Structural studies of Legionella effectors

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.

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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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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.

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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.

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Alveolar Macrophages and Neutrophils Are the Primary Reservoirs for Legionella pneumophila and Mediate Cytosolic Surveillance of Type IV Secretion

Infection and Immunity ◽

10.1128/iai.01891-14 ◽

2014 ◽

Vol 82 (10) ◽

pp. 4325-4336 ◽

Cited By ~ 31

Author(s):

Alan M. Copenhaver ◽

Cierra N. Casson ◽

Hieu T. Nguyen ◽

Thomas C. Fung ◽

Matthew M. Duda ◽

...

Keyword(s):

Immune Response ◽

Alveolar Macrophages ◽

Legionella Pneumophila ◽

Pulmonary Infection ◽

Severe Pneumonia ◽

Effector Proteins ◽

Type Iv Secretion ◽

Host Cells ◽

Content Type ◽

Type Iv

ABSTRACTLegionella pneumophila, an intracellular pathogen responsible for the severe pneumonia Legionnaires' disease, uses itsdot/icm-encoded type IV secretion system (T4SS) to translocate effector proteins that promote its survival and replication into the host cell cytosol. However, by introducing bacterial products into the host cytosol,L. pneumophilaalso activates cytosolic immunosurveillance pathways, thereby triggering robust proinflammatory responses that mediate the control of infection. Thus, the pulmonary cell types thatL. pneumophilainfects not only may act as an intracellular niche that facilitates its pathogenesis but also may contribute to the immune response againstL. pneumophila. The identity of these host cells remains poorly understood. Here, we developed a strain ofL. pneumophilaproducing a fusion protein consisting of β-lactamase fused to the T4SS-translocated effector RalF, which allowed us to track cells injected by the T4SS. Our data reveal that alveolar macrophages and neutrophils both are the primary recipients of T4SS-translocated effectors and harbor viableL. pneumophiladuring pulmonary infection of mice. Moreover, both alveolar macrophages and neutrophils from infected mice produced tumor necrosis factor and interleukin-1α in response to T4SS-sufficient, but not T4SS-deficient,L. pneumophila. Collectively, our data suggest that alveolar macrophages and neutrophils are both an intracellular reservoir forL. pneumophilaand a source of proinflammatory cytokines that contribute to the host immune response againstL. pneumophiladuring pulmonary infection.

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Cryo-EM reveals new species-specific proteins and symmetry elements in the Legionella pneumophila Dot/Icm T4SS

eLife ◽

10.7554/elife.70427 ◽

2021 ◽

Vol 10 ◽

Author(s):

Michael J Sheedlo ◽

Clarissa L Durie ◽

Jeong Min Chung ◽

Louise Chang ◽

Jacquelyn Roberts ◽

...

Keyword(s):

Legionella Pneumophila ◽

Genetic Locus ◽

Opportunistic Pathogen ◽

Effector Proteins ◽

Type Iv ◽

Specific Proteins ◽

Legionnaires Disease ◽

Multiple Conformations ◽

Membrane Spanning ◽

Species Specific

Legionella pneumophila is an opportunistic pathogen that causes the potentially fatal pneumonia known as Legionnaires' Disease. The pathology associated with infection depends on bacterial delivery of effector proteins into the host via the membrane spanning Dot/Icm type IV secretion system (T4SS). We have determined sub-3.0 Å resolution maps of the Dot/Icm T4SS core complex by single particle cryo-EM. The high-resolution structural analysis has allowed us to identify proteins encoded outside the Dot/Icm genetic locus that contribute to the core T4SS structure. We can also now define two distinct areas of symmetry mismatch, one that connects the C18 periplasmic ring (PR) and the C13 outer membrane cap (OMC) and one that connects the C13 OMC with a 16-fold symmetric dome. Unexpectedly the connection between the PR and OMC is DotH, with five copies sandwiched between the OMC and PR to accommodate the symmetry mismatch. Finally, we observe multiple conformations in the reconstructions that indicate flexibility within the structure.

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Legionella pneumophila targets autophagosomes and promotes host autophagy during late infection

10.1101/2021.07.08.451723 ◽

2021 ◽

Author(s):

Rebecca R. Noll ◽

Colleen M. Pike ◽

Stephanie S. Lehman ◽

Chad Williamson ◽

Ramona Neunuebel

Keyword(s):

Legionella Pneumophila ◽

Bacterial Pathogen ◽

Nutrient Release ◽

Effector Proteins ◽

Type Iv Secretion ◽

Host Cells ◽

Effector Protein ◽

Wild Type ◽

Cellular Processes ◽

Type Iv

Autophagy is a fundamental eukaryotic process that mediates clearance of unwanted molecules and facilitates nutrient release. The bacterial pathogen Legionella pneumophila establishes an intracellular niche within phagocytes by manipulating host cellular processes, such as autophagy. Effector proteins translocated by L. pneumophila's Dot/Icm type IV secretion system have been shown to suppress autophagy. However evidence suggests that overall inhibition of autophagy may be detrimental to the bacterium. As autophagy contributes to cellular homeostasis and nutrient acquisition, L. pneumophila may translocate effectors that promote autophagy for these benefits. Here, we show that effector protein Lpg2411 binds phosphatidylinositol-3-phosphate lipids and preferentially binds autophagosomes. Translocated Lpg2411 accumulates late during infection and co-localizes with the autophagy receptor p62 and ubiquitin. Furthermore, autophagy is inhibited to a greater extent in host cells infected with a mutant strain lacking Lpg2411 compared to those infected with wild-type L. pneumophila, indicating that Lpg2411 stimulates autophagy to support the bacterium's intracellular lifestyle.

