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Identification of Proteins Secreted via Vibrio parahaemolyticus Type III Secretion System 1
     Department of Bacterial Infections, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan

    ABSTRACT

    Vibrio parahaemolyticus, a gram-negative marine bacterium, is an important pathogen causing food-borne gastroenteritis or septicemia. Recent genome sequencing of the RIMD2210633 strain (a Kanagawa phenomenon-positive clinical isolate of serotype O3:K6) revealed that the strain has two sets of gene clusters that encode the type III secretion system (TTSS) apparatus. The first cluster, TTSS1, is located on the large chromosome, and the second, TTSS2, is on the small chromosome. Previously, we reported that TTSS1 is involved in the cytotoxicity of the RIMD2210633 strain against HeLa cells. Here, we analyzed proteins secreted via the TTSS apparatus encoded by TTSS1 by using two-dimensional gel electrophoresis and identified the proteins encoded by genes VP1680, VP1686, and VPA450. To investigate the roles of those secreted proteins, we constructed and analyzed a series of deletion mutants. Flow cytometry analysis using fluorescence-activated cell sorting with fluorescein isothiocyanate-labeled annexin V demonstrated that the TTSS1-dependent cell death was by apoptosis. The cytotoxicity to HeLa cells was related to one of the newly identified secreted proteins encoded by VP1680. Adenylate cyclase fusion protein studies proved that the newly identified secreted proteins were translocated into HeLa cells. Thus, these appear to be the TTSS effector proteins in V. parahaemolyticus.

    INTRODUCTION

    Vibrio parahaemolyticus is a halophilic gram-negative bacterium that causes acute gastroenteritis in humans infected through the consumption of raw or inadequately cooked seafood. Clinical manifestations of V. parahaemolyticus infections include diarrhea, abdominal cramps, nausea, vomiting, headaches, fever, chills, and even death (7, 11, 13, 24). Almost all clinical isolates of V. parahaemolyticus show beta-hemolysis in a special blood agar called Wagatsuma agar, and this hemolytic activity is termed the Kanagawa phenomenon (KP). The KP is considered a good marker to differentiate human pathogenic V. parahaemolyticus strains from the nonpathogenic ones (22, 38).

    Recently, the genome sequencing of a KP-positive V. parahaemolyticus strain, RIMD2210633, has been completed (20). One of the most interesting findings was that the strain has two gene clusters, TTSS1 and TTSS2, each encoding distinct type III secretion systems (TTSSs) (14). That was the first report of a TTSS in any Vibrio species. Neither V. cholerae nor V. vulnificus have a TTSS, according to their published genome information (4, 12).

    TTSSs are found in many gram-negative pathogenic bacteria, such as Yersinia spp., Pseudomonas aeruginosa, Salmonella spp., Shigella spp., and enteropathogenic and enterohemorrhagic Escherichia coli. These systems play a key role in infection (14). Such pathogens secrete and translocate functional proteins, referred to as effectors, into the plasma membranes or the cytoplasm of eukaryotic cells via the TTSS. The TTSS apparatus is a needle-like complex composed of about 20 proteins, and its components are highly conserved among the bacteria (2, 6, 17, 40, 42).

    Of the two TTSS-encoding gene clusters of V. parahaemolyticus strain RIMD2210633, TTSS1 (Fig. 1) is particularly similar to those of Yersinia spp. and P. aeruginosa in the number of genes, in their order, and in the identity of each encoded protein (20, 33). TTSS2 is found only in the KP-positive strains (20). Cornelis and Van Gijsegem reported that the TTSSs of animal pathogens can be divided into three major groups: the Ysc-plus-Psc system, the Salmonella pathogenicity island 1-plus-Mxi/Spa system, and the Salmonella pathogenicity island 1-plus-enteropathogenic and -enterohemorrhagic E. coli system (5). The V. parahaemolyticus TTSS1 is of the Ysc-plus-Psc system. However, TTSS2 is not similar to any particular TTSS gene clusters from other bacteria reported to date (20).

