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

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.

scholarly journals Legionella pneumophila targets autophagosomes and promotes host autophagy during late infection

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.

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

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.

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

2018 ◽  
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.

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

2014 ◽  
Vol 82 (10) ◽  
pp. 4325-4336 ◽  
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.

scholarly journals Structural studies of Legionella effectors

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.

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

2019 ◽  
Vol 201 (14) ◽  
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.

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

eLife ◽  
2020 ◽  
Vol 9 ◽  
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.

scholarly journals Targeting of the Small GTPase Rab6A′ by the Legionella pneumophila Effector LidA

2013 ◽  
Vol 81 (6) ◽  
pp. 2226-2235 ◽  
Author(s):  
Yang Chen ◽  
Matthias P. Machner
Keyword(s):  
Legionella Pneumophila ◽  
Small Gtpase ◽  
Effector Proteins ◽  
Host Cells ◽  
Intracellular Replication ◽  
Content Type ◽  
Type Iv ◽  
Gtpase Activating Proteins ◽  
Active Pool ◽  
Cell Cytosol

ABSTRACTWhen the bacteriumLegionella pneumophila, the causative agent of Legionnaires' disease, is phagocytosed by alveolar macrophages, it delivers a large number of effector proteins through its Dot/Icm type IV secretion system into the host cell cytosol. Among those proteins is LidA, an effector that interacts with several host GTPases of the Rab family, including Rab6A′, a regulator of retrograde vesicle trafficking within eukaryotic cells. The effect of LidA on Rab6A′ function and the role of Rab6A′ forL. pneumophilagrowth within host cells has been unclear. Here, we show that LidA preferentially binds Rab6A′ in the active GTP-bound conformation. Rab6 binding occurred through the central region of LidA and followed a stoichiometry for LidA and Rab6A′ of 1:2. LidA maintained Rab6A′ in the active conformation by efficiently blocking the hydrolysis of GTP by Rab6A′, even in the presence of cellular GTPase-activating proteins, suggesting that the function of Rab6A′ must be important for efficient intracellular replication ofL. pneumophila. Accordingly, we found that production of constitutively inactive Rab6A′(T27N) but not constitutively active Rab6A′(Q72L) significantly reduced the ability ofL. pneumophilato initiate intracellular replication in human macrophages. Thus, the presence of an active pool of Rab6 within host cells early during infection is required to support efficient intracellular growth ofL. pneumophila.

scholarly journals Structural analysis of the Legionella pneumophila Dot/Icm Type IV Secretion System Core Complex

2020 ◽  
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

AbstractLegionella 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.

scholarly journals Peptidomimetic Small Molecules Disrupt Type IV Secretion System Activity in Diverse Bacterial Pathogens

mBio ◽  
2016 ◽  
Vol 7 (2) ◽  
Author(s):  
Carrie L. Shaffer ◽  
James A. D. Good ◽  
Santosh Kumar ◽  
K. Syam Krishnan ◽  
Jennifer A. Gaddy ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Small Molecules ◽  
Bacterial Pathogens ◽  
Effector Proteins ◽  
Type Iv Secretion ◽  
Target Cells ◽  
Effector Protein ◽  
Secretion Systems ◽  
Type Iv ◽  
H Pylori

ABSTRACT Bacteria utilize complex type IV secretion systems (T4SSs) to translocate diverse effector proteins or DNA into target cells. Despite the importance of T4SSs in bacterial pathogenesis, the mechanism by which these translocation machineries deliver cargo across the bacterial envelope remains poorly understood, and very few studies have investigated the use of synthetic molecules to disrupt T4SS-mediated transport. Here, we describe two synthetic small molecules (C10 and KSK85) that disrupt T4SS-dependent processes in multiple bacterial pathogens. Helicobacter pylori exploits a pilus appendage associated with the cag T4SS to inject an oncogenic effector protein (CagA) and peptidoglycan into gastric epithelial cells. In H. pylori , KSK85 impedes biogenesis of the pilus appendage associated with the cag T4SS, while C10 disrupts cag T4SS activity without perturbing pilus assembly. In addition to the effects in H. pylori , we demonstrate that these compounds disrupt interbacterial DNA transfer by conjugative T4SSs in Escherichia coli and impede vir T4SS-mediated DNA delivery by Agrobacterium tumefaciens in a plant model of infection. Of note, C10 effectively disarmed dissemination of a derepressed IncF plasmid into a recipient bacterial population, thus demonstrating the potential of these compounds in mitigating the spread of antibiotic resistance determinants driven by conjugation. To our knowledge, this study is the first report of synthetic small molecules that impair delivery of both effector protein and DNA cargos by diverse T4SSs. IMPORTANCE Many human and plant pathogens utilize complex nanomachines called type IV secretion systems (T4SSs) to transport proteins and DNA to target cells. In addition to delivery of harmful effector proteins into target cells, T4SSs can disseminate genetic determinants that confer antibiotic resistance among bacterial populations. In this study, we sought to identify compounds that disrupt T4SS-mediated processes. Using the human gastric pathogen H. pylori as a model system, we identified and characterized two small molecules that prevent transfer of an oncogenic effector protein to host cells. We discovered that these small molecules also prevented the spread of antibiotic resistance plasmids in E. coli populations and diminished the transfer of tumor-inducing DNA from the plant pathogen A. tumefaciens to target cells. Thus, these compounds are versatile molecular tools that can be used to study and disarm these important bacterial machines.

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