Proteomic analysis of the host-pathogen interface in experimental cholera.

The microbial cell surface is a site of critical microbe-host interactions that often control infection outcomes. Defining the set of host proteins present at this interface has been challenging. Here we used a surface-biotinylation approach coupled to quantitative mass spectrometry to identify and quantify both bacterial and host proteins present on the surface of diarrheal fluid-derived Vibrio cholerae in an infant rabbit model of cholera. The V. cholerae surface was coated with numerous host proteins, whose abundance were driven by the presence of cholera toxin, including the C-type lectin SP-D. Mice lacking SP-D had enhanced V. cholerae intestinal colonization, and SP-D production shaped both host and pathogen transcriptomes. Additional host proteins (AnxA1, LPO and ZAG) that bound V. cholerae were also found to recognize distinct taxa of the murine intestinal microbiota, suggesting that these host factors may play roles in intestinal homeostasis in addition to host defense.

Type I interferon remodels lysosome function and modifies intestinal epithelial defense.

Organelle remodeling is critical for cellular homeostasis, but host factors that control organelle function during microbial infection remain largely uncharacterized. Here, a genome-scale CRISPR/Cas9 screen in intestinal epithelial cells with the prototypical intracellular bacterial pathogen Salmonella led us to discover that type I IFN (IFN-I) remodels lysosomes. Even in the absence of infection, IFN-I signaling modified the localization, acidification, protease activity, and proteomic profile of lysosomes. Proteomic and genetic analyses revealed that multiple IFN-I-stimulated genes including IFITM3, SLC15A3, and CNP contribute to lysosome acidification. IFN-I-dependent lysosome acidification was associated with elevated intracellular Salmonella virulence gene expression, rupture of the Salmonella-containing vacuole, and host cell death. Moreover, IFN-I signaling promoted in vivo Salmonella pathogenesis in the intestinal epithelium where Salmonella initiates infection, indicating that IFN-I signaling can modify innate defense in the epithelial compartment. We propose that IFN-I control of lysosome function broadly impacts host defense against diverse viral and microbial pathogens.

Priming and polymerization of a bacterial contractile tail structure.

Contractile tails are composed of an inner tube wrapped by an outer sheath assembled in an extended, metastable conformation that stores mechanical energy necessary for its contraction. Contraction is used to propel the rigid inner tube towards target cells for DNA or toxin delivery. Although recent studies have revealed the structure of the contractile sheath of the type VI secretion system, the mechanisms by which its polymerization is controlled and coordinated with the assembly of the inner tube remain unknown. Here we show that the starfish-like TssA dodecameric complex interacts with tube and sheath components. Fluorescence microscopy experiments in enteroaggregative Escherichia coli reveal that TssA binds first to the type VI secretion system membrane core complex and then initiates tail polymerization. TssA remains at the tip of the growing structure and incorporates new tube and sheath blocks. On the basis of these results, we propose that TssA primes and coordinates tail tube and sheath biogenesis.

Biogenesis and structure of a type VI secretion membrane core complex.

Bacteria share their ecological niches with other microbes. The bacterial type VI secretion system is one of the key players in microbial competition, as well as being an important virulence determinant during bacterial infections. It assembles a nano-crossbow-like structure in the cytoplasm of the attacker cell that propels an arrow made of a haemolysin co-regulated protein (Hcp) tube and a valine-glycine repeat protein G (VgrG) spike and punctures the prey's cell wall. The nano-crossbow is stably anchored to the cell envelope of the attacker by a membrane core complex. Here we show that this complex is assembled by the sequential addition of three type VI subunits (Tss)-TssJ, TssM and TssL-and present a structure of the fully assembled complex at 11.6 Å resolution, determined by negative-stain electron microscopy. With overall C5 symmetry, this 1.7-megadalton complex comprises a large base in the cytoplasm. It extends in the periplasm via ten arches to form a double-ring structure containing the carboxy-terminal domain of TssM (TssMct) and TssJ that is anchored in the outer membrane. The crystal structure of the TssMct-TssJ complex coupled to whole-cell accessibility studies suggest that large conformational changes induce transient pore formation in the outer membrane, allowing passage of the attacking Hcp tube/VgrG spike.