    Many studies on the cytotoxicity to eukaryotic cells of V. parahaemolyticus have been reported to date. Although most of these were related to thermostable direct hemolysin (TDH) or TDH-related hemolysin (25, 34, 43), this bacterium demonstrates cytotoxic activity even in the absence of these hemolysins (1, 19, 32). In a previous study, mutant strains of RIMD2210633 were constructed with deletions in the genes for the TTSS components and the cytotoxicity of the mutant strains to HeLa cells was analyzed (33). Although the cells infected by the parent and TTSS2 mutant strains showed round morphology, including a decrease in cytoplasm and shrunken nuclei, the cells infected by the TTSS1 mutants showed little change in morphology, as did the negative control, uninfected cells. Similar results were obtained using a lactate dehydrogenase (LDH) release assay. These findings revealed that TTSS1 was involved in the cytotoxicity of the RIMD2210633 strain to HeLa cells (33). An additional study using HCT116 cells revealed that cell death caused by TTSS1 was apoptosis (1). On the other hand, TTSS2 was involved in the enterotoxic activity of the organism in a rabbit ileal loop test (33).

    A number of TTSS effector proteins have been reported (5). Although V. parahaemolyticus TTSS1 has an almost full set of genes that are homologous to the Yersinia TTSS components, we could not identify any genes with significant homology to known genes for effector proteins of other bacteria in the proximity of the TTSS1 region at the point of completion of genome sequencing.

    In this study, we investigated the proteins secreted via the TTSS apparatus encoded by TTSS1 by using two-dimensional (2D) gel electrophoresis and identified four TTSS-secreted proteins. We found that one of these secreted proteins, encoded by VP1680, plays a key role in cytotoxicity to eukaryotic cells.

    MATERIALS AND METHODS

    Bacterial strains, plasmids, and culture conditions. All V. parahaemolyticus strains used in this study were obtained from the Laboratory for Culture Collection, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan. V. parahaemolyticus strain RIMD2210633 (KP positive; serotype O3:K6) was used as a parent strain for construction of deletion mutants and for analysis in functional studies. E. coli DH5 and SM10pir strains were used for the general manipulation of plasmids and the mobilization of plasmids into V. parahaemolyticus for construction of mutants, respectively. V. parahaemolyticus strains were usually grown at 37°C with shaking in Luria-Bertani (LB) medium supplemented with 3% NaCl (final concentration). To collect secretion proteins encoded by TTSS1, the strains were grown at 37°C without shaking in heart infusion broth (HIB) supplemented with 1% NaCl (final concentration), 10 mM sodium oxalate, and 10 mM magnesium chloride (HIBox). We used these conditions because a higher level of secretion was observed for the TTSS1 apparatus-secreted proteins under these conditions than under conditions with additive-free HIB. E. coli strains were cultured at 37°C with shaking in LB medium. Antibiotics were used at the following concentrations: ampicillin, 100 μg ml–1; kanamycin, 50 μg ml–1; and chloramphenicol, 20 μg ml–1. Strains and constructed plasmids used in this study are listed in Table 1.

    Construction of deletion mutants. The mutant POR-1 strain with two TDH genes deleted and a vcrD1 mutant were constructed as described previously (32). Other deletion mutants were constructed in a manner similar to that used for the POR-1 and vcrD1 mutants. Briefly, DNA regions upstream and downstream of the desired gene were amplified by PCR using the primers listed in Table 2. Primers 2 and 3 were introduced into a 15-bp overlapped site to connect the 3' end of the upstream fragment with the 5' end of the downstream fragment. A second round of PCR was carried out using these two fragments as templates with primers 1 and 4. The amplified fragment was cloned into the pT7blue T vector (Novagen, Madison, Wis.). The insert was excised by restriction digestion with BamHI and PstI and subcloned into the vector pYAK1, a pir-dependent suicide vector carrying sacB and a chloramphenicol resistance gene. The constructed plasmid was introduced into E. coli strain SM10pir and transferred into V. parahaemolyticus by conjugation.

    Complementation of deleted genes in the mutants. Complementation of deleted genes was carried out as follows. DNA regions including each gene and its promoter were amplified by PCR using the primers listed in Table 2. If we suspected that the gene pairs might form an operon, the whole DNA region between the promoter and the deleted gene was included. Each amplified fragment was cloned into the pT7blue T vector, and the sequence was ascertained. The insert was excised by restriction digestion with BamHI and cloned into the vector pSA19Cm-MCS, a vector constructed from V. parahaemolyticus plasmid pSA19 with a multicloning site derived from pUC119. Each plasmid was introduced into V. parahaemolyticus deletion mutants by electroporation (1.5 kV, 100 , 25 μF).

