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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.
All V. parahaemolyticus strains used in this study were derived from wild-type strain NY-4 (Table (Table1)1) (47). V. parahaemolyticus was grown in LB broth or LB agar supplemented with 2.5% NaCl to stationary phase at 37°C while the culture was shaken (200 rpm). Gene deletion experiments were performed by using Escherichia coli S17 λpir cultured in LB medium. Plasmid pMMB207 (Cmr) was used in complementation and protein expression experiments, and plasmid pDM4 (Cmr) was used for gene deletion experiments. When appropriate, the following antibiotics were added at the indicated concentrations: ampicillin, 100 μg ml−1, and chloramphenicol, 25 μg ml−1 for E. coli and 5 μg ml−1 for V. parahaemolyticus.
HeLa (CCL-2) cells were purchased from the American Type Culture Collection (Manassas, VA). Monolayers of HeLa cells were maintained in DMEM (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS) at 37°C. Passage of HeLa cells was carried out every 1 to 2 days, and the cells were washed once before inoculation.
The complete open reading frame (ORF) of vp1659 was deleted from the chromosome by allelic exchange using a suicide vector, pDM4, carrying flanking regions on both sides of vp1659 (30). Primer pairs (primers 1659-1F and 1659-1R and primers 1659-2F and 1659-2R) (Table (Table2)2) were used to amplify two DNA fragments corresponding to the flanking regions of vp1659 with V. parahaemolyticus strain NY-4 chromosomal DNA as the template. Fragment 1 (approximately 1,000 bp upstream of the start codon of vp1659) was amplified with primers 1659-1F and 1659-1R and digested with XhoI and XbaI. Fragment 2 (approximately 1,000 bp downstream of the stop codon of vp1659) was amplified with primers 1659-2F and 1659-2R and digested with BglII and XbaI. After digestion, these two DNA fragments were simultaneously ligated into the pDM4 suicide vector that was predigested with XhoI and BglII. The resultant plasmid was designated pDM4-1659-1F+2R. This plasmid was transformed into E. coli S17 λpir by electroporation, resulting in strain S17-pDM4-1659-1F+2R. This strain was grown overnight in the presence of chloramphenicol, and an aliquot (1 ml) was mixed with an overnight culture of the NY-4 strain (1 ml) for ~12 h of conjugation at 37°C. Transconjugants were selected from the LB-S agar plates containing both ampicillin and chloramphenicol. We used ampicillin to select against E. coli because the NY-4 strain is naturally resistant to ampicillin. Chloramphenicol was used to select against nontransconjugants. Once pDM4-1659-1F+2R was conjugated from E. coli to V. parahaemolyticus, it integrated into the chromosome of V. parahaemolyticus. The integrated suicide plasmid was excised from the chromosome by growth on LB-S containing 5% sucrose. Once the allelic exchange was completed, clones that were sensitive to chloramphenicol were picked and screened by PCR with primers vp1659_Forward and vp1659_Reverse, which lie adjacent to the vp1659 sequence. One clone with the vp1659 gene deletion was selected and was designated Δvp1659.
The complete sequence of vp1659 with a 6× His tag added at the 3′ terminus of the gene was amplified with primers vp1659-up and vp1659-down (Table (Table2).2). The amplified PCR product was purified before digestion with EcoRI and XbaI. The digested PCR product was ligated into plasmid pMMB207 (digested with the same enzymes), resulting in plasmid pMMB207-RBS-vp1659. This plasmid was transformed into E. coli S17 λpir by electroporation, resulting in strain S17-pMMB207-RBS-vp1659, and was then conjugated from E. coli S17 λpir into the Δvp1659 strain, resulting in the Δvp1659::pvp1659 strain.