Role and Recruitment of the TagL Peptidoglycan-Binding Protein during Type VI Secretion System Biogenesis.

The type VI secretion system (T6SS) is an injection apparatus that uses a springlike mechanism for effector delivery. The contractile tail is composed of a needle tipped by a sharpened spike and wrapped by the sheath that polymerizes in an extended conformation on the assembly platform, or baseplate. Contraction of the sheath propels the needle and effectors associated with it into target cells. The passage of the needle through the cell envelope of the attacker is ensured by a dedicated trans-envelope channel complex. This membrane complex (MC) comprises the TssJ lipoprotein and the TssL and TssM inner membrane proteins. MC assembly is a hierarchized mechanism in which the different subunits are recruited in a specific order: TssJ, TssM, and then TssL. Once assembled, the MC serves as a docking station for the baseplate. In enteroaggregative Escherichia coli, the MC is accessorized by TagL, a peptidoglycan-binding (PGB) inner membrane-anchored protein. Here, we show that the PGB domain is the only functional domain of TagL and that the N-terminal transmembrane region mediates contact with the TssL transmembrane helix. Finally, we conduct fluorescence microscopy experiments to position TagL in the T6SS biogenesis pathway, demonstrating that TagL is recruited to the membrane complex downstream of TssL and is not required for baseplate docking.IMPORTANCE Bacteria use weapons to deliver effectors into target cells. One of these weapons, called the type VI secretion system (T6SS), could be compared to a nano-spear gun using a springlike mechanism for effector injection. By targeting bacteria and eukaryotic cells, the T6SS reshapes bacterial communities and hijacks host cell defenses. In enteroaggregative Escherichia coli, the T6SS is a multiprotein machine that comprises a cytoplasmic tail and a peptidoglycan-anchored trans-envelope channel. In this work, we show that TagL comprises an N-terminal domain that mediates contact with the channel and a peptidoglycan-binding domain that binds the cell wall. We then determine at which stage of T6SS biogenesis TagL is recruited and how TagL absence impacts the assembly pathway.

TssA: The cap protein of the Type VI secretion system tail.

The Type VI secretion system (T6SS) is a multiprotein and mosaic apparatus that delivers protein effectors into prokaryotic or eukaryotic cells. Recent data on the enteroaggregative Escherichia coli (EAEC) T6SS have provided evidence that the TssA protein is a key component during T6SS biogenesis. The T6SS comprises a trans-envelope complex that docks the baseplate, a cytoplasmic complex that represents the assembly platform for the tail. The T6SS tail is structurally, evolutionarily and functionally similar to the contractile tails of bacteriophages. We have shown that TssA docks to the membrane complex, recruits the baseplate complex and initiates and coordinates the polymerization of the inner tube with that of the sheath. Here, we review these recent findings, discuss the variations within TssA-like proteins, speculate on the role of EAEC TssA in T6SS biogenesis and propose future research perspectives.

Characterization of the Porphyromonas gingivalis Type IX Secretion Trans-envelope PorKLMNP Core Complex.

The transport of proteins at the cell surface of Bacteroidetes depends on a secretory apparatus known as type IX secretion system (T9SS). This machine is responsible for the cell surface exposition of various proteins, such as adhesins, required for gliding motility in Flavobacterium, S-layer components in Tannerella forsythia, and tooth tissue-degrading enzymes in the oral pathogen Porphyromonas gingivalis Although a number of subunits of the T9SS have been identified, we lack details on the architecture of this secretion apparatus. Here we provide evidence that five of the genes encoding the core complex of the T9SS are co-transcribed and that the gene products are distributed in the cell envelope. Protein-protein interaction studies then revealed that these proteins oligomerize and interact through a dense network of contacts.

The Type VI Secretion TssEFGK-VgrG Phage-Like Baseplate Is Recruited to the TssJLM Membrane Complex via Multiple Contacts and Serves As Assembly Platform for Tail Tube/Sheath Polymerization.

