Identification and characterization of a type III secretion-associated chaperone in the type III secretion system 1 of Vibrio parahaemolyticus

Vibrio parahaemolyticus causes human gastroenteritis. Genomic sequencing of this organism has revealed that it has two sets of type III secretion systems, T3SS1 and T3SS2, both of which are important for its pathogenicity. However, the mechanism of protein secretion via T3SSs is unknown. A characteristic of many effectors is that they require specific chaperones for efficient delivery via T3SSs; however, no chaperone has been experimentally identified in the T3SSs of V. parahaemolyticus . In this study, we identified candidate T3SS1-associated chaperones from genomic sequence data and examined their roles in effector secretion/translocation and binding to their cognate substrates. From these experiments, we concluded that there is a T3S-associated chaperone, VecA, for a cytotoxic T3SS1-dependent effector, VepA. Further analysis using pulldown and secretion assays characterized the chaperone-binding domain encompassing the first 30–100 amino acids and an amino terminal secretion signal encompassing the first 5–20 amino acids on VepA. These findings will provide a strategy to clarify how the T3SS1 of V. parahaemolyticus secretes its specific effectors.     

副溶血弧菌的Ⅲ型分泌系统

摘要：副溶血弧菌是一种嗜盐性革兰氏阴性短杆菌,主要引起食物中毒性肠胃炎,还可引起水生动物疾病。除了耐热直接溶血毒素和耐热直接溶血相关毒素外,近年发现的副溶血弧菌两套Ⅲ型分泌系统也与该菌的致病性密切相关。Ⅲ型分泌系统1位于染色体1上,主要贡献对宿主细胞的细胞毒性,介导宿主细胞的自体吞噬作用,最后导致细胞死亡。Ⅲ型分泌系统2位于位于染色体2的毒力岛上,具有肠毒性。本文扼要介绍副溶血弧菌Ⅲ型分泌系统的组成、功能及相关转录调控机制。     

Identification of the Vibrio parahaemolyticus type III secretion system 2-associated chaperone VocC for the T3SS2-specific effector VopC

The enteropathogen Vibrio parahaemolyticus possesses two sets of type III secretion systems, T3SS1 and T3SS2. Effector proteins secreted by these T3SSs are delivered into host cells, leading to cell death or diarrhea. However, it is not known how specific effectors are secreted through a specific T3SS when both T3SSs are expressed within bacteria. One molecule thought to determine secretion specificity is a T3SS-associated chaperone; however, no T3SS2-specific chaperone has been identified. Therefore, we screened T3SS2 chaperone candidates by a pull-down assay using T3SS2 effectors fused with glutathione-S-transferase. A secretion assay revealed that the newly identified cognate chaperone VocC for the T3SS2-specific effector VopC was required for the efficient secretion of the substrate through T3SS2. Further experiments determined the chaperone-binding domain and the amino-terminal secretion signal of the cognate effector. These findings, in addition to the previously identified T3SS1-specific chaperone, VecA, provide a strategy to clarify the specificity of effector secretion through T3SSs 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.     

Translocation of VP1686 upregulates RhoB and accelerates phagocytic activity of macrophage through actin remodeling

Here, we report that Vibrio parahaemolyticus induces a rapid remodeling of macrophage actin and activates RhoB GTPase. Mutational analysis revealed that the effects depend on type III secretion system 1 regulated translocation of a V. parahaemolyticus effector protein, VP1686, into the macrophages. Remodeling of actin is shown to be necessary for increased bacterial uptake followed by initiation of apoptosis in macrophages. This provides evidence for functional association of the VP1686 in triggering an eat-me-and-die signal to the host.     

Identification and characterization of VopT, a novel ADP-ribosyltransferase effector protein secreted via the Vibrio parahaemolyticus type III secretion system 2

Vibrio parahaemolyticus strain RIMD2210633 has two sets of genes encoding two separate type III secretion systems (T3SSs), called T3SS1 and T3SS2. T3SS2 has a role in enterotoxicity and is present only in Kanagawa phenomenon-positive strains, which are pathogenic to humans. Accordingly, T3SS2 is considered to be closely related to V. parahaemolyticus human pathogenicity. Despite this, the biological actions of T3SS2 and the identity of the effector protein(s) secreted by this system have not been well understood. Here we report that T3SS2 induces a cytotoxic effect in Caco-2 and HCT-8 cells. Moreover, it was revealed that VPA1327 (vopT), a gene encoded within the proximity of T3SS2, is partly responsible for this cytotoxic effect. The VopT shows approximately 45% and 44% identity with the ADP-ribosyltransferase (ADPRT) domain of ExoT and ExoS, respectively, which are two T3SS-secreted effectors of Pseudomonas aeruginosa. T3SS2 was found to be necessary not only for the secretion, but also for the translocation of the VopT into host cells. We also demonstrate that VopT ADP-ribosylates Ras, a member of the low-molecular-weight G (LMWG) proteins both in vivo and in vitro. These results indicate that VopT is a novel ADPRT effector secreted via V. parahaemolyticus T3SS.     