Strain S17-pMMB207-RBS-vp1659 was transformed into Yersinia enterocolitica (strain JB580v) by conjugation for protein expression. We produced Vp1659 in Y. enterocolitica because a previous study showed that Y. enterocolitica is more likely to yield soluble recombinant proteins (47). Y. enterocolitica containing pMMB207-RBS-vp1659 was grown at 26°C, and an aliquot of the overnight culture (5 ml) was diluted into 500 ml LB medium. After incubation for 2 h with shaking at 26°C, 1 mM isopropyl β-d-thiogalactopyranoside (IPTG) was added. Expression of Vp1659 was induced for 12 h at 26°C with vigorous shaking, before the cells were pelleted. The pellets were resuspended in 15 ml binding buffer (50 mM NaH2PO4, pH 8.0, 0.5 M NaCl, 10 mM imidazole) and sonicated for 15 min. After sonication, the whole-cell lysate was centrifuged at ~3,000 × g for 10 min to remove the insoluble fraction and the supernatant containing soluble proteins was loaded onto a Ni2+ resin column (Invitrogen). After binding of the proteins for 1 h, the column was washed 15 times with washing buffer (50 mM NaH2PO4, pH 8.0, 0.5 M NaCl, 50 mM imidazole). The proteins were eluted with elution buffer (50 mM NaH2PO4, pH 8.0, 0.5 M NaCl, 300 mM imidazole). Protein samples from the elution were electrophoresed by SDS-PAGE and stained with Coomassie blue to check the purity of the protein. Purified Vp1659 was submitted to the Monoclonal Antibody Center at Washington State University (Pullman, WA) for production of polyclonal antibody. The production of the anti-DnaK (Vp0653) polyclonal antibody will be described elsewhere. For antibody absorption, the whole-cell lysate of the Δvp1659 overnight culture was mixed with the anti-Vp1659 polyclonal antibody (1:1) for 12 h. After centrifugation at 15,000 × g for 30 min, the supernatant was used for Western blot analyses. Rabbit anti-VopS polyclonal antibody was generously provided by Kim Orth (Southwestern Medical Center, University of Texas, Dallas, TX).
To collect proteins in the supernatant, an overnight culture of V. parahaemolyticus was diluted into LB-S (1:100) and antibiotics were added as necessary. Diluted samples were grown for 4 h at 37°C with shaking (200 × g). IPTG (1 mM) was added after 1 h of shaking to induce protein expression. The bacterial culture (15 ml) was centrifuged at ~3,000 × g for 15 min, and the supernatant from each sample was passed through a 0.22-μm-pore-size syringe filter to exclude any remaining bacteria. To concentrate the proteins in the supernatant, a final concentration of 10% (vol/vol) trichloroacetic acid was added to the supernatant, and the mixture was incubated on ice for 2 h. After centrifugation at ~12,000 × g and 4°C for 20 min, the pellets were resuspended in ice-cold acetone and the suspension was centrifuged at ~12,000 × g and 4°C for 20 min. Finally, the protein pellets were resuspended in 50 μl 1× loading buffer (40 mM Tris-HCl, pH 8.0, 5% mercaptoethanol, 1% SDS, 5% glycerin, 1% bromphenol blue). After separation of the supernatant proteins by SDS-PAGE, silver staining was performed by using a SilverSNAP stain kit (Pierce, Rockford, IL) to visualize the protein secretion profile.
To collect proteins from whole-cell lysates for the detection of Vp1659, an overnight culture was diluted into LB-S (1:100) or DMEM (1:10) and grown for 4 h with addition of antibiotics and IPTG as needed. The bacterial cultures (15 ml) were centrifuged at ~3,000 × g for 20 min, and the pellets were resuspended in 500 μl phosphate-buffered saline (PBS). The bacterial suspension was sonicated until it became clear and then an equal volume of 2× loading buffer was added. To collect the bacterial proteins after infection, an overnight bacterial culture was rinsed once with DMEM and added to six-well polystyrene culture plates inoculated with ~106 HeLa cells to achieve a multiplicity of infection (MOI) of 100 CFU per cell. Synchronization of the bacterium-host cell contact was facilitated by centrifuging the plates at 600 × g for 5 min. The infected cells were scraped from each well after 4 h of infection and resuspended in 200 μl PBS. The resuspended bacterial HeLa cell mixture was sonicated, and an equal volume of 2× loading buffer was added to each sample for SDS-PAGE. All the samples were boiled for 5 min at 100°C and loaded on a 12% (wt/vol) polyacrylamide gel. After electrophoresis, the proteins were transferred to a nitrocellulose membrane for 12 h using an electroblot apparatus (Bio-Rad, Hercules, CA). Skim milk (5%) in PBS containing 0.1% Tween 20 was used to block the membrane. After 1 h of blocking, the membrane was probed (1:1,000) with polyclonal anti-Vp1659 (this study), anti-Vp1656 (47), or anti-DnaK (described elsewhere) for 1 h at room temperature. As a secondary antibody, an anti-mouse immunoglobulin conjugated to horseradish peroxidase was diluted in PBS with 5% milk at 1:5,000. The membrane was developed by using a Western blotting kit, according to the manufacturer's instructions (Bio-Rad).
