The Chlamydia trachomatis Type III Secretion Chaperone Slc1 Engages Multiple Early Effectors,Including TepP,a Tyrosine-phosphorylated Protein Required for the Recruitment of CrkI-II to Nascent Inclusions and Innate Immune Signaling

Chlamydia trachomatis, the causative agent of trachoma and sexually transmitted infections, employs a type III secretion (T3S) system to deliver effector proteins into host epithelial cells to establish a replicative vacuole. Aside from the phosphoprotein TARP, a Chlamydia effector that promotes actin re-arrangements, very few factors mediating bacterial entry and early inclusion establishment have been characterized. Like many T3S effectors, TARP requires a chaperone (Slc1) for efficient translocation into host cells. In this study, we defined proteins that associate with Slc1 in invasive C. trachomatis elementary bodies (EB) by immunoprecipitation coupled with mass spectrometry. We identified Ct875, a new Slc1 client protein and T3S effector, which we renamed TepP (Translocated early phosphoprotein). We provide evidence that T3S effectors form large molecular weight complexes with Scl1 in vitro and that Slc1 enhances their T3S-dependent secretion in a heterologous Yersinia T3S system. We demonstrate that TepP is translocated early during bacterial entry into epithelial cells and is phosphorylated at tyrosine residues by host kinases. However, TepP phosphorylation occurs later than TARP, which together with the finding that Slc1 preferentially engages TARP in EBs leads us to postulate that these effectors are translocated into the host cell at different stages during C. trachomatis invasion. TepP co-immunoprecipitated with the scaffolding proteins CrkI-II during infection and Crk was recruited to EBs at entry sites where it remained associated with nascent inclusions. Importantly, C. trachomatis mutants lacking TepP failed to recruit CrkI-II to inclusions, providing genetic confirmation of a direct role for this effector in the recruitment of a host factor. Finally, endocervical epithelial cells infected with a tepP mutant showed altered expression of a subset of genes associated with innate immune responses. We propose a model wherein TepP acts downstream of TARP to recruit scaffolding proteins at entry sites to initiate and amplify signaling cascades important for the regulation of innate immune responses to Chlamydia.     

Vp1659 Is a Vibrio parahaemolyticus Type III Secretion System 1 Protein That Contributes to Translocation of Effector Proteins Needed To Induce Cytolysis,Autophagy, and Disruption of Actin Structure in HeLa Cells

