scholarly journals Evolutionary Dissection of the Dot/Icm System Based on Comparative Genomics of 58 Legionella Species

2019 ◽  
Vol 11 (9) ◽  
pp. 2619-2632 ◽  
Author(s):  
Laura Gomez-Valero ◽  
Alvaro Chiner-Oms ◽  
Iñaki Comas ◽  
Carmen Buchrieser
Keyword(s):  
Positive Selection ◽  
Host Cell ◽  
Legionella Pneumophila ◽  
Secretion System ◽  
Effector Proteins ◽  
Structural Constraints ◽  
Recombination Rates ◽  
Host Interaction ◽  
Legionella Species ◽  
Type Ivb Secretion

Abstract The Dot/Icm type IVB secretion system of Legionella pneumophila is essential for its pathogenesis by delivering >300 effector proteins into the host cell. However, their precise secretion mechanism and which components interact with the host cell is only partly understood. Here, we undertook evolutionary analyses of the Dot/Icm system of 58 Legionella species to identify those components that interact with the host and/or the substrates. We show that high recombination rates are acting on DotA, DotG, and IcmX, supporting exposure of these proteins to the host. Specific amino acids under positive selection on the periplasmic region of DotF, and the cytoplasmic domain of DotM, support a role of these regions in substrate binding. Diversifying selection acting on the signal peptide of DotC suggests its interaction with the host after cleavage. Positive selection acts on IcmR, IcmQ, and DotL revealing that these components are probably participating in effector recognition and/or translocation. Furthermore, our results predict the participation in host/effector interaction of DotV and IcmF. In contrast, DotB, DotO, most of the core subcomplex elements, and the chaperones IcmS-W show a high degree of conservation and not signs of recombination or positive selection suggesting that these proteins are under strong structural constraints and have an important role in maintaining the architecture/function of the system. Thus, our analyses of recombination and positive selection acting on the Dot/Icm secretion system predicted specific Dot/Icm components and regions implicated in host interaction and/or substrate recognition and translocation, which will guide further functional analyses.

Creating a customized intracellular niche: subversion of host cell signaling byLegionellatype IV secretion system effectors

2015 ◽  
Vol 61 (9) ◽  
pp. 617-635 ◽  
Author(s):  
Ernest C. So ◽  
Corinna Mattheis ◽  
Edward W. Tate ◽  
Gad Frankel ◽  
Gunnar N. Schroeder
Keyword(s):  
Host Cell ◽  
Legionella Pneumophila ◽  
Current Knowledge ◽  
Secretion System ◽  
Effector Proteins ◽  
Cellular Processes ◽  
Type Iv ◽  
Open Questions ◽  
Exact Function ◽  
Wide Range

The Gram-negative facultative intracellular pathogen Legionella pneumophila infects a wide range of different protozoa in the environment and also human alveolar macrophages upon inhalation of contaminated aerosols. Inside its hosts, it creates a defined and unique compartment, termed the Legionella-containing vacuole (LCV), for survival and replication. To establish the LCV, L. pneumophila uses its Dot/Icm type IV secretion system (T4SS) to translocate more than 300 effector proteins into the host cell. Although it has become apparent in the past years that these effectors subvert a multitude of cellular processes and allow Legionella to take control of host cell vesicle trafficking, transcription, and translation, the exact function of the vast majority of effectors still remains unknown. This is partly due to high functional redundancy among the effectors, which renders conventional genetic approaches to elucidate their role ineffective. Here, we review the current knowledge about Legionella T4SS effectors, highlight open questions, and discuss new methods that promise to facilitate the characterization of T4SS effector functions in the future.

scholarly journals Structural insights into the roles of the IcmS–IcmW complex in the type IVb secretion system of Legionella pneumophila

2017 ◽  
Vol 114 (51) ◽  
pp. 13543-13548 ◽  
Author(s):  
Jianpo Xu ◽  
Dandan Xu ◽  
Muyang Wan ◽  
Li Yin ◽  
Xiaofei Wang ◽  
...  
Keyword(s):  
Legionella Pneumophila ◽  
Hydrophobic Surface ◽  
Adaptor Proteins ◽  
Secretion System ◽  
Effector Proteins ◽  
Host Cells ◽  
Binary Complex ◽  
Type Iv ◽  
Type Ivb Secretion ◽  
Type Ivb Secretion System

