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.     

The role of type III secretion System 2 in Vibrio parahaemolyticus pathogenicity

Vibrio parahaemolyticus, a Gram-negative marine bacterial pathogen, is emerging as a major cause of food-borne illnesses worldwide due to the consumption of raw seafood leading to diseases including gastroenteritis, wound infection, and septicemia. The bacteria utilize toxins and type III secretion system (T3SS) to trigger virulence. T3SS is a multi-subunit needle-like apparatus used to deliver bacterial proteins, termed effectors, into the host cytoplasm which then target various eukaryotic signaling pathways. V. parahaemolyticus carries two T3SSs in each of its two chromosomes, named T3SS1 and T3SS2, both of which play crucial yet distinct roles during infection: T3SS1 causes cytotoxicity whereas T3SS2 is mainly associated with enterotoxicity. Each T3SS secretes a unique set of effectors that contribute to virulence by acting on different host targets and serving different functions. Emerging studies on T3SS2 of V. parahaemolyticus, reveal its regulation, translocation, discovery, characterization of its effectors, and development of animal models to understand the enterotoxicity. This review on recent findings for T3SS2 of V. parahaemolyticus highlights a novel mechanism of invasion that appears to be conserved by other marine bacteria.     

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 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.     

Heme-binding Protein HRG-1 Is Induced by Insulin-like Growth Factor I and Associates with the Vacuolar H+-ATPase to Control Endosomal pH and Receptor Trafficking

Endocytosis and trafficking of receptors and nutrient transporters are dependent on an acidic intra-endosomal pH that is maintained by the vacuolar H⁺-ATPase (V-ATPase) proton pump. V-ATPase activity has also been associated with cancer invasiveness. Here, we report on a new V-ATPase-associated protein, which we identified in insulin-like growth factor I (IGF-I) receptor-transformed cells, and which was separately identified in Caenorhabditis elegans as HRG-1, a member of a family of heme-regulated genes. We found that HRG-1 is present in endosomes but not in lysosomes, and it is trafficked to the plasma membrane upon nutrient withdrawal in mammalian cells. Suppression of HRG-1 with small interfering RNA causes impaired endocytosis of transferrin receptor, decreased cell motility, and decreased viability of HeLa cells. HRG-1 interacts with the c subunit of the V-ATPase and enhances V-ATPase activity in isolated yeast vacuoles. Endosomal acidity and V-ATPase assembly are decreased in cells with suppressed HRG-1, whereas transferrin receptor endocytosis is enhanced in cells that overexpress HRG-1. Cellular uptake of a fluorescent heme analogue is enhanced by HRG-1 in a V-ATPase-dependent manner. Our findings indicate that HRG-1 regulates V-ATPase activity, which is essential for endosomal acidification, heme binding, and receptor trafficking in mammalian cells. Thus, HRG-1 may facilitate tumor growth and cancer progression.     

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.     

The Contribution of Candida albicans Vacuolar ATPase Subunit V1B,Encoded by VMA2, to Stress Response,Autophagy, and Virulence Is Independent of Environmental pH

Candida albicans vacuoles are central to many critical biological processes, including filamentation and in vivo virulence. The V-ATPase proton pump is a multisubunit complex responsible for organellar acidification and is essential for vacuolar biogenesis and function. To study the function of the V₁B subunit of C. albicans V-ATPase, we constructed a tetracycline-regulatable VMA2 mutant, tetR-VMA2. Inhibition of VMA2 expression resulted in the inability to grow at alkaline pH and altered resistance to calcium, cold temperature, antifungal drugs, and growth on nonfermentable carbon sources. Furthermore, V-ATPase was unable to fully assemble at the vacuolar membrane and was impaired in proton transport and ATPase-specific activity. VMA2 repression led to vacuolar alkalinization in addition to abnormal vacuolar morphology and biogenesis. Key virulence-related traits, including filamentation and secretion of degradative enzymes, were markedly inhibited. These results are consistent with previous studies of C. albicans V-ATPase; however, differential contributions of the V-ATPase V_o and V₁ subunits to filamentation and secretion are observed. We also make the novel observation that inhibition of C. albicans V-ATPase results in increased susceptibility to osmotic stress. Notably, V-ATPase inhibition under conditions of nitrogen starvation results in defects in autophagy. Lastly, we show the first evidence that V-ATPase contributes to virulence in an acidic in vivo system by demonstrating that the tetR-VMA2 mutant is avirulent in a Caenorhabditis elegans infection model. This study illustrates the fundamental requirement of V-ATPase for numerous key virulence-related traits in C. albicans and demonstrates that the contribution of V-ATPase to virulence is independent of host pH.     

Characterization of a Red Beet Protein Homologous to the Essential 36-Kilodalton Subunit of the Yeast V-Type ATPase

V-type proton-translocating ATPases (V-ATPases) (EC 3.6.1.3) are electrogenic proton pumps involved in acidification of endomembrane compartments in all eukaryotic cells. V-ATPases from various species consist of 8 to 12 polypeptide subunits arranged into an integral membrane proton pore sector (V₀) and a peripherally associated catalytic sector (V₁). Several V-ATPase subunits are functionally and structurally conserved among all species examined. In yeast, a 36-kD peripheral subunit encoded by the yeast (Saccharomyces cerevisiae) VMA6 gene (Vma6p) is required for stable assembly of the V₀ sector as well as for V₁ attachment. Vma6p has been characterized as a nonintegrally associated V₀ subunit. A high degree of sequence similarity among Vma6p homologs from animal and fungal species suggests that this subunit has a conserved role in V-ATPase function. We have characterized a novel Vma6p homolog from red beet (Beta vulgaris) tonoplast membranes. A 44-kD polypeptide cofractionated with V-ATPase upon gel-filtration chromatography of detergent-solubilized tonoplast membranes and was specifically cross-reactive with anti-Vma6p polyclonal antibodies. The 44-kD polypeptide was dissociated from isolated tonoplast preparations by mild chaotropic agents and thus appeared to be nonintegrally associated with the membrane. The putative 44-kD homolog appears to be structurally similar to yeast Vma6p and occupies a similar position within the holoenzyme complex.     