An overnight culture of V. parahaemolyticus was obtained as described earlier and pelleted by centrifugation at 4,000 × g at room temperature. The bacterial pellets were resuspended in DMEM containing 1% (vol/vol) FBS. Bacterial suspensions (10 μl) were inoculated into each well of a 12-well polystyrene plate containing ~106 HeLa cells/well to achieve an MOI of ~100 CFU per cell. For the lactate dehydrogenase (LDH) release assay, the medium was free of antibiotics before inoculation. The supernatant of HeLa cells after infection was collected at 1, 2, 3, and 4 h; and LDH activity was measured with a cytotoxicity detection kit (Promega, Madison, WI), according to the manufacturer's instructions. The lysis buffer provided in the kit was added to achieve maximum LDH release. The LDH activity in the supernatant of uninfected cells was also measured to obtain a spontaneous LDH value. Percent cytotoxicity was calculated with the following formula: (test LDH release − spontaneous release)/(maximal release − spontaneous release).
Pore formation was analyzed by fluorescence microscopy, as described previously (21, 46). Briefly, monolayers of HeLa cells were grown on coverslips. Each strain of V. parahaemolyticus was inoculated into the appropriate wells, and after 2 h of infection, the coverslips were dipped in PBS containing 25 μg ml−1 ethidium bromide (EtBr) and 5 μg ml−1 acridine orange (AO) for 5 min. The coverslips were rinsed once and observed under a Zeiss confocal microscope (Franceschi Microscopy and Imaging Center, Pullman, WA) using fluorescein isothiocyanate (543 nm) and rhodamine (488 nm) filters. The percentage of EtBr-positive cell was obtained by averaging the data from three fields of HeLa cells (100 cells were counted for each field) infected with different strains.
Mechanical fractionation of infected HeLa cells was performed as described previously (14, 15, 22). Briefly, HeLa cells were seeded in a six-well plate and allowed to grow to confluence. One plate of HeLa cells was infected with each V. parahaemolyticus strain at an MOI of 100 for 2 h. The medium was aspirated and centrifuged at 4,000 × g for 10 min. The bacterial pellets were resuspended in 100 μl PBS, and the suspension was sonicated for 1 min and was used as the bacterial pellet fraction in the Western blot analysis. The infected cells as well as the adherent bacteria were scraped off the six-well plate and resuspended in 400 μl PBS. The cells were then passed through a 27-gauge needle six to eight times, which was expected to lyse the HeLa cells and leave the bacteria intact. The homogenate was centrifuged at 15,000 × g for 15 min, and the supernatant was used as the host-cell membrane and cytoplasm fraction in the Western blot analysis. Western blot analysis was performed by using mouse anti-Vp1659, mouse anti-Vp1656, and rabbit anti-VopS polyclonal antibodies, followed by Immun-Star horseradish peroxidase (HRP) chemiluminescence detection. To separate the cytosol and membrane fraction of HeLa cells infected with the NY-4 strain, the HeLa cells lysed with a needle as described above were centrifuged to remove the intact bacteria and HeLa cell debris. The supernatant was further centrifuged at 30,000 × g for 1 h to obtain the cytosol (supernatant) and membrane (pellet). Both the cytosol and the membrane fractions were probed with anti-VopS, anti-Vp1659, antitubulin (Sigma, St. Louis, MO), and anticalnexin (Santa Cruz Biotechnology, Santa Cruz, CA) antibodies.
Uninfected or infected HeLa cells were washed with PBS two times and fixed with 4% paraformaldehyde for 10 min. The HeLa cells were then blocked in 3% bovine serum albumin for 20 min before they were stained with rhodamine-conjugated phalloidin, according to the manufacturer's instruction (Invitrogen). The HeLa cells, on coverslips, were observed with a Zeiss confocal microscope using rhodamine (488 nm) filters. To detect a mass change in microtubule-associated protein light chain 3 (LC3), which is indicative of autophagy, HeLa cells were infected with the NY-4, Δvp1659, or Δvp1659::pvp1659 strain for 6 h and the whole-cell lysate samples were collected. Western blot analysis was performed using rabbit anti-LC3 (Novus Biologicals, Littleton, CO) and antiactin (Seven Hills Bioreagents, Cincinnati, OH) antibodies, followed by Immun-Star HRP chemiluminescence detection.
Student's t test was used to compare the cytotoxicity and percentage of EtBr-positive cells induced by the NY-4 and Δvp1659::pvp1659 strains and those induced by the Δvp1659 strain. The data were the averages of three independent experiments with two replicates per experiment. A P value of <0.05 was considered a statistically significant difference.