The type IVb secretion system (T4BSS) of Legionella pneumophila is a multiple-component apparatus that delivers ∼300 virulent effector proteins into host cells. The injected effectors modulate host cellular processes to promote bacterial infection and proliferation. IcmS and IcmW are two conserved small, acidic adaptor proteins that form a binary complex to interact with many effectors and facilitate their translocation. IcmS and IcmW can also interact with DotL, an ATPase of the type IV coupling protein complex (T4CP). However, how IcmS–IcmW recognizes effectors, and what the roles of IcmS–IcmW are in T4BSSs are unclear. In this study, we found that IcmS and IcmW form a 1:1 heterodimeric complex to bind effector substrates. Both IcmS and IcmW adopt new structural folds and have no structural similarities with known effector chaperones. IcmS has a compact global structure with an α/β fold, while IcmW adopts a fully α-folded, relatively loose architecture. IcmS stabilizes IcmW by binding to its two C-terminal α-helices. Photocrosslinking assays revealed that the IcmS–IcmW complex binds its cognate effectors via an extended hydrophobic surface, which can also interact with the C terminus of DotL. A crystal structure of the DotL–IcmS–IcmW complex reveals extensive and highly stable interactions between DotL and IcmS–IcmW. Moreover, IcmS–IcmW recruits LvgA to DotL and assembles a unique T4CP. These data suggest that IcmS–IcmW also functions as an inseparable integral component of the DotL–T4CP complex in the bacterial inner membrane. This study provides molecular insights into the dual roles of the IcmS–IcmW complex in T4BSSs.

scholarly journals Molecular architecture of theLegionellaDot/Icm type IV secretion system

2018 ◽  
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.

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

mBio ◽  
2020 ◽  
Vol 11 (2) ◽  
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.

scholarly journals Pore Formation Triggered by Legionella spp. Is an Nlrc4 Inflammasome-Dependent Host Cell Response That Precedes Pyroptosis

2010 ◽  
Vol 78 (3) ◽  
pp. 1403-1413 ◽  
Author(s):  
Tatiana N. Silveira ◽  
Dario S. Zamboni
Keyword(s):  
Host Cell ◽  
Legionella Pneumophila ◽  
Cell Response ◽  
Pore Formation ◽  
De Novo ◽  
Membrane Damage ◽  
Secretion System ◽  
Cell Permeabilization ◽  
Host Cell Response ◽  
Caspase 1

ABSTRACT Legionella pneumophila, the etiological agent of Legionnaires disease, is known to trigger pore formation in bone marrow-derived macrophages (BMMs) by mechanisms dependent on the type IVB secretion system known as Dot/Icm. Here, we used several mutants of L. pneumophila in combination with knockout mice to assess the host and bacterial factors involved in pore formation in BMMs. We found that regardless of Dot/Icm activity, pore formation does not occur in BMMs deficient in caspase-1 and Nlrc4/Ipaf. Pore formation was temporally associated with interleukin-1β secretion and preceded host cell lysis and pyroptosis. Pore-forming ability was dependent on bacterial Dot/Icm but independent of several effector proteins, multiplication, and de novo protein synthesis. Flagellin, which is known to trigger the Nlrc4 inflammasome, was required for pore formation as flaA mutant bacteria failed to induce cell permeabilization. Accordingly, transfection of purified flagellin was sufficient to trigger pore formation independent of infection. By using 11 different Legionella species, we found robust pore formation in response to L. micdadei, L. bozemanii, L. gratiana, L. jordanis, and L. rubrilucens, and this trait correlated with flagellin expression by these species. Together, the results suggest that pore formation is neither L. pneumophila specific nor the result of membrane damage induced by Dot/Icm activity; instead, it is a highly coordinated host cell response dependent on host Nlrc4 and caspase-1 and on bacterial flagellin and type IV secretion system.