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。     

副溶血弧菌拟核相关蛋白H-NS间接抑制VP1687-1686的启动子区转录

为了探讨副溶血性弧菌拟核相关蛋白H-NS对Ⅲ型分泌系统(T3SS) VP1687-1686基因位点的转录调控,本研究提取副溶血弧菌hns突变株(Δhns)和野生株(WT)的总RNA,采用引物延伸实验研究靶基因的转录起始位点,并根据产物的丰度判断H-NS对靶基因的调控关系;采用实时定量RT-PCR研究靶基因mRNA在WT和Δhns中转录丰度,以判定H-NS对靶基因的转录调控关系;将靶基因启动子区域DNA序列克隆至lacZ基因上游,将重组质粒转入WT和Δhns中,得到相应的LacZ菌株,通过LacZ报告基因融合实验研究H-NS对靶基因的调控关系;用PCR扩增靶基因的启动子区DNA序列,并纯化His-H-NS蛋白,通过凝胶阻滞实验(EMSA)研究His-H-NS是否对靶基因启动子区具有直接的结合作用。研究结果显示,T3SS的VP1687-1686只含有一个转录起始位点,位于翻译起始位点上游82 bp处,且H-NS能够抑制其转录活性,但不能直接结合到VP1687-1686区的启动子区。另外,H-NS对calR的转录无调控作用,His-H-NS也不能结合到其启动子区。本研究的结果初步说明,H-NS能够间接抑制VP1687-1686的转录,该抑制机制与CalR无关联。     

VopV, an F-actin-binding type III secretion effector, is required for Vibrio parahaemolyticus-induced enterotoxicity

Vibrio parahaemolyticus, a Gram-negative halophilic bacterium that causes acute gastroenteritis in humans, is characterized by two type III secretion systems (T3SS), namely T3SS1 and T3SS2. T3SS2 is indispensable for enterotoxicity but the effector(s) involved are unknown. Here, we identify VopV as a critical effector that is required to mediate V. parahaemolyticus T3SS2-dependent enterotoxicity. VopV was found to possess multiple F-actin-binding domains and the enterotoxicity caused by VopV correlated with its F-actin-binding activity. Furthermore, a T3SS2-related secretion system and a vopV homologous gene were also involved in the enterotoxicity of a non-O1/non-O139 V. cholerae strain. These results indicate that the F-actin-targeting effector VopV is involved in enterotoxic activity of T3SS2-possessing bacterial pathogens.     

Identification of potential type III secretion proteins via heterologous expression of Vibrio parahaemolyticus DNA

We employed a heterologous secretion assay to identify proteins potentially secreted by type III secretion systems (T3SSs) in Vibrio parahaemolyticus. N-terminal sequences from 32 proteins within T3SS genomic islands and seven proteins from elsewhere in the chromosome included proteins that were recognized for export by the Yersinia enterocolitica flagellar T3SS.     

PipB2 is a substrate of the Salmonella pathogenicity island 1-encoded type III secretion system

Salmonella harbors two type III secretion systems, T3SS1 and T3SS2, encoded on the pathogenicity islands SPI1 and SPI2, respectively. Several effector proteins are secreted through these systems into the eukaryotic host cells. PipB2 is a T3SS2 effector that contributes to the modulation of kinesin-1 motor complex activity. Here, we show that PipB2 is also a substrate of T3SS1. This result was obtained infecting human epithelial HeLa cells for 2 h and was confirmed in murine RAW264.7 macrophages, and rat NRK fibroblasts. Analysis at different time points after infection revealed that translocation of PipB2 is T3SS1-dependent in epithelial cells throughout the infection. In contrast, translocation into macrophages is T3SS1-dependent during invasion but T3SS2-dependent at later time points. The N-terminal 10 amino acid residues contain the signal necessary for translocation through both systems. These results confirm the functional overlap between these virulence-related secretion systems and suggest a new role for the effector PipB2.     

Injection of flagellin into the host cell cytosol by Salmonella enterica serotype Typhimurium

Bacterial flagellins are potent inducers of innate immunity. Three signaling pathways have been implicated in the sensing of flagellins; these involve toll-like receptor 5 (TLR5) and the cytosolic proteins Birc1e/Naip5 and Ipaf. Although the structural basis of TLR5-flagellin interaction is known, little is known about how flagellin enters the host cell cytosol to induce signaling via Birc1e/Naip5 and Ipaf. Here we demonstrate for the first time the translocation of bacterial flagellin into the cytosol of host macrophages by the vacuolar pathogen, Salmonella enterica serotype Typhimurium. Translocation of flagellin into the host cell cytosol was directly demonstrated using beta-lactamase reporter constructs. Flagellin translocation required the Salmonella Pathogenicity Island 1 Type III secretion system (SPI-1 T3SS) but not the flagellar T3SS.     

Acquired type III secretion system determines environmental fitness of epidemic Vibrio parahaemolyticus in the interaction with bacterivorous protists

Genome analyses of marine microbial communities have revealed the widespread occurrence of genomic islands (GIs), many of which encode for protein secretion machineries described in the context of bacteria-eukaryote interactions. Yet experimental support for the specific roles of such GIs in aquatic community interactions remains scarce. Here, we test for the contribution of type III secretion systems (T3SS) to the environmental fitness of epidemic Vibrio parahaemolyticus. Comparisons of V. parahaemolyticus wild types and T3SS-defective mutants demonstrate that the T3SS encoded on genome island VPaI-7 (T3SS-2) promotes survival of V. parahaemolyticus in the interaction with diverse protist taxa. Enhanced persistence was found to be due to T3SS-2 mediated cytotoxicity and facultative parasitism of V. parahaemolyticus on coexisting protists. Growth in the presence of bacterivorous protists and the T3SS-2 genotype showed a strong correlation across environmental and clinical isolates of V. parahaemolyticus. Short-term microcosm experiments provide evidence that protistan hosts facilitate the invasion of T3SS-2 positive V. parahaemolyticus into a coastal plankton community, and that water temperature and productivity further promote enhanced survival of T3SS-2 positive V. parahaemolyticus. This study is the first to describe the fitness advantage of GI-encoded functions in a microbial food web, which may provide a mechanistic explanation for the global spread and the seasonal dynamics of V. parahaemolyticus pathotypes, including the pandemic serotype cluster O3:K6, in aquatic environments.