    2D gel electrophoresis. To collect the secreted proteins, POR-1 or vcrD1 deletion mutants were cultured with HIBox without shaking for 9 h at 37°C and 200-ml bacterial cultures were centrifuged at 16,000 x g for 5 min at 4°C. Supernatants obtained after centrifugation were passed through a 0.2-μm-pore-size membrane filter. Proteins were precipitated by adding trichloroacetic acid to a final concentration of 10% (vol/vol) and incubated on ice for 2 h. The proteins were collected by centrifugation at 17,500 x g for 30 min at 4°C. Pellets were washed twice in 10 ml of ice-cold acetone, dried, and dissolved in 200 μl of rehydration solution containing 8 M urea, 2% (wt/vol) CHAPS {3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate}, 18 mM dithiothreitol, 0.5% (vol/vol) IPG buffer (pH range, 4 to 7; Amersham Biosciences, Piscataway, N.J.), and a trace of bromophenol blue. Protein extracts were either used immediately for 2D gel electrophoresis or stored at –30°C.

    Isoelectric focusing was performed as the first dimension with the IPGphor system and Immobiline Drystrip gel strips (Amersham Biosciences). Aliquots of 200 μl of sample proteins dissolved in the rehydration solution were applied to the strips (pH 4 to 7, 11 cm) and rehydrated for 15 h at 20°C. After rehydration, the proteins were focused for a total of 36 kV · h at 20°C (100 V for 2 h; 500 V for 1 h; 1,000 V for 1 h; 2,000 V for 1 h; 4,000 V for 1 h; 6,000 V for 2 h; and 8,000 V for 2 h). After the first dimension, the strips were soaked in sodium dodecyl sulfate (SDS) equilibration buffer containing 50 mM Tris-HCl (pH 6.8), 6 M urea, 30% glycerol, 2% (wt/vol) SDS, and 1% (wt/vol) dithiothreitol for 15 min. The strips were transferred onto 10% acrylamide gels and subjected to SDS-polyacrylamide gel electrophoresis as the second dimension. The gels were either stained or prepared for membrane transfer. Gel staining was performed with Coomassie brilliant blue G-250 as described by Neuhoff et al. (27). Transfer onto polyvinylidene membranes was carried out electronically using Trans Blot SD semidry transfer cells (Bio-Rad Laboratories, Hercules, Calif.). N-terminal amino acid sequencing was performed using a model 492 Procise (Applied Biosystems, Foster City, Calif.).

    Hemolysis assays. Rabbit erythrocytes (RBC) were diluted into Dulbecco's modified Eagle's medium (DMEM) lacking phenol (GIBCO-BRL, Grand Island, N.Y.) to a 5% final concentration (RBC-DMEM). V. parahaemolyticus strains were cultured overnight, and LB broth was replaced with phosphate-buffered saline (PBS) to an optical density at a wavelength of 600 nm (OD600) of 0.9. Ten microliters of the suspended solution was added to 500 μl of 5% RBC-DMEM and centrifuged at 2,500 x g for 1 min. After 5 h of incubation at 37°C without shaking, the pellet was gently resuspended to facilitate the release of hemoglobin. Cells were repelleted at 12,000 x g for 1 min, and the supernatant was monitored for the presence of released hemoglobin at an optical wavelength of 570 nm. Hemolytic activity was expressed as a percentage of that of the POR-1 strain standard.

    Cytotoxicity assays. For the cytotoxicity assays, HeLa cells (5 x 103 cells) were grown at 37°C under 5% CO2 in air in DMEM (Sigma, St. Louis, Mo.) supplemented with 10% fetal bovine serum (Sigma). Before infection, HeLa cells were washed with PBS (pH 7.2) and incubated further with DMEM lacking phenol red. HeLa cells were infected with bacteria at a multiplicity of infection of 100. At 4 h after infection, the supernatants were collected and the release of LDH was quantified. The release of LDH into the medium was assayed using the CytoTox96 nonradioactive cytotoxicity kit (Promega, Madison, Wis.) according to the manufacturer's instructions. Percent cytotoxicity was calculated using the following equation: (OD490 at experimental release – OD490 at spontaneous release)/(OD490 at maximum release – OD490 at spontaneous release) x 100. The amount of spontaneous release was assumed to be the amount of LDH released from the cytoplasm of uninfected HeLa cells, and the maximum release was the amount of LDH released by total lysis of uninfected HeLa cells.

