expressed, and subjected to crystallization trials. Currently, we have successfully crystallized the N-terminal domain, with crystals diffracting to <2.0Å. Obtaining the crystal structure of lpg1496 and revealing its function will not only lead to a better understanding of the virulence of L. pneumophila, but also contribute to the development of novel therapeutic treatments of Legionnaires' disease.

Download Full-text

Cytosolic recognition of flagellin by mouse macrophages restricts Legionella pneumophila infection

Journal of Experimental Medicine ◽

10.1084/jem.20051659 ◽

2006 ◽

Vol 203 (4) ◽

pp. 1093-1104 ◽

Cited By ~ 347

Author(s):

Ari B. Molofsky ◽

Brenda G. Byrne ◽

Natalie N. Whitfield ◽

Cressida A. Madigan ◽

Etsu T. Fuse ◽

...

Keyword(s):

Cell Death ◽

Legionella Pneumophila ◽

Type Iv Secretion ◽

Listeriolysin O ◽

Nuclear Condensation ◽

Macrophage Response ◽

Caspase 1 ◽

Type Iv ◽

Mouse Macrophages ◽

Bacterial Replication

To restrict infection by Legionella pneumophila, mouse macrophages require Naip5, a member of the nucleotide-binding oligomerization domain leucine-rich repeat family of pattern recognition receptors, which detect cytoplasmic microbial products. We report that mouse macrophages restricted L. pneumophila replication and initiated a proinflammatory program of cell death when flagellin contaminated their cytosol. Nuclear condensation, membrane permeability, and interleukin-1β secretion were triggered by type IV secretion-competent bacteria that encode flagellin. The macrophage response to L. pneumophila was independent of Toll-like receptor signaling but correlated with Naip5 function and required caspase 1 activity. The L. pneumophila type IV secretion system provided only pore-forming activity because listeriolysin O of Listeria monocytogenes could substitute for its contribution. Flagellin monomers appeared to trigger the macrophage response from perforated phagosomes: once heated to disassemble filaments, flagellin triggered cell death but native flagellar preparations did not. Flagellin made L. pneumophila vulnerable to innate immune mechanisms because Naip5+ macrophages restricted the growth of virulent microbes, but flagellin mutants replicated freely. Likewise, after intratracheal inoculation of Naip5+ mice, the yield of L. pneumophila in the lungs declined, whereas the burden of flagellin mutants increased. Accordingly, macrophages respond to cytosolic flagellin by a mechanism that requires Naip5 and caspase 1 to restrict bacterial replication and release proinflammatory cytokines that control L. pneumophila infection.

Download Full-text

The Dot/Icm-translocated effector LegC4 potentiates cytokine-mediated restriction of Legionella within accidental hosts.

10.1101/493197 ◽

2018 ◽

Author(s):

Tshegofatso Ngwaga ◽

Alex J Hydock ◽

Sandhya Ganesan ◽

Stephanie Rochelle Shames

Keyword(s):

Legionella Pneumophila ◽

Defense Mechanisms ◽

Effector Proteins ◽

Type Iv Secretion ◽

Innate Immune ◽

Host Cells ◽

Type Iv ◽

Legionnaires Disease ◽

Ifn Γ ◽

Necrosis Factor

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 (IFN)-γ. Specifically, TNF-mediated signaling was required for LegC4-mediated attenuation of L. pneumophila replication within macrophages. In addition, expression of legC4 was sufficient to restrict L. 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.

Download Full-text

Type IV secretion machinery: molecular architecture and function

Biochemical Society Transactions ◽

10.1042/bst20120332 ◽

2013 ◽

Vol 41 (1) ◽

pp. 17-28 ◽

Cited By ~ 16

Author(s):

Vidya Chandran

Keyword(s):

