Identification and characterization of VopT, a novel ADP-ribosyltransferase effector protein secreted via the Vibrio parahaemolyticus type III secretion system 2
2007; Wiley; Volume: 9; Issue: 11 Linguagem: Inglês
10.1111/j.1462-5822.2007.00980.x
ISSN1462-5822
AutoresToshio Kodama, Mitsuhiro Rokuda, Kwon-Sam Park, Vlademir Vicente Cantarelli, Shigeaki Matsuda, Tetsuya Iida, Takeshi Honda,
Tópico(s)Plant Pathogenic Bacteria Studies
ResumoCellular MicrobiologyVolume 9, Issue 11 p. 2598-2609 Free Access Identification and characterization of VopT, a novel ADP-ribosyltransferase effector protein secreted via the Vibrio parahaemolyticus type III secretion system 2 Toshio Kodama, Corresponding Author Toshio Kodama Department of Bacterial Infections, and *E-mail kodama@biken.osaka-u.ac.jp; Tel. (+81)6 6879 8278; Fax (+81) 6 6879 8277.Search for more papers by this authorMitsuhiro Rokuda, Mitsuhiro Rokuda Department of Bacterial Infections, andSearch for more papers by this authorKwon-Sam Park, Kwon-Sam Park Department of Bacterial Infections, and Department of Food Science and Technology, College of Ocean Science and Technology, Kunsan National University, San 68, Miryong-dong, Kusan, Jeollabuk-do, 573-701, Korea.Search for more papers by this authorVlademir V. Cantarelli, Vlademir V. Cantarelli Department of Bacterial Infections, and Laboratorio Weinmann LTDA, Setor de Microbiologia e Biologia Molecular, Porto Alegre, RS, Brazil.Search for more papers by this authorShigeaki Matsuda, Shigeaki Matsuda Department of Bacterial Infections, andSearch for more papers by this authorTetsuya Iida, Tetsuya Iida Department of Bacterial Infections, and International Research Center for Infectious Diseases, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan.Search for more papers by this authorTakeshi Honda, Takeshi Honda Department of Bacterial Infections, andSearch for more papers by this author Toshio Kodama, Corresponding Author Toshio Kodama Department of Bacterial Infections, and *E-mail kodama@biken.osaka-u.ac.jp; Tel. (+81)6 6879 8278; Fax (+81) 6 6879 8277.Search for more papers by this authorMitsuhiro Rokuda, Mitsuhiro Rokuda Department of Bacterial Infections, andSearch for more papers by this authorKwon-Sam Park, Kwon-Sam Park Department of Bacterial Infections, and Department of Food Science and Technology, College of Ocean Science and Technology, Kunsan National University, San 68, Miryong-dong, Kusan, Jeollabuk-do, 573-701, Korea.Search for more papers by this authorVlademir V. Cantarelli, Vlademir V. Cantarelli Department of Bacterial Infections, and Laboratorio Weinmann LTDA, Setor de Microbiologia e Biologia Molecular, Porto Alegre, RS, Brazil.Search for more papers by this authorShigeaki Matsuda, Shigeaki Matsuda Department of Bacterial Infections, andSearch for more papers by this authorTetsuya Iida, Tetsuya Iida Department of Bacterial Infections, and International Research Center for Infectious Diseases, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan.Search for more papers by this authorTakeshi Honda, Takeshi Honda Department of Bacterial Infections, andSearch for more papers by this author First published: 27 July 2007 https://doi.org/10.1111/j.1462-5822.2007.00980.xCitations: 98AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Summary Vibrio parahaemolyticus strain RIMD2210633 has two sets of genes encoding two separate type III secretion systems (T3SSs), called T3SS1 and T3SS2. T3SS2 has a role in enterotoxicity and is present only in Kanagawa phenomenon-positive strains, which are pathogenic to humans. Accordingly, T3SS2 is considered to be closely related to V. parahaemolyticus human pathogenicity. Despite this, the biological actions of T3SS2 and the identity of the effector protein(s) secreted by this system have not been well understood. Here we report that T3SS2 induces a cytotoxic effect in Caco-2 and HCT-8 cells. Moreover, it was revealed that VPA1327 (vopT), a gene encoded within the proximity of T3SS2, is