scholarly journals Orchestrated delivery of Legionella effectors by the Icm/Dot secretion system

2019 ◽  
Author(s):  
Julie Allombert ◽  
Corentin Jaboulay ◽  
Céline Michard ◽  
Claire Andréa ◽  
Xavier Charpentier ◽  
...  
Keyword(s):  
Host Cell ◽  
Legionella Pneumophila ◽  
Translational Control ◽  
Function Analysis ◽  
Secretion System ◽  
Secretion Systems ◽  
Type Iv ◽  
Set Up ◽  
Type 3 ◽  
Effector Secretion

AbstractLegionella pneumophila uses the Icm/Dot Type IV secretion system (T4SS) to translocate a record number (300) of bacterial effectors into the host cell. Despite recent breakthrough progress in determining the structure and the localization of the secretion machinery, it is still a challenge to understand how the delivery of so many effectors is organized to avoid bottleneck effect and to allow effective manipulation of the host cell by L. pneumophila. Here, we demonstrate that secretion of effectors is ordered and so precisely set up that it lines-up with the delivery timing required for the function of the effectors in the cell. We observe notably that the secretion order of 4 effectors targeting Rab1 is fully consistent with the sequence of their actions on Rab1. Importantly, we show that the timed delivery of an effector is not dependent on its concentration, nor on its picking-up by chaperone proteins. Conversely, this control involves c-di-GMP signaling, as a c-di-GMP synthesizing enzyme, namely the diguanylate cyclase Lpl0780/Lpp0809, significantly contributes to accurate triggering of effector secretion via a post-translational control of the T4SS machinery at the bacterial pole.SignificanceType 3, 4 and 6 secretion systems are multiprotein complex known to be crucial for infectious cycle of many bacterial pathogens. Despite considerable progress on several fronts in structure-function analysis of these systems, one of the blackest boxes in our understanding is the signal that triggers the activation of effectors transfer. This is particularly true for the Icm/Dot T4SS in L. pneumophila that deals with the translocation of a record number of 300 effectors. We demonstrate that Icm/Dot secretion is timely fine-tuned and most importantly, that the complex orchestration of so many effector actions relies at least in part on the defined timing of their translocation into the host cell. Also, we highlight for the first time a post-translational control of a T4SS by c-diGMP signaling.

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.

VirB8: a conserved type IV secretion system assembly factor and drug targetThis paper is one of a selection of papers published in this Special Issue, entitled CSBMCB — Membrane Proteins in Health and Disease.

2006 ◽  
Vol 84 (6) ◽  
pp. 890-899 ◽  
Author(s):  
Christian Baron
Keyword(s):  
Function Analysis ◽  
Model Organism ◽  
Secretion System ◽  
Type Iv Secretion ◽  
Type Iv Secretion System ◽  
Secretion Systems ◽  
Type Iv ◽  
Assembly Factor ◽  
Type Iv Secretion Systems ◽  
Multiple Interactions

Type IV secretion systems are used by many Gram-negative bacteria for the translocation of macromolecules (proteins, DNA, or DNA–protein complexes) across the cell envelope. Among them are many pathogens for which type IV secretion systems are essential virulence factors. Type IV secretion systems comprise 8–12 conserved proteins, which assemble into a complex spanning the inner and the outer membrane, and many assemble extracellular appendages, such as pili, which initiate contact with host and recipient cells followed by substrate translocation. VirB8 is an essential assembly factor for all type IV secretion systems. Biochemical, cell biological, genetic, and yeast two-hybrid analyses showed that VirB8 undergoes multiple interactions with other type IV secretion system components and that it directs polar assembly of the membrane-spanning complex in the model organism Agrobacterium tumefaciens. The availability of the VirB8 X-ray structure has enabled a detailed structure–function analysis, which identified sites for the binding of VirB4 and VirB10 and for self-interaction. Due to its multiple interactions, VirB8 is an excellent model for the analysis of assembly factors of multiprotein complexes. In addition, VirB8 is a possible target for drugs that target its protein–protein interactions, which would disarm bacteria by depriving them of their essential virulence functions.

scholarly journals Identification of a Gene That Affects the Efficiency of Host Cell Infection by Legionella pneumophila in a Temperature-Dependent Fashion

2003 ◽  
Vol 71 (11) ◽  
pp. 6256-6263 ◽  
Author(s):  
Dennis A. Ridenour ◽  
Suat L. G. Cirillo ◽  
Sheng Feng ◽  
Mustapha M. Samrakandi ◽  
Jeffrey D. Cirillo
Keyword(s):  
Host Cell ◽  
Legionella Pneumophila ◽  
Mammalian Cells ◽  
Growth Conditions ◽  
Host Cells ◽  
Secretion Systems ◽  
Cell Infection ◽  
Type Iv ◽  
Low Copy Number ◽  
Type Gene