The Type VI secretion system (T6SS) is a widespread weapon dedicated to the delivery of toxin proteins into eukaryotic and prokaryotic cells. The 13 T6SS subunits assemble a cytoplasmic contractile structure anchored to the cell envelope by a membrane-spanning complex. This structure is evolutionarily, structurally and functionally related to the tail of contractile bacteriophages. In bacteriophages, the tail assembles onto a protein complex, referred to as the baseplate, that not only serves as a platform during assembly of the tube and sheath, but also triggers the contraction of the sheath. Although progress has been made in understanding T6SS assembly and function, the composition of the T6SS baseplate remains mostly unknown. Here, we report that six T6SS proteins-TssA, TssE, TssF, TssG, TssK and VgrG-are required for proper assembly of the T6SS tail tube, and a complex between VgrG, TssE,-F and-G could be isolated. In addition, we demonstrate that TssF and TssG share limited sequence homologies with known phage components, and we report the interaction network between these subunits and other baseplate and tail components. In agreement with the baseplate being the assembly platform for the tail, fluorescence microscopy analyses of functional GFP-TssF and TssK-GFP fusion proteins show that these proteins assemble stable and static clusters on which the sheath polymerizes. Finally, we show that recruitment of the baseplate to the apparatus requires initial positioning of the membrane complex and contacts between TssG and the inner membrane TssM protein.

Architecture and assembly of the Type VI secretion system.

The Type VI secretion system (T6SS) delivers protein effectors to diverse cell types including prokaryotic and eukaryotic cells, therefore it participates in inter-bacterial competition and pathogenesis. The T6SS is constituted of an envelope-spanning complex anchoring a cytoplasmic tubular edifice. This tubular structure is evolutionarily, functionally and structurally related to the tail of contractile phages. It is composed of an inner tube tipped by a spike complex, and engulfed within a sheath-like structure. This structure assembles onto a platform called "baseplate" that is connected to the membrane sub-complex. The T6SS functions as a nano-crossbow: upon contraction of the sheath, the inner tube is propelled towards the target cell, allowing effector delivery. This review focuses on the architecture and biogenesis of this fascinating secretion machine, highlighting recent advances regarding the assembly of the membrane or tail complexes. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.

TssK is a trimeric cytoplasmic protein interacting with components of both phage-like and membrane anchoring complexes of the type VI secretion system.

The Type VI secretion system (T6SS) is a macromolecular machine that mediates bacteria-host or bacteria-bacteria interactions. The T6SS core apparatus assembles from 13 proteins that form two sub-assemblies: a phage-like complex and a trans-envelope complex. The Hcp, VgrG, TssE, and TssB/C subunits are structurally and functionally related to components of the tail of contractile bacteriophages. This phage-like structure is thought to be anchored to the membrane by a trans-envelope complex composed of the TssJ, TssL, and TssM proteins. However, how the two sub-complexes are connected remains unknown. Here we identify TssK, a protein that establishes contacts with the two T6SS sub-complexes through direct interactions with TssL, Hcp, and TssC. TssK is a cytoplasmic protein assembling trimers that display a three-armed shape, as revealed by TEM and SAXS analyses. Fluorescence microscopy experiments further demonstrate the requirement of TssK for sheath assembly. Our results suggest a central role for TssK by linking both complexes during T6SS assembly.

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

The 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.

A Vibrio cholerae BolA-Like Protein Is Required for Proper Cell Shape and Cell Envelope Integrity.