    Detection of apoptotic cells. Aliquots of 105 HeLa cells were infected with the POR-1 strain or one of the mutant strains at a multiplicity of infection of 10 at 37°C for 3 h. Infected cells were gently washed three times with PBS and soaked with PBS including 5 mM EDTA and 10 μg of gentamicin ml–1 for 30 min to detach the cells. Cells were collected by centrifugation. Staining of apoptotic cells and dead cells was demonstrated using the annexin V-fluorescein isothiocyanate (FITC) apoptosis kit (BioVision, Palo Alto, Calif.) according to the manufacturer's instructions. Detection of apoptotic cells was performed by fluorescence-activated cell sorting (FACS) in a FACSCalibur system (Becton Dickinson, Mountain View, Calif.) using the signal detectors FL1 for FITC and FL2 for propidium iodide (PI).

    DNA fragmentation analysis of the infected HeLa cells. Aliquots of 105 HeLa cells were infected with the POR-1 strain or one of the mutant strains at a multiplicity of infection of 10 at 37°C for 7.5 h. Infected cells were gently washed twice with PBS and scraped off with a cell scraper. After centrifugation, DNA was extracted by using a DNeasy tissue kit (QIAGEN, Valencia, Calif.). Extracted DNA was loaded onto a 1.5% agarose gel and electrophoresed at 100 V, followed by ethidium bromide staining.

    Adenylate cyclase reporter gene assays. We constructed secreted-protein gene-cyaA fusion genes as follows. The initial 498 bp of the 5' termini of the genes, tagged by appropriate restriction sites, were amplified by PCR using primers listed in Table 2. The catalytic domain of the cyaA gene (bases 4 to 1,216) was tagged by appropriate restriction sites and amplified by PCR using the Bordetella pertussis cyaA gene as a template and the primers listed in Table 2. The resulting DNA was cloned into pT7blue T vectors and sequenced. The inserts were digested by the appropriate restriction enzymes and used to construct expression plasmids, as shown in Fig. 2. Plasmids were used to transform V. parahaemolyticus by electroporation using the conditions described above. Secretion of the protein-CyaA fusion products was confirmed by Western blotting using anti-CyaA monoclonal antibodies.

    HeLa cells (2 x 105 cells) were infected with V. parahaemolyticus harboring the secreted-protein gene-cyaA fusion gene at a multiplicity of infection of 2.5. After 3 h of infection, the cells were washed five times gently with PBS. The cyclic AMP (cAMP) levels were assayed using a commercial enzyme immunoassay system, cAMP Biotrak EIA (Amersham Biosciences). The cAMP levels were expressed as numbers of picomoles per well because the numbers of cells were almost the same in every well in this short-time infection condition.

    Western blot analysis. Infection with the bacteria harboring the secreted-protein-CyaA fusion gene was performed as described above. After 3 h of infection, cells were scraped off. The whole culture was poured into a tube and centrifuged at 16,000 x g for 5 min at 4°C. The pellets were dissolved in 40 μl of CelLytic B cell lysis reagent (Sigma) and 40 μl of 2x SDS sample buffer. Every eighth part of the sample was applied to SDS-12.5% polyacrylamide gel, and electorphoresis was performed. After electrophoresis, gels were electroblotted onto Immobilon-P transfer membrane (Millipore, Bedford, Mass.) under semidry conditions for 1 h. The membranes were blocked in blocking solution (5% nonfat dry milk powder and 0.1% Tween 20 in Tris-buffered saline) at room temperature for 2 h. Then the membranes were probed with anti-CyaA monoclonal antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, Calif.) for 30 min at room temperature. The second antibodies were anti-mouse immunoglobulin conjugated to horseradish peroxidase (Amersham Biosciences). The blots were developed with the ECL Western blotting kit (Amersham Biosciences) by following the manufacturer's instructions.