ABSTRACT The ability to infect host cells is critical for the survival and replication of intracellular pathogens in humans. We previously found that many genes involved in the ability of Legionella pneumophila to infect macrophages are not expressed efficiently under standard laboratory growth conditions. We have developed an approach using expression of L. pneumophila genes from an exogenous constitutive promoter on a low-copy-number vector that allows identification of genes involved in host cell infection. Through the use of this strategy, we found that expression of a gene, lvhB2, enhances the efficiency of L. pneumophila infection of mammalian cells. The putative protein encoded by lvhB2 has similarity to structural pilin subunits of type IV secretion systems. We confirmed that this gene plays a role in host cell infection by the construction of an in-frame deletion in the L. pneumophila lvhB2 gene and complementation of this mutant with the wild-type gene. The lvhB2 mutant does not display a very obvious defect in interactions with host cells when the bacteria are grown at 37°C, but it has an approximately 100-fold effect on entry and intracellular replication when grown at 30°C. These data suggest that lvhB2 plays an important role in the efficiency of host cell infection by L. pneumophila grown at lower temperatures.

scholarly journals Sel1 Repeat Protein LpnE Is a Legionella pneumophila Virulence Determinant That Influences Vacuolar Trafficking

2007 ◽  
Vol 75 (12) ◽  
pp. 5575-5585 ◽  
Author(s):  
Hayley J. Newton ◽  
Fiona M. Sansom ◽  
Jenny Dao ◽  
Adrian D. McAlister ◽  
Joan Sloan ◽  
...  
Keyword(s):  
Host Cell ◽  
Legionella Pneumophila ◽  
Protein A ◽  
Acanthamoeba Castellanii ◽  
Secretion System ◽  
Cell Trafficking ◽  
Eukaryotic Protein ◽  
Repeat Protein ◽  
Type Iv ◽  
Tetratricopeptide Repeat Protein

ABSTRACT The environmental pathogen Legionella pneumophila possesses five proteins with Sel1 repeats (SLRs) from the tetratricopeptide repeat protein family. Three of these proteins, LpnE, EnhC, and LidL, have been implicated in the ability of L. pneumophila to efficiently establish infection and/or manipulate host cell trafficking events. Previously, we showed that LpnE is important for L. pneumophila entry into macrophages and epithelial cells. In further virulence studies here, we show that LpnE is also required for efficient infection of Acanthamoeba castellanii by L. pneumophila and for replication of L. pneumophila in the lungs of A/J mice. In addition, we found that the role of LpnE in host cell invasion is dependent on the eight SLR regions of the protein. A truncated form of LpnE lacking the two C-terminal SLR domains was unable to complement the invasion defect of an lpnE mutant of L. pneumophila 130b in both the A549 and THP-1 cell lines. The lpnE mutant displayed impaired avoidance of LAMP-1 association, suggesting that LpnE influenced trafficking of the L. pneumophila vacuole, similar to the case for EnhC and LidL. We also found that LpnE was present in L. pneumophila culture supernatants and that its export was independent of both the Lsp type II secretion system and the Dot/Icm type IV secretion system. The fact that LpnE was exported suggested that the protein may interact with a eukaryotic protein. Using LpnE as bait, we screened a HeLa cell cDNA library for interacting partners, using the yeast two-hybrid system. Examination of the protein-protein interaction between LpnE and a eukaryotic protein, obscurin-like protein 1, suggested that LpnE can interact with eukaryotic proteins containing immunoglobulin-like folds via the SLR regions. This investigation has further characterized the contribution of LpnE to L. pneumophila virulence and, more specifically, the importance of the SLR regions to LpnE function.

Structure of the Legionella Dot/Icm type IV secretion system in situ by electron cryotomography

2016 ◽  
Author(s):  
Debnath Ghosal ◽  
Yi-Wei Chang ◽  
Kwangcheol C. Jeong ◽  
Joseph P. Vogel ◽  
Grant J. Jensen
Keyword(s):  
Legionella Pneumophila ◽  
Secretion System ◽  
Type Iv Secretion ◽  
Secretion Systems ◽  
Type Iv ◽  
Type Ivb Secretion ◽  
Structural Preservation ◽  
Electron Cryotomography ◽  
Type Ivb Secretion System