BolA family proteins are conserved in Gram-negative bacteria and many eukaryotes. While diverse cellular phenotypes have been linked to this protein family, the molecular pathways through which these proteins mediate their effects are not well described. Here, we investigated the roles of BolA family proteins in Vibrio cholerae, the cholera pathogen. Like Escherichia coli, V. cholerae encodes two BolA proteins, BolA and IbaG. However, in marked contrast to E. coli, where bolA is linked to cell shape and ibaG is not, in V. cholerae, bolA mutants lack morphological defects, whereas ibaG proved critical for the generation and/or maintenance of the pathogen's morphology. Notably, the bizarre-shaped, multipolar, elongated, and wide cells that predominated in exponential-phase ΔibaGV. cholerae cultures were not observed in stationary-phase cultures. The V. cholerae ΔibaG mutant exhibited increased sensitivity to cell envelope stressors, including cell wall-acting antibiotics and bile, and was defective in intestinal colonization. ΔibaGV. cholerae had reduced peptidoglycan and lipid II and altered outer membrane lipids, likely contributing to the mutant's morphological defects and sensitivity to envelope stressors. Transposon insertion sequencing analysis of ibaG's genetic interactions suggested that ibaG is involved in several processes involved in the generation and homeostasis of the cell envelope. Furthermore, copurification studies revealed that IbaG interacts with proteins containing iron-sulfur clusters or involved in their assembly. Collectively, our findings suggest that V. cholerae IbaG controls cell morphology and cell envelope integrity through its role in biogenesis or trafficking of iron-sulfur cluster proteins.IMPORTANCE BolA-like proteins are conserved across prokaryotes and eukaryotes. These proteins have been linked to a variety of phenotypes, but the pathways and mechanisms through which they act have not been extensively characterized. Here, we unraveled the role of the BolA-like protein IbaG in the cholera pathogen Vibrio cholerae The absence of IbaG was associated with dramatic changes in cell morphology, sensitivity to envelope stressors, and intestinal colonization defects. IbaG was found to be required for biogenesis of several components of the V. cholerae cell envelope and to interact with numerous iron-sulfur cluster-containing proteins and factors involved in their assembly. Thus, our findings suggest that IbaG governs V. cholerae cell shape and cell envelope homeostasis through its effects on iron-sulfur proteins and associated pathways. The diversity of processes involving iron-sulfur-containing proteins is likely a factor underlying the range of phenotypes associated with BolA family proteins.

Tryptophan-mediated Dimerization of the TssL Transmembrane Anchor Is Required for Type VI Secretion System Activity.

The type VI secretion system (T6SS) is a multiprotein complex used by bacteria to deliver effectors into target cells. The T6SS comprises a bacteriophage-like contractile tail structure anchored to the cell envelope by a membrane complex constituted of the TssJ outer-membrane lipoprotein and the TssL and TssM inner-membrane proteins. TssJ establishes contact with the periplasmic domain of TssM whereas the transmembrane segments of TssM and its cytoplasmic domain interact with TssL. TssL protrudes in the cytoplasm but is anchored by a C-terminal transmembrane helix (TMH). Here, we show that TssL TMH dimerization is required for the stability of the protein and for T6SS function. Using the TOXCAT assay and point mutations of the 23 residues of the TssL TMH, we identified Thr194 and Trp199 as necessary for TssL TMH dimerization. NMR hydrogen-deuterium exchange experiments demonstrated the existence of a dimer with the presence of Trp185 and Trp199 at the interface. A structural model based on molecular dynamic simulations shows that TssL TMH dimer formation involves π-π interactions resulting from the packing of the two Trp199 rings at the C-terminus and of the six aromatic rings of Tyr184, Trp185 and Trp188 at the N-terminus of the TMH.

The gp27-like Hub of VgrG Serves as Adaptor to Promote Hcp Tube Assembly.

Contractile injection systems are multiprotein complexes that use a spring-like mechanism to deliver effectors into target cells. In addition to using a conserved mechanism, these complexes share a common core known as the tail. The tail comprises an inner tube tipped by a spike, wrapped by a contractile sheath, and assembled onto a baseplate. Here, using the type VI secretion system (T6SS) as a model of contractile injection systems, we provide molecular details on the interaction between the inner tube and the spike. Reconstitution into the Escherichia coli heterologous host in the absence of other T6SS components and in vitro experiments demonstrated that the Hcp tube component and the VgrG spike interact directly. VgrG deletion studies coupled to functional assays showed that the N-terminal domain of VgrG is sufficient to interact with Hcp, to initiate proper Hcp tube polymerization, and to promote sheath dynamics and Hcp release. The interaction interface between Hcp and VgrG was then mapped using docking simulations, mutagenesis, and cysteine-mediated cross-links. Based on these results, we propose a model in which the VgrG base serves as adaptor to recruit the first Hcp hexamer and initiates inner tube polymerization.