    RESULTS

    Comparative analysis of secreted proteins using 2D gel electrophoresis. To identify the proteins secreted via the TTSS1 apparatus, we performed 2D gel electrophoresis of bacterial culture supernatants and compared the pattern of the parent strain and that of the mutant with the TTSS1 deletion. Because large amounts of the TDH, one of the important toxins produced by V. parahaemolyticus, were secreted into the supernatant constantly, we first constructed a mutant (POR-1, the parent strain) with the two TDH-encoding genes (tdhA and tdhS) deleted (32). Then we deleted the vcrD1 gene, reported as gene number VP1662 (20), a counterpart of Yersinia lcrD with 74% identity, from the POR-1 strain because it was considered to be one of the components of the TTSS1 apparatus. A mutant with a deletion in vcrD1 should be defective in protein secretion via the TTSS1 apparatus (33). To identify the proteins secreted via the TTSS1 apparatus, we performed 2D gel electrophoresis on secreted proteins collected from the culture supernatant of POR-1 or the vcrD1 mutant. Several spots that were not detected in the supernatant of the vcrD1 mutant were identified in that of POR-1 (Fig. 3). Because these proteins were thought to be secreted in a TTSS1-dependent manner, we attempted to determine their N-terminal amino acid sequences. The sequences were LDKIGGTGRGELYGL, VNTTQKISQSPVPDL, ISFGNVSALQAAMPQ, and STIQINSQHRGNLPL. These amino acid sequences corresponded to those of four proteins encoded by VP1656, VP1680, VP1686, and VPA450 (reported gene numbers) (20) with 100% concordance. When we checked the locations of the genes on the genome, VP1680 and VP1686 proved to be in the "hypothetical" region of the TTSS1 gene cluster on chromosome 1 (Fig. 1). VPA450, however, was on chromosome 2, 0.44 Mbp distant from the putative origin of replication of the chromosome and 0.87 to 0.93 Mbp distant from the TTSS2 gene cluster. The hypothetical region consisting of 12 hypothetical genes is located between the TTSS1 component genes in V. parahaemolyticus, but gene clusters like this do not exist in the TTSS clusters of Yersinia spp. or P. aeruginosa (33). VP1656 was located in the TTSS gene cluster on chromosome 1; it had 33% identity to P. aeruginosa popD and 30% identity to Y. enterocolitica yopD. Hence, VP1656 is termed vopD. It has been reported that the Yersinia YopD protein is involved in translocating the effector Yops across the target cell membrane along with YopB, and both YopD and YopB are involved in contact hemolytic activity (28, 35). Thus, we constructed mutants with deletions of vopD and vopB and carried out contact hemolysis and cytotoxicity assays. Both vopD and vopB mutants showed decreased hemolytic activity in response to RBC and decreased cytotoxicity (Fig. 4). Therefore, we speculate that VopD and VopB play roles similar to those of the Yersinia YopD and YopB. VopB, however, could not be detected by 2D gel electrophoresis in this study. This may be because secretion levels of VopB were too low to be detected by SDS-polyacrylamide gel electrophoresis. Alternatively, VopB may be unstable. The product of VPA450 was similar to the Photorhabdus luminescens plu4615 protein, with 93% identity (8). As for VP1680 and VP1686, the genes for the two other secreted proteins, we could not find any motifs or sequences homologous to known DNA sequences. In addition, we carried out the contact hemolysis assay with mutants carrying deletions of newly identified secreted-protein genes VP1680, VP1686, and VPA450. No changes in the hemolytic activity were detected (data not shown).

