Towards a Structural Comprehension of Bacterial Type VI Secretion Systems: Characterization of the TssJ-TssM Complex of an Escherichia coli Pathovar

Type VI secretion systems (T6SS) are trans-envelope machines dedicated to the secretion of virulence factors into eukaryotic or prokaryotic cells, therefore required for pathogenesis and/or for competition towards neighboring bacteria. The T6SS apparatus resembles the injection device of bacteriophage T4, and is anchored to the cell envelope through a membrane complex. This membrane complex is composed of the TssL, TssM and TagL inner membrane anchored proteins and of the TssJ outer membrane lipoprotein. Here, we report the crystal structure of the enteroaggregative Escherichia coli Sci1 TssJ lipoprotein, a two four-stranded β-sheets protein that exhibits a transthyretin fold with an additional α-helical domain and a protruding loop. We showed that TssJ contacts TssM through this loop since a loop depleted mutant failed to interact with TssM in vitro or in vivo. Biophysical analysis of TssM and TssJ-TssM interaction suggest a structural model of the membrane-anchored outer shell of T6SS. Collectively, our results provide an improved understanding of T6SS assembly and encourage structure-aided drug design of novel antimicrobials targeting T6SS

Etude du système de sécrétion de type VI chez Escherichia coli entéro-agrégatif : Caractérisation d'un sous complexe d'ancrage membranaires

Bacterial pathogenesis relies on a subset of mechanisms including adhesion to various matrices, antibiotic resistance, defence and action against surrounding microorganisms, and secretion of virulence factors. Among the secretion systems, the recently identified Type VI secretion system (T6SS) has been shown to be involved in both virulence against eukaryotic cells and inter-bacterial warfare. T6SS are composed of a minimum of 13 proteins called "core components". It is believe to form a macromolecular system that spans the envelope to assemble an extracellular structure composed of the Hcp protein with a trimer of VgrG located at the tip. This model has been built following in silico and structural analyses demonstrating the link between several T6SS subunits and bacteriophage T4 baseplate and tail elements. Other T6SS subunits include membrane proteins. Using enteroaggregative Escherichia coli as a bacterial model, the aim of my work is to understand how this system assembles in the cell envelope. I recently showed that four of these membrane proteins, SciP, SciS, SciN and SciZ make contact to form a complex [1]. These four subunits are critical components of the T6SS. I then delineated the interaction network, demonstrating that SciZ interacts with SciP, and that SciS interacts with both SciP and SciN. Further characterization of these subunits showed that SciN is a lipoprotein associated with the outer membrane [2, 4], whereas SciP and SciS are inner membrane proteins anchored through a single and three transmembrane segments respectively. SciZ is a polytopic inner membrane protein carrying a peptidoglycan-binding motif within its periplasmic domain. Mutagenesis and peptidoglycan binding experiments demonstrated that SciZ anchors the T6SS to the cell wall [1, 3]. Overall, we have identified and characterized a trans-envelope complex anchored in both membrane and to the peptidoglycan layer.Bacterial pathogenesis relies on a subset of mechanisms including adhesion to various matrices, antibiotic resistance, defence and action against surrounding microorganisms, and secretion of virulence factors. Among the secretion systems, the recently identified Type VI secretion system (T6SS) has been shown to be involved in both virulence against eukaryotic cells and inter-bacterial warfare. T6SS are composed of a minimum of 13 proteins called "core components". It is believe to form a macromolecular system that spans the envelope to assemble an extracellular structure composed of the Hcp protein with a trimer of VgrG located at the tip. This model has been built following in silico and structural analyses demonstrating the link between several T6SS subunits and bacteriophage T4 baseplate and tail elements. Other T6SS subunits include membrane proteins. Using enteroaggregative Escherichia coli as a bacterial model, the aim of my work is to understand how this system assembles in the cell envelope. I recently showed that four of these membrane proteins, SciP, SciS, SciN and SciZ make contact to form a complex [1]. These four subunits are critical components of the T6SS. I then delineated the interaction network, demonstrating that SciZ interacts with SciP, and that SciS interacts with both SciP and SciN. Further characterization of these subunits showed that SciN is a lipoprotein associated with the outer membrane [2, 4], whereas SciP and SciS are inner membrane proteins anchored through a single and three transmembrane segments respectively. SciZ is a polytopic inner membrane protein carrying a peptidoglycan-binding motif within its periplasmic domain. Mutagenesis and peptidoglycan binding experiments demonstrated that SciZ anchors the T6SS to the cell wall [1, 3]. Overall, we have identified and characterized a trans-envelope complex anchored in both membrane and to the peptidoglycan layer

Molecular Dissection of the Interface between the Type VI Secretion TssM Cytoplasmic Domain and the TssG Baseplate Component

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Anchoring the type VI secretion system to the peptidoglycan: TssL, TagL, TagP... what else?

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The C‐tail anchored TssL subunit, an essential protein of the enteroaggregative Escherichia coli Sci‐1 Type VI secretion system, is inserted by YidC

International audienceType VI secretion systems (T6SS) are macromolecular complexes present in Gram-negative bacteria. T6SS are structurally similar to the bacteriophage cell-puncturing device and have been shown to mediate bacteria–host or bacteria–bacteria interactions. T6SS assemble from 13 to 20 proteins. In enteroaggregative Escherichia coli (EAEC), one of the subassemblies is composed of four proteins that form a trans-envelope complex: the TssJ outer membrane lipoprotein, the peptidoglycan-anchored inner membrane TagL protein, and two putative inner membrane proteins, TssL and TssM. In this study, we characterized the TssL protein of the EAEC Sci-1 T6SS in terms of localization, topology, and function. TssL is a critical component of the T6SS, anchored to the inner membrane through a single transmembrane segment located at the extreme C-terminus of the protein. We further show that this transmembrane segment is essential for the function of the protein and its proper insertion in the inner membrane is dependent upon YidC and modulated by the Hsp70 homologue DnaK