Vibrio parahaemolyticus harbors two type III secretion systems (T3SSs; T3SS1 and T3SS2), of which T3SS1 is involved in host cell cytotoxicity. T3SS1 expression is positively regulated by ExsA, and it is negatively regulated by ExsD. We compared the secretion profiles of a wild-type strain (NY-4) of V. parahaemolyticus with those of an ExsD deletion mutant (ΔexsD) and with a strain of NY-4 that overexpresses T3SS1 (NY-4:pexsA). From this comparison, we detected a previously uncharacterized protein, Vp1659, which shares some sequence homology with LcrV from Yersinia. We show that vp1659 expression is positively regulated by ExsA and is negatively regulated by ExsD. Vp1659 is specifically secreted by T3SS1 of V. parahaemolyticus, and Vp1659 is not required for the successful extracellular secretion of another T3SS1 protein, Vp1656. Mechanical fractionation showed that Vp1659 is translocated into HeLa cells in a T3SS1-dependent manner and that deletion of Vp1659 does not prevent VopS from being translocated into HeLa cells during infection. Deletion of vp1659 significantly reduces cytotoxicity when HeLa cells are infected by V. parahaemolyticus, while complementation of the Δvp1659 strain restores cytotoxicity. Differential staining showed that Vp1659 is required to induce membrane permeability in HeLa cells. We also show evidence that Vp1659 is required for actin rearrangement and the induction of autophagy. On the basis of these data, we conclude that Vp1659 is a T3SS1-associated protein that is a component of the secretion apparatus and that it is necessary for the efficient translocation of effector proteins into epithelial cells.As a marine pathogen, Vibrio parahaemolyticus is frequently isolated from seafood products such as oysters and shrimp (19, 45). The main symptoms of V. parahaemolyticus infection in humans include diarrhea, nausea, and vomiting. In addition to the gastrointestinal infection, necrotizing fasciitis and septic shock are reportedly associated with V. parahaemolyticus infection (37). V. parahaemolyticus can also cause wound infections after contact with contaminated water (6, 7, 16, 37).V. parahaemolyticus is able to adhere to and invade epithelial cells (1, 38, 43). Pili are involved in the adherence to the intestinal epithelium (32), but it is not clear what factors are required for V. parahaemolyticus to invade epithelial cells. Hemolysins are considered primary factors involved in the pathogenesis of V. parahaemolyticus. For example, a thermostable direct hemolysin (tdh) mutant strain loses the ability to cause fluid accumulation in the intestinal lumen (33), while deletion of a tdh-related gene (trh) results in the complete loss of hemolysis and the partial loss of fluid accumulation in a rabbit intestinal ligation model (42). Recent studies show that the disruption of epithelial tight junctions, which is a hallmark of bacterial dissemination into the circulatory system and subsequent septicemia, is independent of the thermostable direct hemolysin, suggesting that additional factors are required for the pathogenesis of V. parahaemolyticus (27).A broad range of Gram-negative bacteria employ type III secretion systems (T3SSs) to export virulence-related proteins into the extracellular milieu and/or to deliver these proteins directly into host cells (5, 12, 13). T3SSs are composed of three parts: a secretion apparatus, translocators, and effectors (17, 18). The secretion apparatus and translocators are encoded by ca. 25 genes that are conserved and usually located in a genomic island. Genes that encode effectors are less conserved and can be found distal from the T3SS islands. The secretion apparatus serves to secrete both effectors and translocators from bacterial cells, and translocators help the effectors cross into the eukaryotic cells, where they can disrupt normal host cell signal functions.Two distinct T3SSs (T3SS1 and T3SS2) were identified in the genome of V. parahaemolyticus (28). On the basis of the sequence similarity and gene organization, T3SS1 was classified as a member of the Ysc family of secretion systems, while T3SS2 was classified as a member of the Inv-Mxi-Spa family (40). Functional analysis shows that deletion of T3SS1 decreases cytotoxicity against HeLa cells, while deletion of T3SS2 diminishes intestinal fluid accumulation (35). Interestingly, in some strains, T3SS2 can be involved in the cytotoxic effect specifically against Caco-2 and HCT-8 cells (23). One study showed that T3SS1 of V. parahaemolyticus induces autophagy, but blocking autophagy does not completely mitigate cytotoxicity, indicating that other T3SS1-induced mechanisms contribute to cell death (3, 4). Recent work from our laboratory showed that V. parahaemolyticus induces cell rounding, pore formation, and membrane damage, consistent with the induction of an oncosis pathway (46). Importantly, treatment of infected cells with an osmoprotectant (polyethylene glycol 3350) significantly reduced cytotoxicity, indicating that oncosis is the primary mechanism by which T3SS1 of V. parahaemolyticus causes cell death for in vitro cultures (46). Nevertheless, it is unknown which effector protein(s) is involved in cell cytotoxicity. By comparing the secretion protein profiles of wild-type and T3SS1 mutant strains, four T3SS1 proteins have been identified (34). Among these, Vp1680 is translocated into host cells and is required for the induction of autophagy during infection of HeLa cells (3, 34). Recent studies showed that VopS is able to prevent the interaction of Rho GTPase with its downstream factors by a new modification mechanism, called AMPylation (44), and this prevents the assembly of actin fibers. Two proteins (VopT and VopL) have been identified as T3SS2 substrates (23, 26). VopT is a member of ADP-ribosyltransferase and is partially responsible for the cytotoxic effect specific to Caco-2 and HCT-8 cells (23). VopL induces the assembly of actin stress fibers (26) and is potentially responsible for the internalization of V. parahaemolyticus into Caco-2 cells (1). Many other potential effector proteins are encoded proximal to T3SS1 and T3SS2 apparatus genes, but these have not been functionally characterized. The function of structural genes has not been extensively studied for either T3SS1 or T3SS2 in V. parahaemolyticus.T3SSs are expressed after contact with host cells or when cells are grown under inducing conditions (17). Expression of T3SS1 in V. parahaemolyticus is induced when bacteria are grown in tissue culture medium (Dulbecco''s minimal essential medium [DMEM]), although the secretion of one substrate (Vp1656) was not detected under this condition, probably due to the low detection sensitivity (47). T3SS1 genes are not expressed when bacteria are grown in LB medium supplemented with 2.5% NaCl (LB-S). Disruption of the exsD gene or overexpression of exsA results in the constitutive expression of T3SS1 genes and the secretion of Vp1656 even when bacteria are grown in LB-S (47). For the present study, we took advantage of these regulatory mechanisms and compared the proteins secreted by the NY-4 (wild type), ΔexsD, ΔexsD::pexsD (exsD complement), and NY-4:pexsA strains. We identified two proteins (VopS and Vp1659) that are present in the supernatants of the ΔexsD and NY-4:pexsA strains but that are absent in the supernatants of the NY-4 and ΔexsD::pexsD strains. Herein we demonstrate that Vp1659 is secreted into the extracellular milieu and is translocated into HeLa cells by T3SS1. Functional analysis is consistent with the hypothesis that Vp1659 plays a role in actin rearrangement and induction of cytotoxicity and autophagy.