Regulation of Vibrio parahaemolyticus T3SS2 gene expression and function of T3SS2 effectors that modulate actin cytoskeleton

Vibrio parahaemolyticus is a leading causative agent of seafood‐borne gastroenteritis worldwide. Most clinical isolates from patients with diarrhoea possess two sets of genes for the type III secretion system (T3SS) on each chromosome (T3SS1 and T3SS2). T3SS is a protein secretion system that delivers effector proteins directly into eukaryotic cells. The injected effectors modify the normal cell functions by altering or disrupting the normal cell signalling pathways. Of the two sets of T3SS genes present in V. parahaemolyticus, T3SS2 is essential for enterotoxicity in several animal models. Recent studies have elucidated the biological activities of several T3SS2 effectors and their roles in virulence. This review focuses on the regulation of T3SS2 gene expression and T3SS2 effectors that specifically target the actin cytoskeleton.     

Advances on Vibrio parahaemolyticus research in the postgenomic era

Vibrio parahaemolyticus is a leading cause of seafood-borne bacterial gastroenteritis in humans. Since its discovery in 1950, this bacterium has been isolated in widespread outbreaks and in sporadic cases of gastroenteritis worldwide. Although the exotoxin, thermostable direct hemolysin, had been the focus of extensive research on the pathogenicity of V. parahaemolyticus, the whole-genome sequencing of a clinical isolate, RIMD2210633 strain, was a breakthrough in this field. The possession of two sets of gene clusters for type III secretion systems (T3SS1 and T3SS2) was unveiled by that genome project. T3SS is a protein export apparatus that delivers bacterial proteins, called effectors, directly into the host's cytosol, to disrupt host cell function. The subsequent studies have established that T3SS2, which is encoded in an 80 kb pathogenicity island called V. parahaemolyticus pathogenicity island (Vp-PAI), is closely related to enteropathogenicity. Recent functional analyses of Vp-PAI-encoded genes revealed the sophisticated mechanisms in V. parahaemolyticus for sensing the intestinal environment and host cell contact, and a dozen T3SS2-exported proteins encoded in Vp-PAI. In this review, we summarize recent advances in V. parahaemolyticus research regarding the control of the expression of Vp-PAI-encoded genes, structural components and the secretory regulation of T3SS2, and the biological activities of T3SS2-exported effectors. Thus, Vp-PAI-encoded T3SS2 becomes an important key in the postgenomic era to shed light on the enteropathogenic mechanism of V. parahaemolyticus.     

Type III effector VopC mediates invasion for Vibrio species

Vibrio spp. are associated with infections caused by contaminated food and water. A type III secretion system (T3SS2) is a shared feature of all clinical isolates of V. parahaemolyticus and some V. cholerae strains. Despite its being responsible for enterotoxicity, no molecular mechanism has been determined for the T3SS2-dependent pathogenicity. Here, we show that although Vibrio spp. are typically thought of as extracellular pathogens, the T3SS2 of Vibrio mediates host cell invasion, vacuole formation, and replication of intracellular bacteria. The catalytically active effector VopC is critical for Vibrio T3SS2-mediated invasion. There are other marine bacteria encoding VopC homologs associated with a T3SS; therefore, we predict that these bacteria are also likely to use T3SS-mediated invasion as part of their pathogenesis mechanisms. These findings suggest a new molecular paradigm for Vibrio pathogenicity and modify our view of the roles of T3SS effectors that are translocated during infection.     

Generation and Characterization of a scFv Antibody Against T3SS Needle of Vibrio parahaemolyticus

Vibrio parahaemolyticus, a halophilic gram-negative bacterium, is a food-borne pathogen that largely inhabits marine and estuarine environments, and poses a serious threat to human and animal health all over the world. The hollow “needle” channel, a specific assemble of T3SS which exists in most of gram-negative bacteria, plays a key role in the transition of virulence effectors to host cells. In this study, needle protein VP1694 was successfully expressed and purified, and the fusion protein Trx-VP1694 was used to immunize Balb/c mice. Subsequently, a phage single-chain fragment variable antibody (scFv) library was constructed, and a specific scFv against VP1694 named scFv-FA7 was screened by phage display panning. To further identify the characters of scFv, the soluble expression vector pACYC-scFv-skp was constructed and the soluble scFv was purified by Ni²⁺ affinity chromatography. ELISA analysis showed that the scFv-FA7 was specific to VP1694 antigen, and its affinity constant was 1.07 × 10⁸ L/mol. These results offer a molecular basis to prevent and cure diseases by scFv, and also provide a new strategy for further research on virulence mechanism of T3SS in V. parahaemolyticus by scFv.     