    Cytotoxicity to HeLa cells. One of the characteristic effects of TTSS1 is cytotoxicity to eukaryotic cells (33). We performed assays of the cytotoxicity to HeLa cells using mutants carrying deletions of the newly identified secreted-protein genes to determine whether any of them play a role in this cytotoxicity. In this study, mutant strains lacking each of the three proteins encoded by VP1680, VP1686, and VPA450 and mutants with deletions of the hypothetical region were constructed and exposed to HeLa cells for 4 h, and then cytotoxicity was assayed by detecting LDH release from the cells by using a commercial kit (CytoTox96; Promega). Mutant strains constructed with a deletion in the hypothetical region are depicted in Fig. 5A in detail. The wild-type strain RIMD2210633 and POR-1 had cytotoxicity levels of 80 to 85% (Fig. 5B). Decreased cytotoxicity (to 30%) was observed for the vcrD1 mutant, as reported previously (33). Interestingly, there was a considerable decrease in cytotoxicity for the mutants with a deletion in VP1680, the h-3 region (which includes VP1680 and its neighboring gene VP1682), and the h-1 region (the entire hypothetical region), although little change was observed for the mutants with deletions in VP1686, VPA450, the h-2 region, and the h-4 region (Fig. 5B). These results suggest that VP1680 is involved in cytotoxicity against HeLa cells. Moreover, the VP1680-encoded protein may be a principal cytotoxic effector of the V. parahaemolyticus TTSS1, because the cytotoxicity of the VP1680 mutant was indistinguishable from that of the vcrD1 mutant.

    TTSS1-dependent cell death was apoptotic. To determine whether the TTSS1-dependent death of HeLa cells was apoptosis, we collected the infected HeLa cells and performed flow cytometry analysis by FACS using FITC-conjugated annexin V apoptosis kits (BioVision). HeLa cells were infected with the POR-1 strain or with one of the other mutants for 3 h. After infection, cells were washed, collected, and stained with annexin V-FITC and PI. Annexin V has a strong natural affinity for phosphatidylserine, which is translocated to the cell surface from the inner face of the plasma membrane soon after apoptosis is initiated (16). However, PI stains all dead cells. Therefore, we counted the proportion of FITC-positive PI-negative cells as early apoptotic cells (16). The ratio was high for POR-1, the vcrD2 mutant, and RIMD2212472. On the other hand, the ratio was low for the vcrD1 mutant and the VP1680 mutant and for PBS (the negative control) (Fig. 6). These results indicate that V. parahaemolyticus induces TTSS1-dependent apoptotic cell death in HeLa cells and that this acute apoptosis was caused mainly by the VP1680 protein.

    In addition, we extracted the DNA from the infected HeLa cells and confirmed the DNA fragmentation (Fig. 7). Obvious DNA fragmentation was observed in cells infected with POR-1 and the vcrD2 mutant, and obviously less fragmentation was observed in cells infected with the VP1680 mutant. No fragmentation was observed in uninfected cells and cells infected with the vcrD1 mutant. These results indicate that the apoptosis was caused by the secreted proteins via TTSS1. The VP1680 protein was the major one to cause apoptosis, although other secreted proteins also may be involved.

    Translocation of the identified proteins into HeLa cells via the TTSS1 apparatus. To determine whether newly identified secreted proteins encoded by VP1680, VP1686, and VPA450 are translocated into eukaryotic cells, we used the calmodulin-dependent adenylate cyclase reporter system (41). An adenylate cyclase from Bordetella, CyaA, is activated upon binding of calmodulin, an abundant intracellular protein found within eukaryotic, but not prokaryotic, cells (3). In this study, we constructed fusions between cyaA and the genes encoding the TTSS1 secreted proteins; the fusion genes were then introduced into V. parahaemolyticus strains. These strains were then incubated with HeLa cells. We examined the translocation of the TTSS1 secreted proteins into eukaryotic cells by measuring the cAMP levels of the infected HeLa cells. We constructed fusions between 498-bp regions of the 5' terminus of each gene for TTSS1 secreted proteins and a DNA fragment encoding the catalytic domain of CyaA (including 404 N-terminal amino acids). The constructed fusion genes were cloned into pSA19Cm-ONO, yielding the plasmids pVP1680N-cyaA, pVP1686N-cyaA, and pVPA450N-cyaA. In these plasmids, the promoter of the vopN-vopD operon was cloned to express the genes. Each plasmid was introduced into the V. parahaemolyticus POR-1 and vcrD1 mutants.

    HeLa cells were infected with these V. parahaemolyticus strains for 3 h, and we examined the production of the gene products by Western blotting using anti-CyaA monoclonal antibodies. Similar amounts of the fusion proteins were observed in all strains irrespective of the kind of protein or the ability of secretion (Fig. 8). In the assays for intracellular cAMP concentrations in infected HeLa cells, a large increase in intracellular cAMP levels was detected for the POR-1 strain harboring pVP1686N-cyaA, whereas the vcrD1 mutant harboring the fusion gene showed a lower increase (Table 3). A slight increase was detected for the POR-1 strain harboring pVPA450N-cyaA. On the other hand, no significant difference was obtained for the strains harboring pVP1680N-cyaA. However, when the VP1680-cyaA gene, which contained the full length of the VP1680 gene, was used, higher cAMP levels were observed for the POR-1 strain than for the vcrD1 mutant.