partly responsible for this cytotoxic effect. The VopT shows approximately 45% and 44% identity with the ADP-ribosyltransferase (ADPRT) domain of ExoT and ExoS, respectively, which are two T3SS-secreted effectors of Pseudomonas aeruginosa. T3SS2 was found to be necessary not only for the secretion, but also for the translocation of the VopT into host cells. We also demonstrate that VopT ADP-ribosylates Ras, a member of the low-molecular-weight G (LMWG) proteins both in vivo and in vitro. These results indicate that VopT is a novel ADPRT effector secreted via V. parahaemolyticus T3SS. Introduction Vibrio parahaemolyticus is a Gram-negative bacterium that causes acute gastroenteritis after the consumption of contaminated raw or undercooked seafood (Blake et al., 1980). Most of the clinical isolates of V. parahaemolyticus that are isolated from diarrhoeal patients show β-haemolysis on a special blood agar (Wagatsuma agar) (Sakazaki et al., 1968). This haemolysis is called the Kanagawa phenomenon (KP) and is induced by thermostable direct haemolysin (TDH) (Shirai et al., 1990; Honda and Iida, 1993). Purified TDH causes a number of biological effects, including erythrocyte lysis, cardiotoxicity, disruption of the microtubule, induction of chloride ion secretion and production of fluid accumulation in the ileal loop model (Honda et al., 1976a,b; Sakurai et al., 1976; Goshima et al., 1977; Honda and Iida, 1993; Tang et al., 1995; 1997; Fabbri et al., 1999; Raimondi et al., 2000; Naim et al., 2001; Takahashi et al., 2001). Therefore, it has been considered that TDH is an important virulence factor produced by this bacterium. Recently, it has been reported that not only TDH, but also other unknown virulence factor(s) may be involved in the cytotoxicity and enterotoxicity caused by KP-positive strains of V. parahaemolyticus (Park et al., 2004a; Lynch et al., 2005). Whole genome sequencing of the KP-positive V. parahaemolyticus strain, RIMD2210633, revealed the presence of two sets of genes encoding two separate type III secretion systems (T3SSs), on chromosomes 1 and 2, and these were termed T3SS1 and T3SS2 respectively (Makino et al., 2003). The T3SS1 gene cluster is present in both KP-positive and KP-negative strains, while the T3SS2 gene cluster is unique to KP-positive strains (Park et al., 2004b). A functional analysis of these two T3SSs revealed that T3SS1 is involved in the cytotoxicity observed in HeLa cells, while T3SS2 is involved in enterotoxicity (Park et al., 2004b). Therefore, T3SS2 is considered to be related to the pathogenicity of V. parahaemolyticus to humans. Type III secretion system can be found in many Gram-negative pathogenic bacteria, such as Yersinia spp., Pseudomonas aeruginosa, Salmonella spp., Shigella spp., and enteropathogenic and enterohaemorrhagic Escherichia coli (Hueck et al., 1998). T3SS is a multisubunit molecular machine that can deliver bacterial proteins, called effectors, directly into the plasma membrane or the cytoplasm of infected host cells. While the genes for the T3SS apparatus are highly conserved among bacterial species, the effectors secreted by these T3SSs tend to be unique to each pathogen. Generally, following translocation into host cells, the effectors modify host cell function by disrupting the normal cell signalling processes. Despite being closely related with the observed pathogenicity to humans, there are many unclear points about the detailed mechanism of action of T3SS2, especially because suitable assay systems to analyse the biological activity of T3SS2 in vitro are still lacking. In this study, we found that T3SS2 is directly involved in the cytotoxic effect observed in some cultured cell lines. In addition, we demonstrated that VPA1327 (vopT), encoded within the proximity of T3SS2, is partly responsible for this cytotoxic effect. We also demonstrated that VopT is secreted and translocated into host cells through T3SS2, and acts as an ADP-ribosyltransferase (ADPRT) effector