Type VI Secretion System Toxins Horizontally Shared between Marine Bacteria

The type VI secretion system (T6SS) is a widespread protein secretion apparatus used by Gram-negative bacteria to deliver toxic effector proteins into adjacent bacterial or host cells. Here, we uncovered a role in interbacterial competition for the two T6SSs encoded by the marine pathogen Vibrio alginolyticus. Using comparative proteomics and genetics, we identified their effector repertoires. In addition to the previously described effector V12G01_02265, we identified three new effectors secreted by T6SS1, indicating that the T6SS1 secretes at least four antibacterial effectors, of which three are members of the MIX-effector class. We also showed that the T6SS2 secretes at least three antibacterial effectors. Our findings revealed that many MIX-effectors belonging to clan V are “orphan” effectors that neighbor mobile elements and are shared between marine bacteria via horizontal gene transfer. We demonstrated that a MIX V-effector from V. alginolyticus is a functional T6SS effector when ectopically expressed in another Vibrio species. We propose that mobile MIX V-effectors serve as an environmental reservoir of T6SS effectors that are shared and used to diversify antibacterial toxin repertoires in marine bacteria, resulting in enhanced competitive fitness.     

副溶血弧菌Ⅲ型分泌系统（T3SS）效应蛋白及其对宿主细胞的操控

副溶血弧菌是典型的食源性病原菌,也是全球范围内引起肠胃炎的主要病原菌。针筒状的Ⅲ型分泌系统(T3SS)为该菌主要的毒力因子,细菌感染时可将其效应蛋白直接注射至宿主细胞中,通过效应蛋白操纵宿主细胞,介导毒力的发挥。多数临床分离的副溶血弧菌含有2套T3SSs,其中T3SS1分泌的效应蛋白主要通过诱导细胞自噬、变圆和裂解等过程来发挥其细胞毒性,而T3SS2分泌的效应蛋白则主要通过破坏细胞骨架和操控细胞信号传导来发挥肠毒性。本文主要对副溶血弧菌T3SSs的组成和目前已发现的效应蛋白及其对宿主细胞的操控进行介绍。该研究不仅对深入了解该菌的致病机制有重要意义,而且也为宿主细胞信号转导机制研究提供新视角。     

副溶血弧菌的毒力基因表达调控的分子机制

副溶血弧菌是革兰氏阴性嗜盐细菌,是海洋脊椎动物和无脊椎动物中主要致病菌,也是引起人类急性肠胃炎、败血症和坏死性筋膜炎等疾病的主要病原体。在过去,由副溶血弧菌引起的致病感染在世界范围内有不断增加的趋势。副溶血弧菌的致病性与其自身产生的多种毒力因子有关,这些毒力因子包括粘附因子、脂多糖、溶血素、III型分泌系统、VI型分泌系统、铁摄取系统、蛋白酶、外膜蛋白等。然而,这些毒力因子的表达都受到环境因子以及宿主体内信号因子的调控。副溶血弧菌通过感知外界生存环境的各种信号因子,从而激活体内不同的信号通路,进而诱导不同的毒力因子的表达。本文主要对副溶血弧菌毒力因子表达调控的分子机制进行综述,为更好地理解宿主与病原体的相互作用对副溶血弧菌的致病机制的影响,以及为今后预防和治疗由副溶血弧菌所引起的疾病提供理论参考。     

Binding to type I collagen is essential for the infectivity of Vibrio parahaemolyticus to host cells

Vibrio parahaemolyticus is a globally present marine bacterium that often leads to acute gastroenteritis. Two type III secretion systems (T3SSs), T3SS1 and T3SS2, are important for host infection. Type I collagen is a component of the extracellular matrix and is abundant in the small intestine. However, whether type I collagen serves as the cellular receptor for V. parahaemolyticus infection of host cells remains enigmatic. In this study, we discovered that type I collagen is not only important for the attachment of V. parahaemolyticus to host cells but is also involved in T3SS1‐dependent cytotoxicity. In addition, 2 virulence factors, MAM7 and VpadF enable V. parahaemolyticus to interact with type I collagen and mediate T3SS2‐dependent host cell invasion. Type I collagen, the collagen receptor α1 integrin, and its downstream factor phosphatidylinositol 3‐kinase (PI3K) are responsible for V. parahaemolyticus invasion of host cells. Further biochemical studies revealed that VpadF mainly relies on the C‐terminal region for type I collagen binding and MAM7 relies on mce domains to bind to type I collagen. As MAM7 and/or VpadF homologues are widely distributed in the genus Vibrio, we propose that Vibrios have evolved a unique strategy to infect host cells by binding to type I collagen.     