AbstractType IV secretion systems (T4SSs) are large macromolecular machines that translocate protein and DNA and are involved in the pathogenesis of multiple human diseases. Here, using electron cryotomography (ECT), we report the in situ structure of the Dot/Icm type IVB secretion system (T4BSS) utilized by the human pathogen Legionella pneumophila. This is the first structure of a type IVB secretion system, and also the first structure of any T4SS in situ. While the Dot/Icm system shares almost no sequence homology with type IVA secretion systems (T4ASSs), its overall structure shows remarkable similarities to two previously imaged T4ASSs, suggesting shared aspects of mechanism. However, compared to one of these, the negative-stain reconstruction of the purified T4ASS from the R388 plasmid, it is approximately twice as long and wide and exhibits several additional large densities, reflecting type-specific elaborations and potentially better structural preservation in situ.

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

2010 ◽  
Vol 192 (22) ◽  
pp. 6001-6016 ◽  
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.

scholarly journals Protein Kinase LegK2 Is a Type IV Secretion System Effector Involved in Endoplasmic Reticulum Recruitment and Intracellular Replication of Legionella pneumophila

2011 ◽  
Vol 79 (5) ◽  
pp. 1936-1950 ◽  
Author(s):  
Eva Hervet ◽  
Xavier Charpentier ◽  
Anne Vianney ◽  
Jean-Claude Lazzaroni ◽  
Christophe Gilbert ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Kinase ◽  
Host Cell ◽  
Legionella Pneumophila ◽  
Secretion System ◽  
Type Iv Secretion ◽  
Type Iv Secretion System ◽  
Intracellular Replication ◽  
Content Type ◽  
Type Iv

ABSTRACTLegionella pneumophilais the etiological agent of Legionnaires' disease. Crucial to the pathogenesis of this intracellular pathogen is its ability to subvert host cell defenses, permitting intracellular replication in specialized vacuoles within host cells. The Dot/Icm type IV secretion system (T4SS), which translocates a large number of bacterial effectors into host cell, is absolutely required for rerouting theLegionellaphagosome. ManyLegionellaeffectors display distinctive eukaryotic domains, among which are protein kinase domains.In silicoanalysis andin vitrophosphorylation assays identified five functional protein kinases, LegK1 to LegK5, encoded by the epidemicL. pneumophilaLens strain. Except for LegK5, theLegionellaprotein kinases are all T4SS effectors. LegK2 plays a key role in bacterial virulence, as demonstrated by gene inactivation. ThelegK2mutant containing vacuoles displays less-efficient recruitment of endoplasmic reticulum markers, which results in delayed intracellular replication. Considering that a kinase-dead substitution mutant oflegK2exhibits the same virulence defects, we highlight here a new molecular mechanism, namely, protein phosphorylation, developed byL. pneumophilato establish a replicative niche and evade host cell defenses.

scholarly journals Analysis of Dot/Icm Type IVB Secretion System Subassemblies by Cryoelectron Tomography Reveals Conformational Changes Induced by DotB Binding

mBio ◽  
2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Donghyun Park ◽  
David Chetrit ◽  
Bo Hu ◽  
Craig R. Roy ◽  
Jun Liu
Keyword(s):  
High Resolution ◽  
Legionella Pneumophila ◽  
Secretion System ◽  
Type Iv Secretion ◽  
Structural Basis ◽  
Secretion Systems ◽  
Inner Membrane ◽  
Content Type ◽  
Type Iv ◽  
Type Iv Secretion Systems

ABSTRACT Type IV secretion systems (T4SSs) are sophisticated nanomachines used by many bacterial pathogens to translocate protein and DNA substrates across a host cell membrane. Although T4SSs have important roles in promoting bacterial infections, little is known about the biogenesis of the apparatus and the mechanism of substrate transfer. Here, high-throughput cryoelectron tomography (cryo-ET) was used to visualize Legionella pneumophila T4SSs (also known as Dot/Icm secretion machines) in both the whole-cell context and at the cell pole. These data revealed the distribution patterns of individual Dot/Icm machines in the bacterial cell and identified five distinct subassembled intermediates. High-resolution in situ structures of the Dot/Icm machine derived from subtomogram averaging revealed that docking of the cytoplasmic DotB (VirB11-related) ATPase complex onto the DotO (VirB4-related) ATPase complex promotes a conformational change in the secretion system that results in the opening of a channel in the bacterial inner membrane. A model is presented for how the Dot/Icm apparatus is assembled and for how this machine may initiate the transport of cytoplasmic substrates across the inner membrane. IMPORTANCE Many bacteria use type IV secretion systems (T4SSs) to translocate proteins and nucleic acids into target cells, which promotes DNA transfer and host infection. The Dot/Icm T4SS in Legionella pneumophila is a multiprotein nanomachine that is known to translocate over 300 different protein effectors into eukaryotic host cells. Here, advanced cryoelectron tomography and subtomogram analysis were used to visualize the Dot/Icm machine assembly and distribution in a single L. pneumophila cell. Extensive classification and averaging revealed five distinct intermediates of the Dot/Icm machine at high resolution. Comparative analysis of the Dot/Icm machine and subassemblies derived from wild-type cells and several mutants provided a structural basis for understanding mechanisms that underlie the assembly and activation of the Dot/Icm machine.