protein. Results T3SS1- and T3SS2-dependent cytotoxicity induced by V. parahaemolyticus Because the histology of ileal loop of rabbits infected by V. parahaemolyticus showed denudation and disruption of the surface epithelium (Park et al., 2004b), we tested whether T3SS2 contributes to this phenomenon using cultured cell lines. In accordance with a previous report, the parental strain POR-1 was cytotoxic, not only to HeLa cells, but also to J774, T84 and HT-29 cells. This cytotoxicity was dramatically decreased when cells were infected with POR-2 (ΔvcrD1), a T3SS1 apparatus gene-deficient strain, as well as with ΔvcrD1/ΔvcrD2, which is both T3SS1 and T3SS2 deficient (Fig. 1A and B). On the other hand, the cytotoxic effect of POR-3 (ΔvcrD2) was indistinguishable from that of POR-1 (Fig. 1A). These results indicated that T3SS1, but not T3SS2, plays a role in the observed cytotoxic effect against these cell lines. On the other hand, T3SS2-dependent cytotoxic activity was observed with Caco-2 and HCT-8 cells. As shown in Fig. 1C, compared with POR-1, the cytotoxicity of the POR-2 against Caco-2 cells was almost null 3 h after infection. However, it was nearly identical to that of POR-1 6 h after the infection with these strains. This cytotoxicity was dramatically decreased when cells were infected with ΔvcrD1/ΔvcrD2, which is both T3SS1 and T3SS2 deficient (Fig. 1C). Similar results were also observed against HCT-8 cells (Fig. 1D). This T3SS2-dependent cytotoxicity was not observed at all in the cell lines used in Fig. 1B. These data indicated that T3SS2 induces cytotoxicity against selected cell lines. Figure 1Open in figure viewerPowerPoint T3SS1- and T3SS2-dependent cytotoxicity induced by Vibrio parahaemolyticus.A. T3SS1-dependent cytotoxicity of V. parahaemolyticus. HeLa cells were infected with bacteria at an moi of 10. After infection, cytotoxicity was assayed by measuring total cellular LDH release into the cellular supernatant. POR-1 (filled squares, solid line), POR-2 (ΔvcrD1) strain (filled circles, dashed line), POR-3 (ΔvcrD2) strain (open circles, dashed line), ΔvcrD1/ΔvcrD2 strain (open squares, dashed line).B. T3SS1-dependent cytotoxicity against HeLa or J774 or T84 or HT-29 cells. The amount of LDH release of the cells was measured 6 h after infection with POR-1 (a) or POR-2 (b) or ΔvcrD1/ΔvcrD2 (c).C. T3SS2-dependent cytotoxicity of V. parahaemolyticus. The amount of LDH released in the supernatant of infected Caco-2 cells was determined. Parent strain (filled squares, solid line), POR-2 strain (filled circles, dashed line), POR-3 strain (open circles, dashed line), ΔvcrD1/ΔvcrD2 strain (open squares, dashed line).D. T3SS-2-dependent cytotoxicity against Caco-2 or HCT-8 cells. The amount of LDH released by the cells was measured 6 h after infection with POR-1 (a) or POR-2 (b) or ΔvcrD1/ΔvcrD2 (c). VopT inhibits yeast cell growth and has cytotoxic activity against Caco-2 cells In order to identify the effector protein responsible for T3SS2-dependent cytotoxicity, we searched for candidates within the pathogenicity island (PAI) region of chromosome 2 (Makino et al., 2003), as most of the T3SS effectors were found to be encoded within the same region of their secretion system genes. This resulted in three potential candidates, VopP (VPA1346), which is homologous to the Yersinia enterocolitica YopP and Yersinia pestis YopJ (36% identity), VopC (VPA1321), which is homologous to the E. coli cytotoxic necrotizing factor (CNF; 38% identity), and VopT (VPA1327), which is homologous to the Pseudomonas exoenzyme T (ExoT; 45% identity). It has been observed that an effector responsible for T3SS1-dependent cytotoxicity inhibited the growth of yeast when expressed in yeast cells (T. Ono, unpubl. data). Therefore, we tried to determine whether these putative effectors had any effect on the growth of yeast strains, which expressed these proteins under the control of a galactose-inducible promoter. When yeasts harbouring VopC plasmid