Interaction between the Type III Effector VopO and GEF-H1 Activates the RhoA-ROCK Pathway

Vibrio parahaemolyticus is an important pathogen that causes food-borne gastroenteritis in humans. The type III secretion system encoded on chromosome 2 (T3SS2) plays a critical role in the enterotoxic activity of V. parahaemolyticus. Previous studies have demonstrated that T3SS2 induces actin stress fibers in various epithelial cell lines during infection. This stress fiber formation is strongly related to pathogenicity, but the mechanisms that underlie T3SS2-dependent actin stress fiber formation and the main effector have not been elucidated. In this study, we identified VopO as a critical T3SS2 effector protein that activates the RhoA-ROCK pathway, which is an essential pathway for the induction of the T3SS2-dependent stress fiber formation. We also determined that GEF-H1, a RhoA guanine nucleotide exchange factor (GEF), directly binds VopO and is necessary for T3SS2-dependent stress fiber formation. The GEF-H1-binding activity of VopO via an alpha helix region correlated well with its stress fiber-inducing capacity. Furthermore, we showed that VopO is involved in the T3SS2-dependent disruption of the epithelial barrier. Thus, VopO hijacks the RhoA-ROCK pathway in a different manner compared with previously reported bacterial toxins and effectors that modulate the Rho GTPase signaling pathway.     

Vibrio parahaemolyticus Effector Proteins Suppress Inflammasome Activation by Interfering with Host Autophagy Signaling

Bacterial pathogens utilize pore-forming toxins or sophisticated secretion systems to establish infection in hosts. Recognition of these toxins or secretion system by nucleotide-binding oligomerization domain leucine-rich repeat proteins (NLRs) triggers the assembly of inflammasomes, the multiprotein complexes necessary for caspase-1 activation and the maturation of inflammatory cytokines such as IL-1β or IL-18. Here we demonstrate that both the NLRP3 and NLRC4 inflammasomes are activated by thermostable direct hemolysins (TDHs) and type III secretion system 1 (T3SS1) in response to V. parahaemolyticus infection. Furthermore, we identify T3SS1 secreted effector proteins, VopQ and VopS, which induce autophagy and the inactivation of Cdc42, respectively, to prevent mainly NLRC4 inflammasome activation. VopQ and VopS interfere with the assembly of specks in infected macrophages. These data suggest that bacterial effectors interfere with inflammasome activation and contribute to bacterial evasion from the host inflammatory responses.     

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.     

Vibrio Type III Effector VPA1380 Is Related to the Cysteine Protease Domain of Large Bacterial Toxins

Vibrio parahaemolyticus is a Gram-negative halophilic bacterium and one of the leading causes of food-borne gastroenteritis. Its genome harbors two Type III Secretion Systems (T3SS1 and T3SS2), but only T3SS2 is required for enterotoxicity seen in animal models. Effector proteins secreted from T3SS2 have been previously shown to promote colonization of the intestinal epithelium, invasion of host cells, and destruction of the epithelial monolayer. In this study, we identify VPA1380, a T3SS2 effector protein that is toxic when expressed in yeast. Bioinformatic analyses revealed that VPA1380 is highly similar to the inositol hexakisphosphate (IP6)-inducible cysteine protease domains of several large bacterial toxins. Mutations in conserved catalytic residues and residues in the putative IP6-binding pocket abolished toxicity in yeast. Furthermore, VPA1380 was not toxic in IP6 deficient yeast cells. Therefore, our findings suggest that VPA1380 is a cysteine protease that requires IP6 as an activator.     

Presence of genes for type III secretion system 2 in <Emphasis Type="Italic">Vibrio</Emphasis><Emphasis Type="Italic">mimicus</Emphasis> strains

Background  

Vibrios, which include more than 100 species, are ubiquitous in marine and estuarine environments, and several of them e.g. Vibrio cholerae, V. parahaemolyticus, V. vulnificus and V. mimicus, are pathogens for humans. Pathogenic V. parahaemolyticus strains possess two sets of genes for type III secretion system (T3SS), T3SS1 and T3SS2. The latter are critical for virulence of the organism and be classified into two distinct phylogroups, T3SS2α and T3SS2β, which are reportedly also found in pathogenic V. cholerae non-O1/non-O139 serogroup strains. However, whether T3SS2-related genes are present in other Vibrio species remains unclear.     