A previous study showed that the T3SS1 genes in V. parahaemolyticus NY-4 are not expressed when the bacteria are grown in LB-S (47). The T3SS1 genes are expressed, however, when either the exsD gene is disrupted or the exsA gene is expressed in trans in the NY-4 strain while it is cultured in LB-S. We compared the proteins in the supernatant of the NY-4 (wild type), ΔexsD, ΔexsD::pexsD, and NY-4:pexsA strains of V. parahaemolyticus growing in LB-S. The supernatant proteins were separated by SDS-gel electrophoresis and silver stained. Two proteins were present in the supernatant of the ΔexsD and NY-4:pexsA strains but were not visible in the supernatant of the NY-4 and ΔexsD::pexsD strains (Fig. (Fig.11 A). These two protein bands were excised from the gel and submitted for protein identification by matrix-assisted laser desorption ionization tandem mass spectrometry (MALDI MS/MS; Laboratory for Biotechnology and Bioanalysis, Washington State University, Pullman, WA). Approximately 150 amino acid residues were detected for the smaller protein band, and this sequence corresponded to 39% of the deduced composition of VopS (Vp1686). Approximately 163 amino acid residues were detected for the larger protein, and this sequence corresponded to 27% of the deduced composition of Vp1659. To confirm if the larger protein band was Vp1659, we generated recombinant Vp1659 protein and polyclonal antibody. Western blot results showed the presence of a clear band (67 kDa) for the secreted proteins originating from the ΔexsD and NY-4:pexsA strains, but no band was evident for the NY-4 or ΔexsD::pexsD strain (Fig. (Fig.1B).1B). The results of this experiment confirmed previous reports that VopS is a secreted protein (2, 34) and identified a novel secreted protein, Vp1659.
To determine if the expression of vp1659 is influenced by culture conditions, strain NY-4 was grown in LB-S or DMEM or in contact with HeLa cells, and Vp1659 was detected in whole-cell lysates using a Western blot assay. The expression of vp1659 was undetectable when the NY-4 strain was grown in LB-S (Fig. (Fig.22 A, upper panel, lane 1). When NY-4 was grown in DMEM, a protein band (Fig. (Fig.2A,2A, upper panel, lane 2) with a similar size to that of the purified Vp1659 (Fig. (Fig.2A,2A, upper panel, lane 4) was detected by polyclonal anti-Vp1659 antibody. For protein samples from NY-4 after contact with HeLa cells for 4 h, a similarly sized protein band (Fig. (Fig.2A,2A, upper panel, lane 3) was detected by polyclonal anti-Vp1659 antibody. More importantly, the band from NY-4 detected after contact with HeLa cells was more intense than that detected from NY-4 grown in DMEM alone (compare lane 2 and lane 3, Fig. Fig.2A,2A, upper panel). Because contact with HeLa cells was confounded by the presence of DMEM, we speculated that contact with HeLa cells had an additional role in facilitating the expression of vp1659, thereby producing a more intense band, and this is consistent with the findings of a previously reported promoter fusion assay (47). The V. parahaemolyticus DnaK chaperone protein was detected by anti-DnaK polyclonal antibody and served as a loading control (Fig. (Fig.2A,2A, lower panel). To confirm that the growth conditions employed in our study did not affect the expression of DnaK, we loaded the protein samples extracted from similar numbers of CFU of bacteria grown under different conditions and probed with anti-DnaK polyclonal antibody. The results showed that a protein band with a similar intensity was detected in the wild-type strain grown in LB-S, in DMEM, or in contact with HeLa cells (Fig. (Fig.2B).2B). In addition, the ΔexsA and ΔexsD strains yielded protein bands of similar intensity (Fig. (Fig.2B2B).
ExsA is an AraC-like transcriptional factor that controls the transcription of T3SS1 genes in V. parahaemolyticus (47). Vp1659 was not detected in whole-cell lysates when the ΔexsA strain was grown in LB-S or DMEM (Fig. (Fig.33 A, lanes 2 and 3). In trans expression of exsA in the ΔexsA strain restored the synthesis of Vp1659 when V. parahaemolyticus was grown in LB-S or DMEM (Fig. (Fig.3A,3A, lanes 4 and 5). Furthermore, the in trans expression of exsA in NY-4 activated the synthesis of Vp1659 when V. parahaemolyticus was grown in LB-S or DMEM (Fig. (Fig.3A,3A, lanes 6 and 7). Qualitatively, it was also clear that overexpression of exsA produced more protein than the amount produced by the wild-type strain under inducing conditions (Fig. (Fig.3B,3B, lane 1). These results indicate that ExsA is necessary for the expression of vp1659.