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.     

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.     

The position of lysosomes within the cell determines their luminal pH

We examined the luminal pH of individual lysosomes using quantitative ratiometric fluorescence microscopy and report an unappreciated heterogeneity: peripheral lysosomes are less acidic than juxtanuclear ones despite their comparable buffering capacity. An increased passive (leak) permeability to protons, together with reduced vacuolar H⁺–adenosine triphosphatase (V-ATPase) activity, accounts for the reduced acidifying ability of peripheral lysosomes. The altered composition of peripheral lysosomes is due, at least in part, to more limited access to material exported by the biosynthetic pathway. The balance between Rab7 and Arl8b determines the subcellular localization of lysosomes; more peripheral lysosomes have reduced Rab7 density. This in turn results in decreased recruitment of Rab-interacting lysosomal protein (RILP), an effector that regulates the recruitment and stability of the V1G1 component of the lysosomal V-ATPase. Deliberate margination of lysosomes is associated with reduced acidification and impaired proteolytic activity. The heterogeneity in lysosomal pH may be an indication of a broader functional versatility.     

Silencing of Atp6v1c1 Prevents Breast Cancer Growth and Bone Metastasis

Previous studies have shown that Atp6v1c1, a regulator of the assembly of the V0 and V1 domains of the V-ATPase complex, is up-regulated in metastatic oral tumors. Despite these studies, the function of Atp6v1c1 in tumor growth and metastasis is still unknown. Atp6v1c1''s expression in metastatic oral squamous cell carcinoma indicates that Atp6v1c1 has an important function in cancer growth and metastasis. We hypothesized that elevated expression of Atp6v1c1 is essential to cancer growth and metastasis and that Atp6v1c1 promotes cancer growth and metastasis through activation of V-ATPase activity. To test this hypothesis, a Lentivirus-mediated RNAi knockdown approach was used to study the function of Atp6v1c1 in mouse 4T1 mammary tumor cell proliferation and migration in vitro and cancer growth and metastasis in vivo. Our data revealed that silencing of Atp6v1c1 in 4T1 cancer cells inhibited lysosomal acidification and severely impaired 4T1 cell growth, migration, and invasion through Matrigel in vitro. We also show that Atp6v1c1 knockdown with Lenti-c1s3, a lentivirus targeting Atp6v1c1 for shRNA mediated knockdown, can significantly inhibit 4T1 xenograft tumor growth, metastasis, and osteolytic lesions in vivo. Our study demonstrates that Atp6v1c1 may promote breast cancer growth and bone metastasis through regulation of lysosomal V-ATPase activity, indicating that Atp6v1c1 may be a viable target for breast cancer therapy and silencing of Atp6v1c1 may be an innovative therapeutic approach for the treatment and prevention of breast cancer growth and metastasis.     

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.     

Exotoxin S secreted by internalized Pseudomonas aeruginosa delays lytic host cell death

The Pseudomonas aeruginosa toxin ExoS, secreted by the type III secretion system (T3SS), supports intracellular persistence via its ADP-ribosyltransferase (ADPr) activity. For epithelial cells, this involves inhibiting vacuole acidification, promoting vacuolar escape, countering autophagy, and niche construction in the cytoplasm and within plasma membrane blebs. Paradoxically, ExoS and other P. aeruginosa T3SS effectors can also have antiphagocytic and cytotoxic activities. Here, we sought to reconcile these apparently contradictory activities of ExoS by studying the relationships between intracellular persistence and host epithelial cell death. Methods involved quantitative imaging and the use of antibiotics that vary in host cell membrane permeability to selectively kill intracellular and extracellular populations after invasion. Results showed that intracellular P. aeruginosa mutants lacking T3SS effector toxins could kill (permeabilize) cells when extracellular bacteria were eliminated. Surprisingly, wild-type strain PAO1 (encoding ExoS, ExoT and ExoY) caused cell death more slowly, the time extended from 5.2 to 9.5 h for corneal epithelial cells and from 10.2 to 13.0 h for HeLa cells. Use of specific mutants/complementation and controls for initial invasion showed that ExoS ADPr activity delayed cell death. Triggering T3SS expression only after bacteria invaded cells using rhamnose-induction in T3SS mutants rescued the ExoS-dependent intracellular phenotype, showing that injected effectors from extracellular bacteria were not required. The ADPr activity of ExoS was further found to support internalization by countering the antiphagocytic activity of both the ExoS and ExoT RhoGAP domains. Together, these results show two additional roles for ExoS ADPr activity in supporting the intracellular lifestyle of P. aeruginosa; suppression of host cell death to preserve a replicative niche and inhibition of T3SS effector antiphagocytic activities to allow invasion. These findings add to the growing body of evidence that ExoS-encoding (invasive) P. aeruginosa strains can be facultative intracellular pathogens, and that intracellularly secreted T3SS effectors contribute to pathogenesis.