The Serine Protease EspC from Enteropathogenic Escherichia coli Regulates Pore Formation and Cytotoxicity Mediated by the Type III Secretion System

Type III secretion systems (T3SSs) are specialized macromolecular machines critical for bacterial virulence, and allowing the injection of bacterial effectors into host cells. The T3SS-dependent injection process requires the prior insertion of a protein complex, the translocon, into host cell membranes consisting of two-T3SS hydrophobic proteins, associated with pore-forming activity. In all described T3SS to date, a hydrophilic protein connects one hydrophobic component to the T3SS needle, presumably insuring the continuum between the hollow needle and the translocon. In the case of Enteropathogenic Escherichia coli (EPEC), the hydrophilic component EspA polymerizes into a filament connecting the T3SS needle to the translocon composed of the EspB and EspD hydrophobic proteins. Here, we identify EspA and EspD as targets of EspC, a serine protease autotransporter of Enterobacteriaceae (SPATE). We found that in vitro, EspC preferentially targets EspA associated with EspD, but was less efficient at proteolyzing EspA alone. Consistently, we found that EspC did not regulate EspA filaments at the surface of primed bacteria that was devoid of EspD, but controlled the levels of EspD and EspA secreted in vitro or upon cell contact. While still proficient for T3SS-mediated injection of bacterial effectors and cytoskeletal reorganization, an espC mutant showed increased levels of cell-associated EspA and EspD, as well as increased pore formation activity associated with cytotoxicity. EspP from enterohaemorrhagic E. coli (EHEC) also targeted translocator components and its activity was interchangeable with that of EspC, suggesting a common and important function of these SPATEs. These findings reveal a novel regulatory mechanism of T3SS-mediated pore formation and cytotoxicity control during EPEC/EHEC infection.     

Salmonella effectors: important players modulating host cell function during infection

Salmonella enterica serovar Typhimurium (S. Typhimurium) is a Gram-negative facultative food-borne pathogen that causes gastroenteritis in humans. This bacterium has evolved a sophisticated machinery to alter host cell function critical to its virulence capabilities. Central to S. Typhimurium pathogenesis are two Type III secretion systems (T3SS) encoded within pathogenicity islands SPI-1 and SPI-2 that are responsible for the secretion and translocation of a set of bacterial proteins termed effectors into host cells with the intention of altering host cell physiology for bacterial entry and survival. Thus, once delivered by the T3SS, the secreted effectors play critical roles in manipulating the host cell to allow for bacteria invasion, induction of inflammatory responses, and the assembly of an intracellular protective niche created for bacterial survival and replication. Emerging evidence indicates that these effectors are modular proteins consisting of distinct functional domains/motifs that are utilized by the bacteria to activate intracellular signalling pathways modifying host cell function. Also, recently reported are the dual functionality of secreted effectors and the concept of 'terminal reassortment'. Herein, we highlight some of the nascent concepts regarding Salmonella effectors in the context of infection.     

The Vibrio parahaemolyticus effector VopC mediates Cdc42‐dependent invasion of cultured cells but is not required for pathogenicity in an animal model of infection

Vibrio parahaemolyticus is a Gram‐negative marine bacterium that causes acute gastroenteritis in humans. The virulence of V. parahaemolyticus is dependent upon a type III secretion system (T3SS2). One effector for T3SS2, VopC, is a homologue of the catalytic domain of cytotoxic necrotizing factor (CNF), and was recently reported to be a Rho family GTPase activator and to be linked to internalization of V. parahaemolyticus by non‐phagocytic cultured cells. Here, we provide direct evidence that VopC deamidates Rac1 and CDC42, but not RhoA, in vivo. Our results alsosuggest that VopC, through its activation of Rac1, contributes to formation of actin stress fibres in infected cells. Invasion of host cells, which occurs at a low frequency, does not seem linked to Rac1 activation, but instead appears to require CDC42. Finally, using an infant rabbit model of V. parahaemolyticus infection, we show that the virulence of V. parahaemolyticus is not dependent upon VopC‐mediated invasion. Genetic inactivation of VopC did not impair intestinal colonization nor reduce signs of disease, including fluid accumulation, diarrhoea and tissue destruction. Thus, although VopC can promote host cell invasion, such internalization is not a critical step of the disease process, consistent with the traditional view of V. parahaemolyticus as an extracellular pathogen.