ExsD is a negative regulator for T3SS1 (47), and therefore, we determined if expression of Vp1659 is also affected by ExsD. Vp1659 was synthesized in the ΔexsD strain when V. parahaemolyticus was grown under noninducing conditions (in LB-S) or inducing conditions (in DMEM) (Fig. (Fig.3B,3B, lanes 1 and 2), indicating that ExsD negatively affects the expression of Vp1659. Complementation of the ΔexsD strain with a wild-type exsD gene in trans prevented vp1659 expression when the bacteria were grown in LB-S or DMEM (Fig. (Fig.3B,3B, lanes 3 and 4). When NY-4 was provided with an exsD gene in trans, vp1659 expression was blocked when the bacteria were grown in LB-S or DMEM (Fig. (Fig.3B,3B, lanes 5 and 6). Thus, overexpression of exsD represses the expression of vp1659 and inducing growth conditions do not override the inhibition from the in trans expression of exsD.
To further verify that Vp1659 is secreted by T3SSs in V. parahaemolyticus, we examined the supernatant of the NY-4, ΔvcrD (T3SS1 negative [T3SS1−]), ΔescV (T3SS2 negative [T3SS2−]), and ΔvcrD escV (T3SS1− T3SS2−) strains. To induce the expression of T3SS1 genes, NY-4 and each mutant strain were transformed with an exsA plasmid for the in trans expression of this gene. Western blot analysis showed that Vp1659 is synthesized in all the strains (Fig. (Fig.4,4, upper panel), while Vp1659 is present only in the supernatant of the NY-4:pexsA and ΔescV::pexsA strains (Fig. (Fig.4,4, lower panel). These results demonstrated that Vp1659 is secreted specifically through T3SS1.
Because the gene encoding Vp1659 is located in the T3SS1 genomic island, it is possible that Vp1659 is a structural protein required for the assembly of an intact secretion apparatus. To test this possibility, vp1659 was disrupted and plasmid pexsA was transformed into both the NY-4 and the Δvp1659 strains to induce the expression of T3SS1 genes. Vp1656 is known to be secreted specifically by T3SS1, and it may play a role in hemolytic activity (34). The results showed that Vp1656 was synthesized and secreted in both strains (Fig. (Fig.55 A), indicating that Vp1659 is not required for the secretion of Vp1656. As a confirmation, we deleted vp1659 in the ΔexsD strain to produce a ΔexsD vp1659 strain. Vp1656 was both synthesized (Fig. (Fig.5B)5B) and secreted (Fig. (Fig.5C)5C) in both the ΔexsD and ΔexsD vp1659 strains. We mutated vp1656 in the ΔexsD strain to produce a ΔexsD vp1656 strain. Polyclonal anti-Vp1656 antibody did not detect a protein band consistent with Vp1656, indicating that the protein detected in these experiments was Vp1656 (Fig. (Fig.5C).5C). DnaK served as a loading control for these experiments (Fig. 5B and C). These results showed that Vp1659 is not a structural protein required for the assembly of a functional secretion apparatus, although the results of this experiment do not eliminate the possibility that Vp1659 is part of a terminal tip or translocator apparatus.
We employed a mechanical fractionation approach to determine if Vp1659 is associated with the host cell membrane or cytosol (host membrane/cytosol fraction) after infection. Vp1659 was detected in the bacterial pellet of the NY-4, ΔvcrD, ΔescV, and ΔvcrD escV strains (Fig. (Fig.66 A, 1st row), indicating that all these V. parahaemolyticus strains synthesized Vp1659. Vp1659 was detected in the membrane/cytosol fraction of HeLa cells infected with the NY-4 or ΔescV strain (Fig. (Fig.6A,6A, 2nd row, lanes 1 and 3). In contrast, Vp1659 was not detected in this fraction of HeLa cells infected with the ΔvcrD or ΔvcrD escV strain (Fig. (Fig.6A,6A, 2nd row, lanes 2 and 4). These results indicated that Vp1659 was translocated into the host cell membrane or cytosol through the T3SS1 of V. parahaemolyticus. To determine if the Vp1659 detected here was from bacterial lysis during the mechanical fractionation, we determined if Vp1656 was present in this fraction. Vp1656 is homologous to a translocator protein, YopD, in Yersinia, and we were not able to detect Vp1656 in the membrane/cytosol fraction of HeLa cells infected with the NY-4, ΔvcrD, ΔescV, and ΔvcrD escV strains (Fig. (Fig.6A,6A, 3rd and 4th rows). While Vp1656 might be expected in this fraction, given that it is homologous to YopD, our results indicate that it is not secreted into the host cell cytosol. Thus, assuming that this finding does not represent an assay sensitivity issue, Vp1656 is a useful negative control for bacterial lysis (Fig. (Fig.6A).6A). We further determined if Vp1659 is localized in the cytosol or membrane fraction by ultracentrifugation. The membrane/cytosol fraction of HeLa cells infected with NY-4 was centrifuged at 30,000 × g for 1 h to obtain the cytosol fraction (supernatant) and the membrane fraction (pellet). Vp1659 was detected in the cytosol fraction but not in the membrane fraction (Fig. (Fig.6B).6B). Tubulin, a cytosol marker, was exclusively present in the cytosol, while calnexin, a membrane marker, was exclusively present in the membrane fraction (Fig. (Fig.6B),6B), indicating that these two fractions were separated successfully by ultracentrifugation and were not cross contaminated.