    These results suggest that the TTSS1 secreted proteins identified in this study were translocated into HeLa cells via the TTSS1 apparatus, although the secretion levels might be different among the proteins.

    TTSS1 is also active in an environmental strain. Almost all V. parahaemolyticus strains appear to have the TTSS1 gene cluster, including environmental isolates (20, 33). Some of these strains were as highly cytotoxic as the clinical strains under the conditions used here. The environmental strain, RIMD2212472, was isolated from sea mud off the coast of Japan and showed high cytotoxicity. Since both vcrD1 and VP1680 were detected in this strain by PCR amplification and sequencing, we constructed vcrD1 and VP1680 deletion mutants from it. We performed the LDH release assay using those strains to determine whether TTSS1 also plays a key role in cytotoxicity in the environmental strains. Both the vcrD1 and VP1680 mutants showed considerable decreases in cytotoxicity, and the gene-complemented mutant strains showed restored levels of LDH release (Fig. 9). These results indicate that the high cytotoxicity of this environmental strain is also caused by TTSS1 and that VP1680 plays an important role in this cytotoxicity.

    DISCUSSION

    Two sets of the TTSS gene cluster were found in the clinical isolates of V. parahaemolyticus (20). One of these, TTSS1, located on chromosome 1, is involved in cytotoxicity to HeLa cells (33). V. parahaemolyticus TTSS1 has almost all of the component genes that have been reported for the TTSS regions of Yersinia spp. and P. aeruginosa. However, no homologue of the effector protein genes has been found in the TTSS1 region.

    In this study, we performed 2D gel electrophoresis assays using culture supernatants of POR-1 and the vcrD1 mutant to determine the proteins secreted by the TTSS1 apparatus. In the supernatant of the vcrD1 mutant, at least four spots were absent compared with that of the parent strain. One of these proteins was VopD, which is a counterpart of PopD and YopD (33 and 30% identity, respectively), and the other three spots were shown to be the proteins encoded by VP1680, VP1686, and VPA450. Thus, we identified four new TTSS-secreted proteins of V. parahaemolyticus in this study. VP1680 and VP1686 are located in the hypothetical region in TTSS1 on chromosome 1, and VPA450 is on chromosome 2. It is interesting that proteins secreted via the TTSS1 apparatus appear to be encoded not only on chromosome 1, but also on chromosome 2. In several TTSS-possessing bacteria, the genes encoding TTSS-secreted proteins are indeed located outside the gene clusters encoding the TTSS apparatus (14). We could not find any motifs or homologues for the proteins in databases, except that encoded by VPA450, which was similar to the Photorhabdus luminescens plu4615 protein with 93% identity (8). The hypothetical region is located between the TTSS component genes, and all the genes in the region have no homology to any known genes. Because we used 2D gel electrophoresis to identify secreted proteins, some additional proteins that were secreted in small amounts or whose pI values were beyond the scope of the range of 2D gels used here (pI 4 to 7) may exist. For example, the protein encoded by VP1683 might be a TTSS1 apparatus-secreted protein because of its gene organization. However, the predicted pI of the VP1683 protein (pI 9.8) indicates that this protein was too alkaline to be detected in this study.

    When we looked around the hypothetical region, we found small genes located between the genes for TTSS1 apparatus-secreted proteins. These genes were VP1682, VP1684, and VP1687 and were located immediately upstream of the genes VP1680, VP1683, and VP1686, respectively. Every small gene had similar properties, such as an acidic pI (VP1682, pI 4.7; VP1684, pI 4.2; and VP1687, pI 4.0) and low molecular mass (VP1682, 17 kDa; VP1684, 19 kDa; and VP1687, 16 kDa). These properties suggested an association with TTSS chaperones (45). In addition, VPA450 corresponds to a similar small gene, VPA451 (pI 4.2, 17 kDa), upstream. Many TTSS chaperones, such as SycE for YopE and SycH for YopH, and their important roles have been described previously (44, 45). The products of these small genes may be chaperones of the V. parahaemolyticus TTSS1 apparatus-secreted proteins. More studies are needed to elucidate this relationship.