were plated onto galactose-containing medium to induce the expression of VopC, the yeast cells were able to grow as well as the control yeast strain (Fig. 2A). In contrast, yeast cells expressing VopP or VopT were unable to grow on galactose-containing medium. VopP is homologous to Yersinia YopP/J, which acts to block the expression of cytokines and antiapoptotic factors by preventing activation of the superfamily of MAPK kinase (Orth et al., 1999; Yoon et al., 2003). It has been reported that VopP is also able to inhibit both the mammalian and yeast MAPK signalling pathways (Trosky et al., 2004). VopT is homologous to two ADP-ribosylating toxins, ExoT and ExoS, of P. aeruginosa. The ADPRT activity of ExoS was shown to result in the modification of multiple substrates, including the low-molecular-weight G (LMWG) proteins Ras, RalA, and certain Rab proteins, Rac1, and Cdc42, leading to cell death (Kaufman et al., 2000). Therefore, we next tried to determine whether these effector candidates were involved in T3SS2-dependent cytotoxicity against Caco-2 cells. As shown in Fig. 2B, the cytotoxic effect of ΔvcrD1/ΔvopC and ΔvcrD1/ΔvopP on infected cells remained at similar levels as that of ΔvcrD1. On the other hand, the cytotoxic effect of ΔvcrD1/ΔvopT on infected cells was significantly decreased (P > 0.01) compared with the other three strains mentioned above. The decrease in cytotoxicity observed for ΔvcrD1/ΔvopT was fully restored by an in trans complementation with the wild-type vopT gene (Fig. 4D). Similar results were observed using HCT-8 cells (Fig. 2C). Taken together, these results suggest that vopT is at least one of the genes responsible for T3SS2-dependent cytotoxicity. Figure 2Open in figure viewerPowerPoint VopT has both growth inhibition activity against yeast and cytotoxicity against Caco-2 cells.A. VopT inhibits growth when expressed in yeast. The yeast cells were transformed with a galactose-inducible vector alone or vector containing VopP or VopC or VopT (left). Growth assay demonstrates the effect of the effector candidates' expression on yeast growth on media containing glucose (middle) and galactose (right).B. VopT is responsible for T3SS2-dependent cytotoxicity against Caco-2 cells. Caco-2 cells were infected with isogenic mutants of the POR-2 strain for 6 h. Cytotoxicity was evaluated by the amount of LDH released. Asterisk indicates statistically significant differences from the results obtained with POR-2 strain (P < 0.01).C. VopT is responsible for T3SS2-dependent cytotoxicity against HCT-8 cells. HCT-8 cells were infected with ΔvcrD1/ΔvopT strain for 6 h. Cytotoxicity was evaluated by the amount of LDH released. The double asterisk indicates a statistically significant differences from the results obtained with the POR-2 strain (P < 0.05). Figure 4Open in figure viewerPowerPoint ADP-ribosyltransferase (ADPRT) domain of VopT is necessary for yeast cell growth arrest and cytotoxicity against Caco-2 cells.A. Alignment of the catalytic residues and cofactor-binding residues of ExoS, ExoT and VopT. Areas of the conserved catalytic and cofactor-binding residues are boxed.B. Effect of mutation within ADPRT residues of VopT on inhibition of yeast cell growth. Yeast, containing empty vector or galactose-inducible wild-type VopT (VopT) or various ADPRT mutants of VopT, was grown on glucose- (middle) or galactose- (right) containing medium.C. Expression and secretion of wild-type and ADPRT mutant VopT. Western blot analysis of bacterial pellets (top) and secreted proteins (bottom) of ΔvcrD1/ΔvopT strain expressing wild-type or mutant VopT strains. Blot was probed with anti-FLAG antibody.D. Effect of mutation within ADPRT residues of VopT on toxicity against Caco-2 cells. Caco-2 cells were infected with ΔvcrD1/ΔvopT strain expressing wild-type (WT) or mutant VopT. The amount of LDH release was determined after 6 h of infection. The asterisk indicates a statistically significant difference from the results obtained with