To further determine if Vp1659 plays a role in the translocation of other known effectors, we infected HeLa cells with the NY-4 and Δvp1659 strains and determined the translocation of VopS. VopS was detected in the bacterial pellet of both the NY-4 and Δvp1659 strains (Fig. (Fig.77 A, 1st row), indicating that both wild-type and Vp1659 mutant V. parahaemolyticus strains synthesized VopS at similar levels. VopS was detected in the membrane/cytosol fraction of HeLa cells infected with both the NY-4 and Δvp1659 strains (Fig. (Fig.7A,7A, 2nd row). The presence of VopS in the membrane/cytosol fraction of HeLa cells was not caused by bacterial lysis during the mechanical fractionation because Vp1656 was present in the bacterial pellets (Fig. (Fig.7A,7A, 3rd row) but was absent in the membrane/cytosol fraction of HeLa cells infected with either NY-4 or Δvp1659 strains (Fig. (Fig.7A,7A, 4th row). Tubulin served as a control to ensure that similar concentrations of proteins were loaded (Fig. (Fig.7A,7A, 5th row). When we separated the cytosol and membrane fractions by ultracentrifugation, we found that VopS was localized in the cytosol of HeLa cells infected with strain NY-4 (Fig. (Fig.7B).7B). These results indicate that the loss of Vp1659 does not prevent the translocation of VopS into the cytosol.
Because T3SS1 is required for the cytotoxicity against HeLa cells (46), we determined if Vp1659 plays any role in this process. HeLa cells were infected with the NY-4, Δvp1659, and Δvp1659::pvp1659 strains; and cytotoxicity was quantified by the LDH assay after 1, 2, 3, 4, and 8 h of infection. NY-4 caused cytotoxicity against HeLa cells in a time-dependent manner, with the maximum LDH release occurring at between 2 and 3 h and nearly complete cell death being observed after 4 h of infection. The cell lysis phenotype was clearly delayed for the Δvp1659 strain, with only 30% of the HeLa cells apparently being dead after 4 h of infection (Fig. (Fig.88 A). The Δvp1659::pvp1659 complement strain recovered the full cytotoxicity phenotype, with approximately 90% of the HeLa cells being lysed after infection for 4 h (Fig. (Fig.8A).8A). The cytotoxicity induced by NY-4 or the Δvp1659::pvp1659 strain was significantly higher than that induced by the Δvp1659 strain after 4 h of infection (P < 0.05). As a control, analysis of whole-cell lysates confirmed that VopS was synthesized at equivalent levels by the NY-4, Δvp165, and Δvp1659::pvp1659 strains in these experiments (data not shown). Microscope analysis showed that HeLa cells infected with NY-4 and the Δvp1659::pvp1659 strain exhibited rounding and detachment from the bottom of the well after 2 h, while HeLa cells infected with the Δvp1659 strain maintained a uniform monolayer and a normal appearance compared with the appearance of the uninfected cells (data not shown).
Because the T3SS1 in strain NY-4 is involved in pore formation in the cell membrane of HeLa cells (46), we determined if Vp1659 is required for this process. Infected cells were stained with a membrane-impermeant dye (EtBr) and a membrane-permeant dye (AO). When AO penetrates the cell, it exhibits green fluorescence, whereas the combination of both AO and EtBr results in yellow or red fluorescence, thereby confirming membrane permeability. After 2 h of infection with NY-4 and the Δvp1659::pvp1659 strain, a significant number of HeLa cells displayed red under a fluorescence microscope, indicating that both EtBr and AO entered the cells (data not shown). Quantitative analysis showed that EtBr-positive cells accounted for approximately 35% of the cells for cultures infected with the NY-4 or Δvp1659::pvp1659 strain (Fig. (Fig.8B),8B), while less than 1% of cells for cultures infected with the Δvp1659 strain were EtBr positive. These results showed that Vp1659 is required directly or indirectly for pore formation in the cell membrane, leading to the uptake of the membrane-impermeant dye (EtBr) (46).