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

Journal of Bacteriology ◽

10.1128/jb.00755-18 ◽

2019 ◽

Vol 201 (14) ◽

Cited By ~ 3

Author(s):

Tshegofatso Ngwaga ◽

Alex J. Hydock ◽

Sandhya Ganesan ◽

Stephanie R. Shames

Keyword(s):

Legionella Pneumophila ◽

Defense Mechanisms ◽

Effector Proteins ◽

Host Cells ◽

Content Type ◽

Host Restriction ◽

Type Iv ◽

Legionnaires Disease ◽

Ifn Γ ◽

Necrosis Factor

ABSTRACTLegionella pneumophilais ubiquitous in freshwater environments, where it replicates within unicellular protozoa. However,L. pneumophilais 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. pneumophilautilizes 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 ofL. pneumophila. We found previously that the effector LegC4 is important forL. pneumophilareplication 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 attenuatesL. pneumophilareplication. We found that LegC4 enhanced restriction ofL. pneumophilareplication within macrophages activated with tumor necrosis factor (TNF) or interferon gamma (IFN-γ). In addition, expression oflegC4was sufficient to restrictLegionella longbeachaereplication within TNF- or IFN-γ-activated macrophages. Thus, this study demonstrates that LegC4 contributes toL. pneumophilaclearance from healthy hosts by potentiating cytokine-mediated host defense mechanisms.IMPORTANCELegionellaspp. are natural pathogens of protozoa and accidental pathogens of humans. Innate immunity in healthy individuals effectively controlsLegionellainfection 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 ofLegionellaby 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.

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Legionella pneumophila Strain 130b Possesses a Unique Combination of Type IV Secretion Systems and Novel Dot/Icm Secretion System Effector Proteins

Journal of Bacteriology ◽

10.1128/jb.00778-10 ◽

2010 ◽

Vol 192 (22) ◽

pp. 6001-6016 ◽

Cited By ~ 81

Author(s):

Gunnar N. Schroeder ◽

Nicola K. Petty ◽

Aurélie Mousnier ◽

Clare R. Harding ◽

Adam J. Vogrin ◽

...

Keyword(s):

Legionella Pneumophila ◽

Draft Genome ◽

Severe Pneumonia ◽

Gene Clusters ◽

Secretion System ◽

Effector Proteins ◽

Type Iv Secretion ◽

Secretion Systems ◽

Type Iv ◽

Core Set

ABSTRACT Legionella pneumophila is a ubiquitous inhabitant of environmental water reservoirs. The bacteria infect a wide variety of protozoa and, after accidental inhalation, human alveolar macrophages, which can lead to severe pneumonia. The capability to thrive in phagocytic hosts is dependent on the Dot/Icm type IV secretion system (T4SS), which translocates multiple effector proteins into the host cell. In this study, we determined the draft genome sequence of L. pneumophila strain 130b (Wadsworth). We found that the 130b genome encodes a unique set of T4SSs, namely, the Dot/Icm T4SS, a Trb-1-like T4SS, and two Lvh T4SS gene clusters. Sequence analysis substantiated that a core set of 107 Dot/Icm T4SS effectors was conserved among the sequenced L. pneumophila strains Philadelphia-1, Lens, Paris, Corby, Alcoy, and 130b. We also identified new effector candidates and validated the translocation of 10 novel Dot/Icm T4SS effectors that are not present in L. pneumophila strain Philadelphia-1. We examined the prevalence of the new effector genes among 87 environmental and clinical L. pneumophila isolates. Five of the new effectors were identified in 34 to 62% of the isolates, while less than 15% of the strains tested positive for the other five genes. Collectively, our data show that the core set of conserved Dot/Icm T4SS effector proteins is supplemented by a variable repertoire of accessory effectors that may partly account for differences in the virulences and prevalences of particular L. pneumophila strains.

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

Infection and Immunity ◽

10.1128/iai.01382-05 ◽

2006 ◽

Vol 74 (6) ◽

pp. 3285-3295 ◽

Cited By ~ 68

Author(s):

Esteban Fernandez-Moreira ◽

Juergen H. Helbig ◽

Michele S. Swanson

Keyword(s):

Legionella Pneumophila ◽

Surface Composition ◽

Membrane Vesicles ◽

Type Iv Secretion ◽

Phagosome Maturation ◽

Developmentally Regulated ◽

E Coli ◽

Type Iv ◽

Mouse Macrophages ◽

Legionnaires Disease

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.

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

eLife ◽

10.7554/elife.59530 ◽

2020 ◽

Vol 9 ◽

Cited By ~ 2

Author(s):

Clarissa L Durie ◽

Michael J Sheedlo ◽

Jeong Min Chung ◽

Brenda G Byrne ◽

Min Su ◽

...

Keyword(s):

Legionella Pneumophila ◽

Structural Features ◽

Secretion System ◽

Effector Proteins ◽

Type Iv Secretion ◽

Host Cells ◽

Type Iv Secretion System ◽

Core Complex ◽

The Core ◽

Type Iv

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.

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