the POR-2 strain (P < 0.05) and the double asterisk indicates a highly significant difference from the results obtained with the POR-2 strain (P < 0.01). VopT is secreted via T3SS2 The V. parahaemolyticus strain RIMD2210633 possesses two sets of genes for two separate T3SSs and that they recognize and secrete distinct proteins (Park et al., 2004b). We attempted to confirm that VopT is really secreted via T3SS2. As shown in Fig. 3, although FLAG-tagged VopT was expressed at similar levels in bacterial pellets (top), this protein was only detected in the supernatants from POR-1 or POR-2. VopT was not detected in the supernatant of the T3SS2-related mutant strains, such as POR-3 and ΔvcrD1/ΔvcrD2 (Fig. 3, bottom), indicating that VopT was specifically secreted via T3SS2. Figure 3Open in figure viewerPowerPoint VopT is specifically secreted by T3SS2. Western blot analysis of bacterial pellets (top) and secreted proteins (bottom) of the parent strain and T3SS-related mutant strains harbouring pvopT-FLAG and vector control. The blot was probed with anti-FLAG antibody. sup, supernatant. VopT is translocated into host cells by T3SS2 To determine whether VopT was translocated into host cells by the T3SS2, a plasmid expressing VopT C-terminally fused with the catalytic domain of adenylate cyclase toxin (pSA-vopT-cyaA) was constructed, and used to transform V. parahaemolyticus strains. Caco-2 cells were infected with the obtained transformants and the level of intracellular cAMP was determined by a specific ELISA system. The level of intracellular cAMP induced by the parent and POR-2 strains expressing VopT-cyaA in infected cells were above the upper detection limit of the assay, whereas the T3SS2-related mutant strains (POR-3 and ΔvcrD1/ΔvcrD2) all induced cAMP production at low levels, indicating that these T3SS2 mutants were unable to translocate VopT-cyaA into the infected cells (Table 1). Taken together, these results strongly suggest that VopT is translocated into host cells via T3SS2. Table 1. Intracellular cAMP concentrations in infected cells (pmol cAMP per well). Cell line POR-1 POR-2 POR-3 ΔvcrD1/ΔvcrD2 Caco-2 > 3200 > 3200 206.7 ± 40.4 323.3 ± 49.4 HCT-8 > 3200 > 3200 156.7 ± 55.1 296.7 ± 40.4 HeLa > 3200 > 3200 169.7 ± 81.8 201.7 ± 115.1 J774 > 3200 > 3200 313.3 ± 160.4 383.3 ± 102.1 HT-29 > 3200 > 3200 121.7 ± 53.5 233.3 ± 115.0 T84 > 3200 > 3200 196.7 ± 115.9 220.0 ± 75.5 ADPRT domain of VopT is necessary for cytotoxicity ExoS and ExoT, which are two T3SS-secreted effectors of P. aeruginosa, are closely related bifunctional proteins (75% identity at the amino acid level), containing a GTPase-activating protein (GAP) domain in the N-terminus and a 14-3-3-dependent ADPRT domain in the C-terminus. Amino acid sequence alignment showed that VopT has 45% identity with the C-terminal ADPRT domain of ExoT. The ADPRT activity of ExoS and ExoT is dependent on the binding of eukaryotic host protein(s) termed FAS (for factor-activating ExoS), a member of the 14-3-3 protein family (Coburn and Gill, 1991; Fu et al., 1993). The catalytic residues of ADPRT and the FAS-binding residues of ExoS and ExoT are identical to VopT (Fig. 4A). Therefore, we constructed three different VopT mutants, containing mutations in the catalytic domain, VopTE186,188A, in the FAS-binding residues, VopT230−234G, and in both residues, VopTE188,188A/230−234G. Then we tested the inhibitory growth effect of these mutations on yeast cells expressing these constructs. As shown in Fig. 4B, expression of wild-type VopT resulted in total inhibition of the yeast cell growth, while those yeast cells expressing VopTE186,188A showed partial inhibition of growth, forming smaller colonies on galactose-containing medium. Conversely, yeast strains expressing VopT230−234G and VopTE188,188A/230−234G were able to form colonies as large as those strains expressing the control vector. Next, we tried to determine their effect of VopT mutants on the cytotoxicity against Caco-2 cells. Although