scholarly journals Live-Cell Imaging of Phosphoinositide Dynamics and Membrane Architecture during Legionella Infection

mBio ◽  
2014 ◽  
Vol 5 (1) ◽  
Author(s):  
Stephen Weber ◽  
Maria Wagner ◽  
Hubert Hilbi
Keyword(s):  
Host Cell ◽  
Legionella Pneumophila ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Effector Proteins ◽  
Host Cells ◽  
Wild Type ◽  
Host Cell Membrane ◽  
Content Type ◽  
Temporal And Spatial

ABSTRACTThe causative agent of Legionnaires’ disease,Legionella pneumophila, replicates in amoebae and macrophages in a distinct membrane-bound compartment, theLegionella-containing vacuole (LCV). LCV formation is governed by the bacterial Icm/Dot type IV secretion system that translocates ~300 different “effector” proteins into host cells. Some of the translocated effectors anchor to the LCV membrane via phosphoinositide (PI) lipids. Here, we use the soil amoebaDictyostelium discoideum, producing fluorescent PI probes, to analyze the LCV PI dynamics by live-cell imaging. Upon uptake of wild-type or Icm/Dot-deficientL. pneumophila, PtdIns(3,4,5)P3transiently accumulated for an average of 40 s on early phagosomes, which acquired PtdIns(3)Pwithin 1 min after uptake. Whereas phagosomes containing ΔicmTmutant bacteria remained decorated with PtdIns(3)P, more than 80% of wild-type LCVs gradually lost this PI within 2 h. The process was accompanied by a major rearrangement of PtdIns(3)P-positive membranes condensing to the cell center. PtdIns(4)Ptransiently localized to early phagosomes harboring wild-type or ΔicmT L. pneumophilaand was cleared within minutes after uptake. During the following 2 h, PtdIns(4)Psteadily accumulated only on wild-type LCVs, which maintained a discrete PtdIns(4)Pidentity spatially separated from calnexin-positive endoplasmic reticulum (ER) for at least 8 h. The separation of PtdIns(4)P-positive and ER membranes was even more pronounced for LCVs harboring ΔsidC-sdcAmutant bacteria defective for ER recruitment, without affecting initial bacterial replication in the pathogen vacuole. These findings elucidate the temporal and spatial dynamics of PI lipids implicated in LCV formation and provide insight into host cell membrane and effector protein interactions.IMPORTANCEThe environmental bacteriumLegionella pneumophilais the causative agent of Legionnaires’ pneumonia. The bacteria form in free-living amoebae and mammalian immune cells a replication-permissive compartment, theLegionella-containing vacuole (LCV). To subvert host cell processes, the bacteria secrete the amazing number of ~300 different proteins into host cells. Some of these proteins bind phosphoinositide (PI) lipids to decorate the LCV. PI lipids are crucial factors involved in host cell membrane dynamics and LCV formation. UsingDictyosteliumamoebae producing one or two distinct fluorescent probes, we elucidated the dynamic LCV PI pattern in high temporal and spatial resolution. Notably, the endocytic PI lipid PtdIns(3)Pwas slowly cleared from LCVs, thus incapacitating the host cell’s digestive machinery, while PtdIns(4)Pgradually accumulated on the LCV, enabling critical interactions with host organelles. The LCV PI pattern underlies the spatiotemporal configuration of bacterial effector proteins and therefore represents a crucial aspect of LCV formation.

scholarly journals The Legionella Kinase LegK2 Targets the ARP2/3 Complex To Inhibit Actin Nucleation on Phagosomes and Allow Bacterial Evasion of the Late Endocytic Pathway

mBio ◽  
2015 ◽  
Vol 6 (3) ◽  
Author(s):  
Céline Michard ◽  
Daniel Sperandio ◽  
Nathalie Baïlo ◽  
Javier Pizarro-Cerdá ◽  
Lawrence LeClaire ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Host Cell ◽  
Legionella Pneumophila ◽  
Actin Polymerization ◽  
Secretion System ◽  
Endocytic Pathway ◽  
Specific Chemical ◽  
Actin Remodeling ◽  
Content Type