V. parahaemolyticus is known to alter the host cell actin structure, but HeLa cells infected with the Δvp1659 strain do not exhibit obvious cell rounding (data not shown). Consequently, we confirmed that filamentous actin was disrupted in HeLa cells infected by the wild-type strain of V. parahaemolyticus (Fig. (Fig.99 B) for 2 h. V. parahaemolyticus with the deletion of vp1659 did not affect the filamentous actin structure (Fig. (Fig.9C),9C), while complementation of the Δvp1659 strain with in trans vp1659 expression restored the ability of V. parahaemolyticus to disrupt the actin structure (Fig. (Fig.9D).9D). Filamentous actin was undisturbed in our negative control HeLa cells (Fig. (Fig.9A).9A). These results indicate that Vp1659 is required for the disruption of the filamentous actin structure in HeLa cells. It is also notable that there were no obvious changes for the actin structure in HeLa cells even after 8 h of infection with the Δvp1659 strain (Fig. (Fig.9E9E).
When V. parahaemolyticus infects HeLa cells, it induces the conversion of microtubule-associated protein light chain 3 I (LC3-I) into LC3-II (3, 4), which is a key marker of autophagy. The LC3-I in HeLa cells infected with NY-4 was completely converted into LC3-II (Fig. (Fig.10,10, lane 1). LC3-I from HeLa cells infected with the Δvp1659 strain was partially converted into LC3-II (Fig. (Fig.10,10, lane 2), but this result appeared to be no different from that for the uninfected control (Fig. (Fig.10,10, lane 3). The rate of conversion of LC3-I into LC3-II in HeLa cells infected with the Δvp1659::pvp1659 strain was increased compared to that for the uninfected control (Fig. (Fig.10,10, lane 4). These results indicate that Vp1659 is required for the induction of autophagy by V. parahaemolyticus.
In this study, we overexpressed the T3SS1 of V. parahaemolyticus and detected two proteins, VopS (Vp1686) and Vp1659. The gene encoding Vp1659 is found in the T3SS1 genomic island, and it is annotated as a hypothetical protein (28). When we generated a deletion mutant for vp1659, we immediately discovered that host cell death was attenuated (see below), and consequently, we initiated a systematic series of experiments to characterize how Vp1659 contributes to this phenotype. We initially demonstrated that synthesis of Vp1659 is regulated in a manner consistent with that for a T3SS1 protein, where ExsA and ExsD serve as positive and negative regulators, respectively. Deletion of vp1659 had no apparent affect on the expression of Vp1656 or the secretion of Vp1656 into the extracellular milieu, indicating that Vp1659 is not required to move proteins through the T3SS1 secretion apparatus. Interestingly, mechanical fractionation and ultracentrifugation experiments followed by Western blotting showed that Vp1659 is translocated into the cytosol of HeLa cells; VopS is also translocated even when vp1659 is deleted. At this point in our investigation, it appeared to be possible that Vp1659 was a potential effector protein that contributed directly to the oncosis phenotype that is produced by V. parahaemolyticus infection (46).
Subsequent phenotypic studies showed that deletion of vp1659 not only attenuated cell cytolysis and membrane permeabilization but also blocked actin rearrangement in the host cell and apparently interfered with the autophagy pathway. Actin rearrangement is linked to VopS (vp1686) (44), which belongs to the Fic (filamentation induced by cyclic AMP) protein family. VopS is both necessary and sufficient to induce actin rearrangement by alteration of GTPase via a novel mechanism called AMPylation (44). There is no evidence directly linking actin rearrangement to host cell lysis, because deletion of VopS does not reduce cytotoxicity (34). Autophagy is initiated by VopQ (vp1680) via a PI3-kinase-independent pathway (3). While autophagy clearly contributes to host cell lysis, it does not explain this phenotype entirely (3). Importantly, while VopS is sufficient to cause actin rearrangement and cell rounding and VopQ is required to induce autophagy, there is no evidence that these pathways are linked in any manner or with oncosis. Consequently, the only way that deletion of vp1659 could block multiple and independent pathways is if Vp1659 is part of the tip of the injectisome or translocator apparatus, whereupon its absence either significantly reduces or eliminates translocation. Judging by the fact that VopS is translocated even when vp1659 is deleted and that VopS is sufficient to induce actin rearrangement, we surmise that the loss of this phenotype with the vp1659 deletion mutant is due to an insufficient quantity of VopS being translocated into the HeLa cells to affect the actin structure.