    In this study, VP1680 was clearly involved in the TTSS1-dependent cytotoxicity to eukaryotic cells. Cytotoxicity is the major expressed phenotype for V. parahaemolyticus TTSS1. The cell death appeared to be via apoptosis, which was demonstrated by a FACS assay and DNA fragmentation observation. However, the results of these two experiments for the VP1680 deletion mutant were somewhat discrepant. Few apoptotic cells were detected in the FACS assay, but weak DNA fragmentation was detected by electrophoresis. One possible reason for these differences may be the difference in infection times. Some other effector proteins related to apoptosis may be secreted via the TTSS1 apparatus at a later period of the infection. The mechanisms of apoptosis are well studied in relation to several TTSS effector proteins, such as Yersinia YopJ andYopP (9, 23, 30, 31, 36, 37, 39, 47). Further study is required to elucidate the mechanism of the apoptosis caused by the VP1680 protein.

    The roles of the other effectors have not been elucidated in this study, and we are continuing to study them. It is interesting that homologues of both the VPA450 protein and its putative chaperone, the VPA451 protein, exist in Photorhabdus luminescens, a pathogen of insects. It will be interesting to determine the common characteristics between the two bacteria.

    We demonstrated that the newly identified TTSS1 secreted proteins are all translocated into HeLa cells by using the adenylate cyclase fusion assay. These proteins are the first identified proteins of V. parahaemolyticus that are secreted via the TTSS1 apparatus and then translocated into the eukaryotic cell cytoplasm. In this study, the VP1686 protein caused a significant increase in intracellular cAMP levels whereas the VP1680 and VPA450 proteins caused only minor increases. This might reflect the different levels of translocation of the proteins, while the production levels of these strains were similar.

    All the V. parahaemolyticus strains so far examined, including both clinical and environmental isolates, have the TTSS1 genes (20, 33). In this study, we showed that TTSS1 plays an important role in cytotoxicity to HeLa cells, not only in clinical strains, but also in the environmental strain RIMD2212472. VP1680 seems to have a major role in the cytotoxicity of the environmental isolates also. To elucidate whether every environmental isolate possesses VP1680, we examined the presence of VP1680 in seven environmental isolates by PCR. This gene was present in all seven strains selected for this study, and DNA sequencing of the amplicons revealed that the extent of sequence variation among the gene products in environmental isolates was no greater than six amino acids (data not shown). VP1680 may be an essential gene also among the environmental strains.

    In a previous study, it was demonstrated that cytotoxicity is caused by TTSS1 and that enterotoxicity is caused by TTSS2 (33). TTSS2 is found only in the KP-positive strains, whereas TTSS1 is found in all strains, including environmental strains. Thus, TTSS2 is likely to be relevant to pathogenicity for humans. On the other hand, we wonder whether TTSS1 is really involved in pathogenicity. We do not know the answer at the moment, but it is possible that TTSS1 alone is not sufficient to cause disease in humans and that it synergistically acts in pathogenesis when TTSS2 is present. TTSS1 may act in defense against attacks of some mammalian cells, such as those in the human intestine. Further work is required to test this hypothesis. Some bacteria, such as P. aeruginosa, Aeromonas hydrophila, and Aeromonas salmonicida, have TTSSs in both clinical and environmental isolates (10, 46). TTSS is a tool of bacteria to interact intimately with eukaryotic cells. TTSS1 might be used in a natural host (15, 18). Although little is known about the life cycle of V. parahaemolyticus in its natural environment, the fact that all V. parahaemolyticus strains have the TTSS1 genes suggests that they may have a stage or stages in their natural life cycles in which they interact intimately with particular eukaryotic cells.

    ACKNOWLEDGMENTS

    This work was supported by the Research for the Future Program (grant 00L01411) of the Japan Society for the Promotion of Science and Grants-in-Aid for Scientific Research on Priority Areas and Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

    We thank the staff of Kansai International Airport Quarantine Station for the V. parahaemolyticus strains.

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