The SciZ protein anchors the enteroaggregative Escherichia coli Type VI secretion system to the cell wall

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SciN Is an Outer Membrane Lipoprotein Required for Type VI Secretion in Enteroaggregative Escherichia coli▿

Enteroaggregative Escherichia coli (EAEC) is a pathogen implicated in several infant diarrhea or diarrheal outbreaks in areas of endemicity. Although multiple genes involved in EAEC pathogenesis have been identified, the overall mechanism of virulence is not well understood. Recently, a novel secretion system, called type VI secretion (T6S) system (T6SS), has been identified in EAEC and most animal or plant gram-negative pathogens. T6SSs are multicomponent cell envelope machines responsible for the secretion of at least two putative substrates, Hcp and VgrG. In EAEC, two copies of T6S gene clusters, called sci-1 and sci-2, are present on the pheU pathogenicity island. In this study, we focused our work on the sci-1 gene cluster. The Sci-1 apparatus is probably composed of all, or a subset of, the 21 gene products encoded on the cluster. Among these subunits, some are shared by all T6SSs identified to date, including a ClpV-type AAA+ ATPase (SciG) and an IcmF (SciS) and an IcmH (SciP) homologue, as well as a putative lipoprotein (SciN). In this study, we demonstrate that sciN is a critical gene necessary for T6S-dependent secretion of the Hcp-like SciD protein and for biofilm formation. We further show that SciN is a lipoprotein, as shown by the inhibition of its processing by globomycin and in vivo labeling with [3H]palmitic acid. SciN is tethered to the outer membrane and exposed in the periplasm. Sequestration of SciN at the inner membrane by targeting the +2 residue responsible for lipoprotein localization (Gly2Asp) fails to complement an sciN mutant for SciD secretion and biofilm formation. Together, these results support a model in which SciN is an outer membrane lipoprotein exposed in the periplasm and essential for the Sci-1 apparatus function

Rotation of Vibrio fischeri Flagella Produces Outer Membrane Vesicles That Induce Host Development

00001 ăWOS:000380818500004International audienceUsing the squid-vibrio association, we aimed to characterize the mechanism through which Vibrio fischeri cells signal morphogenesis of the symbiotic light-emitting organ. The symbiont releases two cell envelope molecules, peptidoglycan (PG) and lipopolysaccharide (LPS) that, within 12 h of light organ colonization, act in synergy to trigger normal tissue development. Recent work has shown that outer membrane vesicles (OMVs) produced by V. fischeri are sufficient to induce PG-dependent morphogenesis; however, the mechanism(s) of OMV release by these bacteria has not been described. Like several genera of both beneficial and pathogenic bacteria, V. fischeri cells elaborate polar flagella that are enclosed by an extension of the outer membrane, whose function remains unclear. Here, we present evidence that along with the well-recognized phenomenon of blebbing from the cell's surface, rotation of this sheathed flagellum also results in the release of OMVs. In addition, we demonstrate that most of the development-inducing LPS is associated with these OMVs and that the presence of the outer membrane protein OmpU but not the LPS O antigen on these OMVs is important in triggering normal host development. These results also present insights into a possible new mechanism of LPS release by pathogens with sheathed flagella. IMPORTANCE Determining the function(s) of sheathed flagella in bacteria has been challenging, because no known mutation results only in the loss of this outer membrane-derived casing. Nevertheless, the presence of a sheathed flagellum in such host-associated genera as Vibrio, Helicobacter, and Brucella has led to several proposed functions, including physical protection of the flagella and masking of their immunogenic flagellins. Using the squid-vibrio light organ symbiosis, we demonstrate another role, that of V. fischeri cells require rotating flagella to induce apoptotic cell death within surface epithelium, which is a normal step in the organ's development. Further, we present evidence that this rotation releases apoptosis-triggering lipopolysaccharide in the form of outer membrane vesicles. Such release may also occur by pathogens but with different outcomes for the host

Architecture and assembly of the Type VI secretion system

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Structural characterization and oligomerization of the TssL protein, a component shared by bacterial type VI and type IVb secretion systems.

International audienceThe Type VI secretion system (T6SS) is a macromolecular system distributed in Gram-negative bacteria, responsible for the secretion of effector proteins into target cells. The T6SS has a broad versatility as it can target both eukaryotic and prokaryotic cells. It is therefore involved in host pathogenesis or killing neighboring bacterial cells to colonize a new niche. At the architecture level, the T6SS core apparatus is composed of 13 proteins, which assemble in two subcomplexes. One of these subcomplexes, composed of subunits that share structural similarities with bacteriophage tail and baseplate components, is anchored to the cell envelope by the membrane subcomplex. This latter is constituted of at least three proteins, TssL, TssM, and TssJ. The crystal structure of the TssJ outer membrane lipoprotein and its interaction with the inner membrane TssM protein have been recently reported. TssL and TssM share sequence homology and characteristics with two components of the Type IVb secretion system (T4bSS), IcmH/DotU and IcmF, respectively. In this study, we report the crystal structure of the cytoplasmic domain of the TssL inner membrane protein from the enteroaggregative Escherichia coli Sci-1 T6SS. It folds as a hook-like structure composed of two three-helix bundles. Two TssL molecules associate to form a functional complex. Although the TssL trans-membrane segment is the main determinant of self-interaction, contacts between the cytoplasmic domains are required for TssL function. Based on sequence homology and secondary structure prediction, we propose that the TssL structure is the prototype for the members of the TssL and IcmH/DotU families