The gene encoding Vp1659 is located within the T3SS1 island of V. parahaemolyticus, and it is annotated as a 67-kDa hypothetical protein. Analysis of protein homology (BLASTp queries) indicates similarity between the C terminus of Vp1659 and LcrV from Yersinia (37 kDa) and PcrV from Pseudomonas (32 kDa) (33% and 35%, respectively). Bioinformatics analysis (http://myhits.isb-sib.ch/cgi-bin/motif_scan) shows that Vp1659 (GenBank accession number NP_798038.1) includes some functional domains that are not present in LcrV (GenBank accession number YP_001004069.1), although the matches were very limited (e value, >10−3). Amino acids 329 to 607 share close sequence homology with LcrV (e value, 1.3 × 10−12).
LcrV from Yersinia has been linked to several phenotypes. One study showed that LcrV can be translocated into host cells and that this translocation process is not dependent on the Ysc apparatus (11). Translocated LcrV is colocalized with endosomal proteins during the early infection, and during later infection, LcrV is colocalized with lysosomal, mitochondrial, and Golgi proteins (9). LcrV has also been shown to localize on the surface of the bacterial cells, and treatment with anti-LcrV antibody can prevent the translocation of Yop effectors into the host cell cytosol (36). Another study showed that treatment with anti-LcrV antibody does not block the delivery of Yops into the host cells, although this treatment can protect against experimental plague (10). Deletion of lcrV blocked the movement of Yersina effector proteins into the cytotosol of HeLa cells (8, 24), suggesting that LcrV may compose part of the translocator apparatus. A recent study showed that LcrV of Yersinia forms a distinct structure at the tip of the secretion needle to assist the pore formation and translocation of effector proteins into host cells (31). Similarly, deletion of pcrV in Pseudomonas does not affect secretion of other T3SS substrates, but this does abolish translocation of effectors into fibroblasts (15). These results are consistent with our findings, and we hypothesize that Vp1659 in V. parahaemolyticus composes part of the secretion apparatus and that the efficiency of effector translocation is compromised in the absence of Vp1659. Considering that Vp1659 has a mass approximately 2-fold larger than the masses of the LcrV and PcrV homologues and that Vp1659 is translocated into the host cell cytosol, it may have another role as an effector protein, but there is no direct evidence of additional functions at this time.
It is important to note that in an earlier study we provisionally rejected the conclusion that V. parahaemolyticus strain NY-4 induces autophagy in host cells (46). This conclusion was based on staining with monodansylcadaverine to reveal autophagic bodies, and as might be expected, stain-based assays can lack analytical sensitivity. Burdette et al. (3) used a much more sensitive Western blot assay to show that V. parahaemolyticus induces the conversion of LC3-I to LC3-II, and this conversion is considered a hallmark of autophagy (20, 25). In the present study, we also employed the LC3 Western blot assay and confirmed that strain NY-4 also initiates autophagy in HeLa cells.
In conclusion, by overexpressing T3SS1, we identified a T3SS1 protein, Vp1659, which can be detected in the cytosol fraction of HeLa cells after infection. vp1659 expression is positively regulated by ExsA and negatively regulated by ExsD. Deletion of vp1659 does not affect the secretion of Vp1656 or completely block the translocation of VopS, but deletion of Vp1659 limits the ability of V. parahaemolyticus to cause cell membrane permeabilization, actin depolymerization, and autophagy. The mechanism by which Vp1659 induces these phenotypes is probably through the loss of efficient translocation of relevant effector proteins into host cells. Consequently, Vp1659 is probably a component of the secretion apparatus tip or translocator used by V. parahaemolyticus during infection.
We acknowledge the excellent technical assistance from Lisa Orfe, Stacey LaFrentz, Dan Erwin, Seth Nydam, and Pat Friel and services provided through the Washington State University Monoclonal Antibody Center.
This project was supported in part by the National Institutes of Health, U.S. Department of Health and Human Services, under contract number No1-AI-30055 and by the Agricultural Animal Health Program, College of Veterinary Medicine, Washington State University.
Published ahead of print on 23 April 2010.