scholarly journals Rapid Escape of the dot/icm Mutants of Legionella pneumophila into the Cytosol of Mammalian and Protozoan Cells

2007 ◽  
Vol 75 (7) ◽  
pp. 3290-3304 ◽  
Author(s):  
Maëlle Molmeret ◽  
Marina Santic’ ◽  
Rexford Asare ◽  
Reynold A. Carabeo ◽  
Yousef Abu Kwaik
Keyword(s):  
Legionella Pneumophila ◽  
Brefeldin A ◽  
High Resolution Electron Microscopy ◽  
Secretion System ◽  
Type Iv Secretion ◽  
Golgi Vesicle ◽  
Secretion Systems ◽  
Vesicle Traffic ◽  
Type Iv ◽  
Resolution Electron

ABSTRACT The Legionella pneumophila-containing phagosome evades endocytic fusion and intercepts endoplasmic reticulum (ER)-to-Golgi vesicle traffic, which is believed to be mediated by the Dot/Icm type IV secretion system. Although phagosomes harboring dot/icm mutants are thought to mature through the endosomal-lysosomal pathway, colocalization studies with lysosomal markers have reported contradictory results. In addition, phagosomes harboring the dot/icm mutants do not interact with endocytosed materials, which is inconsistent with maturation of the phagosomes in the endosomal-lysosomal pathway. Using multiple strategies, we show that the dot/icm mutants defective in the Dot/Icm structural apparatus are unable to maintain the integrity of their phagosomes and escape into the cytoplasm within minutes of entry into various mammalian and protozoan cells in a process independent of the type II secretion system. In contrast, mutants defective in cytoplasmic chaperones of Dot/Icm effectors and rpoS, letA/S, and letE regulatory mutants are all localized within intact phagosomes. Importantly, non-dot/icm L. pneumophila mutants whose phagosomes acquire late endosomal-lysosomal markers are all located within intact phagosomes. Using high-resolution electron microscopy, we show that phagosomes harboring the dot/icm transporter mutants do not fuse to lysosomes but are free in the cytoplasm. Inhibition of ER-to-Golgi vesicle traffic by brefeldin A does not affect the integrity of the phagosomes harboring the parental strain of L. pneumophila. We conclude that the Dot/Icm transporter is involved in maintaining the integrity of the L. pneumophila phagosome, independent of interception of ER-to-Golgi vesicle traffic, which is a novel function of type IV secretion systems.

scholarly journals LidA, a Translocated Substrate of the Legionella pneumophila Type IV Secretion System, Interferes with the Early Secretory Pathway

2005 ◽  
Vol 73 (7) ◽  
pp. 4370-4380 ◽  
Author(s):  
Isabelle Derré ◽  
Ralph R. Isberg
Keyword(s):  
Host Cell ◽  
Legionella Pneumophila ◽  
Mammalian Cells ◽  
Secretory Pathway ◽  
Close Contact ◽  
Secretion System ◽  
Type Iv Secretion ◽  
Type Iv Secretion System ◽  
Early Secretory Pathway ◽  
Type Iv

ABSTRACT Legionella pneumophila uses a type IV secretion system to deliver effector molecules into the host cell and establish its replication vacuole. In this study, we investigated the role of LidA, a translocated substrate associated with the surface of the L. pneumophila-containing vacuole. LidA is secreted into the host cell throughout the replication cycle of the bacteria and associates with compartments of the early secretory pathway. When overexpressed in mammalian cells or yeast, LidA interferes with the early secretory pathway, probably via a domain predicted to be rich in coiled-coil structure. Finally, during intracellular replication, the replication vacuoles are in close contact with the endoplasmic reticulum-Golgi intermediate compartment and the Golgi apparatus, suggesting a positive correlation between intracellular growth and association of the vacuole with compartments of the early secretory pathway. We propose that LidA is involved in the recruitment of early secretory vesicles to the L. pneumophila-containing vacuole and that the vacuole associates with the secretory pathway to facilitate this process.

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