ABSTRACTLegionella pneumophila, the etiological agent of legionellosis, replicates within phagocytic cells. Crucial to biogenesis of the replicative vacuole is the Dot/Icm type 4 secretion system, which translocates a large number of effectors into the host cell cytosol. Among them is LegK2, a protein kinase that plays a key role inLegionellainfection. Here, we identified the actin nucleator ARP2/3 complex as a target of LegK2. LegK2 phosphorylates the ARPC1B and ARP3 subunits of the ARP2/3 complex. LegK2-dependent ARP2/3 phosphorylation triggers global actin cytoskeleton remodeling in cells, and it impairs actin tail formation byListeria monocytogenes, a well-known ARP2/3-dependent process. During infection, LegK2 is addressed to theLegionella-containing vacuole surface and inhibits actin polymerization on the phagosome, as revealed by legK2 gene inactivation. Consequently, LegK2 prevents late endosome/lysosome association with the phagosome and finally contributes to remodeling of the bacterium-containing phagosome into a replicative niche. The inhibition of actin polymerization by LegK2 and its effect on endosome trafficking are ARP2/3 dependent since it can be phenocopied by a specific chemical inhibitor of the ARP2/3 complex. Thus, LegK2-ARP2/3 interplay highlights an original mechanism of bacterial virulence with an unexpected role in local actin remodeling that allows bacteria to control vesicle trafficking in order to escape host defenses.IMPORTANCEDeciphering the individual contribution of each Dot/Icm type 4 secretion system substrate to the intracellular life-style ofL. pneumophilaremains the principal challenge in understanding the molecular basis ofLegionellavirulence. Our finding that LegK2 is a Dot/Icm effector that inhibits actin polymerization on theLegionella-containing vacuole importantly contributes to the deciphering of the molecular mechanisms evolved byLegionellato counteract the endocytic pathway. Indeed, our results highlight the essential role of LegK2 in preventing late endosomes from fusing with the phagosome. More generally, this work is the first demonstration of local actin remodeling as a mechanism used by bacteria to control organelle trafficking. Further, by characterizing the role of the bacterial protein kinase LegK2, we reinforce the concept that posttranslational modifications are key strategies used by pathogens to evade host cell defenses.

scholarly journals Extra! Extracellular Effector Delivery into Host Cells via the Type 3 Secretion System

mBio ◽  
2017 ◽  
Vol 8 (3) ◽  
Author(s):  
Melissa M. Kendall
Keyword(s):  
Host Cell ◽  
Secretion System ◽  
Effector Proteins ◽  
Host Cells ◽  
Alternative Mechanism ◽  
Host Cell Membrane ◽  
Type Three Secretion System ◽  
Host Signaling ◽  
Type 3 Secretion System ◽  
Type 3

ABSTRACT The type three secretion system (T3SS) is critical for the virulence of diverse bacterial pathogens. Pathogens use the T3SS to deliver effector proteins into host cells and manipulate host signaling pathways. The prevailing mechanism is that effectors translocate from inside the T3SS directly into the host cell. Recent studies reveal an alternative mechanism of effector translocation, in which an effector protein located outside the bacterial cell relies on the T3SS for delivery into host cells. Tejeda-Dominguez et al. (F. Tejeda-Dominguez, J. Huerta-Cantillo, L. Chavez-Dueñas, and F. Navarro-Garcia, mBio 8:e00184-17, 2017, https://doi.org/10.1128/mBio.00184-17 !) demonstrate that the EspC effector of enteropathogenic Escherichia coli is translocated by binding to the outside of the T3SS and subsequently gains access to the host cell cytoplasm through the T3SS pore embedded within the host cell membrane. This work reveals a novel mechanism of translocation that is likely relevant for a variety of other pathogens that use the T3SS as part of their virulence arsenal.

scholarly journals Host Pathways Important for Coxiella burnetii Infection Revealed by Genome-Wide RNA Interference Screening

mBio ◽  
2013 ◽  
Vol 4 (1) ◽  
Author(s):  
Justin A. McDonough ◽  
Hayley J. Newton ◽  
Scott Klum ◽  
Rachel Swiss ◽  
Hervé Agaisse ◽  
...  
Keyword(s):  
Coxiella Burnetii ◽  
Secretion System ◽  
Effector Proteins ◽  
Host Factors ◽  
Content Type ◽  
Host Determinants ◽  
Genome Wide ◽  
Type Ivb Secretion ◽  
Type Ivb Secretion System ◽  
Bacterial Effector Proteins