Defining Assembly Pathways by Fluorescence Microscopy.

Bacterial secretion systems are among the largest protein complexes in prokaryotes and display remarkably complex architectures. Their assembly often follows clearly defined pathways. Deciphering these pathways not only reveals how bacteria accomplish building these large functional complexes but can provide crucial information on the interactions and subcomplexes within secretion systems, their distribution within bacteria, and even functional insights. The emergence of fluorescent proteins has provided a new powerful tool for biological imaging, and the use of fluorescently labeled components presents an interesting method to accurately define the biogenesis of macromolecular complexes. Here we describe the use of this method to decipher the assembly pathway of bacterial secretion systems.

Fusion Reporter Approaches to Monitoring Transmembrane Helix Interactions in Bacterial Membranes.

In transenvelope multiprotein machines such as bacterial secretion systems, protein-protein interactions not only occur between soluble domains but might also be mediated by helix-helix contacts in the inner membrane. Here we describe genetic assays commonly used to test interactions between transmembrane α-helices in their native membrane environment. These assays are based on the reconstitution of dimeric regulators allowing the control of expression of reporter genes. We provide detailed protocols for the TOXCAT and GALLEX assays used to monitor homotypic and heterotypic transmembrane helix-helix interactions.

Type VI secretion TssK baseplate protein exhibits structural similarity with phage receptor-binding proteins and evolved to bind the membrane complex.

The type VI secretion system (T6SS) is a multiprotein machine widespread in Gram-negative bacteria that delivers toxins into both eukaryotic and prokaryotic cells. The mechanism of action of the T6SS is comparable to that of contractile myophages. The T6SS builds a tail-like structure made of an inner tube wrapped by a sheath, assembled under an extended conformation. Contraction of the sheath propels the inner tube towards the target cell. The T6SS tail is assembled on a platform-the baseplate-which is functionally similar to bacteriophage baseplates. In addition, the baseplate docks the tail to a trans-envelope membrane complex that orients the tail towards the target. Here, we report the crystal structure of TssK, a central component of the T6SS baseplate. We show that TssK is composed of three domains, and establish the contribution of each domain to the interaction with TssK partners. Importantly, this study reveals that the N-terminal domain of TssK is structurally homologous to the shoulder domain of phage receptor-binding proteins, and the C-terminal domain binds the membrane complex. We propose that TssK has conserved the domain of attachment to the virion particle but has evolved the reception domain to use the T6SS membrane complex as receptor.

Structure-Function Analysis of the TssL Cytoplasmic Domain Reveals a New Interaction between the Type VI Secretion Baseplate and Membrane Complexes.

The type VI secretion system (T6SS) is a multiprotein complex that delivers toxin effectors in both prokaryotic and eukaryotic cells. It is constituted of a long cytoplasmic structure-the tail-made of stacked Hcp hexamers and wrapped by a contractile sheath. Contraction of the sheath propels the inner tube capped by the VgrG spike protein toward the target cell. This tubular structure is built onto an assembly platform-the baseplate-that is composed of the TssEFGK-VgrG subunits. During the assembly process, the baseplate is recruited to a trans-envelope complex comprising the TssJ outer membrane lipoprotein and the TssL and TssM inner membrane proteins. This membrane complex serves as a docking station for the baseplate/tail and as a channel for the passage of the inner tube during sheath contraction. The baseplate is recruited to the membrane complex through multiple contacts including interactions of TssG and TssK with the cytoplasmic loop of TssM and of TssK with the cytoplasmic domain of TssL, TssLCyto. Here, we show that TssLCyto interacts also with the TssE baseplate subunit. Based on the available TssLCyto structures, we targeted conserved regions and specific features of TssLCyto in enteroaggregative Escherichia coli. By using bacterial two-hybrid analysis and co-immunoprecipitation, we further show that the disordered L3-L4 loop is necessary to interact with TssK and that the L6-L7 loop mediates the interaction with TssE, whereas the TssM cytoplasmic loop binds the conserved groove of TssLCyto. Finally, competition assays demonstrated that these interactions are physiologically important for T6SS function.

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

Type 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.