Virulence Factors ◽

Legionella Pneumophila ◽

Antibiotic Resistance Gene ◽

Type Iv Secretion ◽

Molecular Architecture ◽

Paradigm Shifts ◽

Secretion Systems ◽

Membrane Pore ◽

Type Iv ◽

Monomeric Proteins

Bacteria have evolved several secretion machineries to bring about transport of various virulence factors, nutrients, nucleic acids and cell-surface appendages that are essential for their pathogenesis. T4S (Type IV secretion) systems are versatile secretion systems found in various Gram-negative and Gram-positive bacteria and in few archaea. They are large multisubunit translocons secreting a diverse array of substrates varying in size and nature from monomeric proteins to nucleoprotein complexes. T4S systems have evolved from conjugation machineries and are implicated in antibiotic resistance gene transfer and transport of virulence factors in Legionella pneumophila causing Legionnaires’ disease, Brucella suis causing brucellosis and Helicobacter pylori causing gastroduodenal diseases. The best-studied are the Agrobacterium tumefaciens VirB/D4 and the Escherichia coli plasmid pKM101 T4S systems. Recent structural advances revealing the cryo-EM (electron microscopy) structure of the core translocation assembly and high-resolution structure of the outer-membrane pore of T4S systems have made paradigm shifts in the understanding of T4S systems. The present paper reviews the advances made in biochemical and structural studies and summarizes our current understanding of the molecular architecture of this mega-assembly.

Download Full-text

Isolation of the Dot/Icm Type IV Secretion System Core Complex from Legionella pneumophila

Methods in Molecular Biology - Legionella ◽

10.1007/978-1-4939-9048-1_15 ◽

2019 ◽

pp. 241-247 ◽

Cited By ~ 2

Author(s):

Tomoko Kubori ◽

Hiroki Nagai

Keyword(s):

Legionella Pneumophila ◽

Secretion System ◽

Type Iv Secretion ◽

Type Iv Secretion System ◽

Core Complex ◽

Type Iv

Download Full-text

Coxiella burnetii express type IV secretion system proteins that function similarly to components of the Legionella pneumophila Dot/Icm system

Molecular Microbiology ◽

10.1046/j.1365-2958.2003.03626.x ◽

2003 ◽

Vol 49 (4) ◽

pp. 965-976 ◽

Cited By ~ 105

Author(s):

Dario S. Zamboni ◽

Susan McGrath ◽

Michel Rabinovitch ◽

Craig R. Roy

Keyword(s):

Coxiella Burnetii ◽

Legionella Pneumophila ◽

Secretion System ◽

Type Iv Secretion ◽

Type Iv Secretion System ◽

Type Iv

Download Full-text

IcmF and DotU Are Required for Optimal Effector Translocation and Trafficking of the Legionella pneumophila Vacuole

Infection and Immunity ◽

10.1128/iai.72.10.5972-5982.2004 ◽

2004 ◽

Vol 72 (10) ◽

pp. 5972-5982 ◽

Cited By ~ 39

Author(s):

Susan M. VanRheenen ◽

Guillaume Duménil ◽

Ralph R. Isberg

Keyword(s):

Legionella Pneumophila ◽

Severe Form ◽

Secretion System ◽

Type Iv Secretion ◽

Type Iv Secretion System ◽

Intracellular Growth ◽

Macrophage Infection ◽

Type Iv ◽

Bacterial Mutants ◽

Bacterial Replication

ABSTRACT The gram-negative bacterium Legionella pneumophila causes a severe form of pneumonia called Legionnaires' disease, characterized by bacterial replication within alveolar macrophages. Prior to intracellular replication, the vacuole harboring the bacterium must first escape trafficking to the host lysosome, a process that is dependent on the Dot/Icm type IV secretion system. To identify genes required for intracellular growth, bacterial mutants were isolated that were delayed in escape from the macrophage but which retain a minimally functional Dot/Icm machinery. The mutations were found in eight distinct genes, including three genes known to be required for optimal intracellular growth. Two of these genes, icmF and dotU, are located at one end of a cluster of genes that encode the type IV secretion system, yet both icmF and dotU lack orthologs in other type IV translocons. DotU protein is degraded in the early postexponential phase in wild-type L. pneumophila and at all growth phases in an icmF mutant. IcmF contains an extracytoplasmic domain(s) based on accessibility to a membrane-impermeant amine-reactive reagent. In the absence of either gene, L. pneumophila targets inappropriately to LAMP-1-positive compartments during macrophage infection, is defective in the formation of replicative vacuoles, and is impaired in the translocation of the effector protein SidC. Therefore, although IcmF and DotU do not appear to be part of the core type IV secretion system, these proteins are necessary for an efficiently functioning secretion apparatus.

Download Full-text