ABSTRACTCoxiella burnetiiis an intracellular pathogen that replicates within a lysosome-like vacuole. A Dot/Icm type IVB secretion system is used byC. burnetiito translocate effector proteins into the host cytosol that likely modulate host factor function. To identify host determinants required forC. burnetiiintracellular growth, a genome-wide screen was performed using gene silencing by small interfering RNA (siRNA). Replication ofC. burnetiiwas measured by immunofluorescence microscopy in siRNA-transfected HeLa cells. Newly identified host factors included components of the retromer complex, which mediates cargo cycling between the endocytic pathway and the Golgi apparatus. Reducing the levels of the retromer cargo-adapter VPS26-VPS29-VPS35 complex or retromer-associated sorting nexins abrogatedC. burnetiireplication. Several genes, when silenced, resulted in enlarged vacuoles or an increased number of vacuoles withinC. burnetii-infected cells. Silencing of theSTX17gene encoding syntaxin-17 resulted in a striking defect in homotypic fusion of vacuoles containingC. burnetii, suggesting a role for syntaxin-17 in regulating this process. Lastly, silencing host genes needed forC. burnetiireplication correlated with defects in the translocation of Dot/Icm effectors, whereas, silencing of genes that affected vacuole morphology, but did not impact replication, did not affect Dot/Icm translocation. These data demonstrate thatC. burnetiivacuole maturation is important for creating a niche that permits Dot/Icm function. Thus, genome-wide screening has revealed host determinants involved in sequential events that occur duringC. burnetiiinfection as defined by bacterial uptake, vacuole transport and acidification, activation of the Dot/Icm system, homotypic fusion of vacuoles, and intracellular replication.IMPORTANCEQ fever in humans is caused by the bacteriumCoxiella burnetii. Infection withC. burnetiiis marked by its unique ability to replicate within a large vacuolar compartment inside cells that resembles the harsh, acidic environment of a lysosome. Central to its pathogenesis is the delivery of bacterial effector proteins into the host cell cytosol by a Dot/Icm type IVB secretion system. These proteins can interact with and manipulate host factors, thereby leading to creation and maintenance of the vacuole that the bacteria grow within. Using high-throughput genome-wide screening in human cells, we identified host factors important for several facets ofC. burnetiiinfection, including vacuole transport and membrane fusion events that promote vacuole expansion. In addition, we show that maturation of theC. burnetiivacuole is necessary for creating an environment permissive for the Dot/Icm delivery of bacterial effector proteins into the host cytosol.

scholarly journals Identification of a Specific Chaperone for SptP, a Substrate of the Centisome 63 Type III Secretion System ofSalmonella typhimurium

1998 ◽  
Vol 180 (13) ◽  
pp. 3393-3399 ◽  
Author(s):  
Yixin Fu ◽  
Jorge E. Galán
Keyword(s):  
Actin Cytoskeleton ◽  
Host Cell ◽  
Type Iii Secretion ◽  
Tyrosine Phosphatase ◽  
Secretion System ◽  
Effector Proteins ◽  
Loss Of Function ◽  
Secretion Systems ◽  
Type Iii ◽  
Type Iii Protein Secretion

ABSTRACT Salmonella typhimurium uses of a type III protein secretion system encoded at centisome 63 of its chromosome to deliver effector molecules into the host cell. These proteins stimulate host cell responses such as reorganization of the actin cytoskeleton and activation of transcription factors. One of these effector proteins is SptP, a tyrosine phosphatase that causes disruption of the host cell actin cytoskeleton. A characteristic feature of many substrates of type III secretion systems is their association with specific cytoplasmic chaperones which appears to be required for secretion and/or translocation of these proteins into the host cell. We report here the identification of SicP, a 13-kDa acidic polypeptide that is encoded immediately upstream of sptP. A loss-of-function mutation in sicP resulted in drastically reduced levels of SptP but did not affect sptP expression, indicating that SicP exerts its effect posttranscriptionally. Pulse-chase experiments demonstrated that the loss of SicP leads to increased degradation of SptP. In addition, we show that SicP binds to SptP directly and that the binding site is located between residues 15 and 100 of the tyrosine phosphatase. Taken together, these results indicate that SicP acts as a specific chaperone for SptP.

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