the expression of these proteins in the supernatants and bacterial pellets were at similar levels (Fig. 4C), the cytotoxicity of the complemented VopT-deletion mutant was partly (VopTE186,188A), or hardly (VopT230−234G and VopTE186,188A/230−234G) recovered (Fig. 4D). The difference in the cytotoxic effect of the VopT mutants against Caco-2 cells correlated well with the inhibitory effect on yeast cell growth (Fig. 4B). These results strongly suggest that both the ADPRT and FAS-binding domains of VopT are critical for the biological activity of VopT. ADP ribosylation activity of VopT in vivo and in vitro The ADPRT activity of ExoS was shown to result in modification of multiple substrates, including the LMWG proteins. Therefore, we checked that ADP ribosylation of several LMWG proteins, such as Ras, Cdc42, Rab3, Rab4, Rab5, Rab8, Rad11, Rad27, Rac1, RalA, Ran, Rap1, Rap2 and RhoA, by bacterially translocated VopT. ADP ribosylation was assessed based on altered protein mobility on SDS-PAGE. As far as we investigated, only Ras was modified in V. parahaemolyticus-infected Caco-2 cells (data not shown). Next, we tried to determine the effect of VopT mutants on the Ras modification. Although the expression of these proteins in the supernatants and bacterial pellets was at similar levels (Fig. 4C), Ras modification was observed only in Caco-2 cells infected with the wild-type VopT-expressing V. parahaemolyticus (Fig. 5A). None of the VopT mutants, containing mutations in the catalytic domain, VopTE186,188A, in the FAS-binding residues, VopT230−234G, and in both residues, VopTE188,188A/230−234G, altered Ras mobility on SDS-PAGE (Fig. 5A). ExoS interacts with 14-3-3 proteins in the FAS-binding residues and this interaction is necessary for the ADP ribosylation activity. To address the question of whether the FAS-binding residues of VopT is important for interaction with 14-3-3, purified wild-type VopT or VopT mutants were precipitated with GST-14-3-3 or GST alone. As a result, wild-type VopT and VopTE186,188A, which is containing mutations in the catalytic domain, interacted specifically with 14-3-3, whereas the VopT mutants, containing mutations in the FAS-binding residues (VopT230−234G and VopTE188,188A/230−234G) did not (Fig. 5B). Next, we checked ADPRT activity of VopT in vitro. The ADP-ribosylated Ras was detected in the presence of wild-type VopT, 14-3-3, and [32P]-NAD (Fig. 5C, lane 1), whereas none of ADP-rebosylated Ras was detected without 14-3-3 (lane 2) or [32P]-NAD (lane 3) or VopT (lane 4). [32P]-ADP ribosylation of Ras by wild-type VopT was inhibited by addition of 10-fold excess cold NAD, which is not labelled with 32P (lane 5). All VopT mutants (VopTE186,188A, VopT230−234G, VopTE186,188A/230−234G) did not have this activity (lane 6–8). These results indicated that VopT is an ADPRT effector that targets Ras in V. parahaemolyticus-infected cells and that the catalytic residues of ADPRT and the FAS-binding residues of VopT are necessary for this activity. Figure 5Open in figure viewerPowerPoint VopT has ADP ribosylation activity against Ras both in vivo and in vitro.A. Bacterially translocated VopT modifies Ras in vivo. Caco-2 cells were infected with V. parahaemolyticusΔvcrD1/ΔvopT strain expressing wild-type (WT) or mutant VopT for 6 h. The infected cells were lysed and were separated by SDS-PAGE (12%), and then probed with anti-Ras antibody. The mobility of modified and unmodified Ras is indicated.B. VopT interacts directly with 14-3-3 via FAS-binding motif. Purified wild-type or mutant VopT were pulled down with purified GST-14-3-3 or GST alone. Samples were separated by SDS-PAGE, and then probed with anti-VopT antibody (upper panel) and anti-GST antibody (lower panel).C. [32P]-ADP ribosylation of Ras by VopT in vitro. Ras was incubated with or without purified VopTs, 14-3-3 and [32P]-NAD. [32P]-ADP ribosylation of Ras was detected by autoradiography. Discussion The genome sequence of a clinical strain of V. parahaemolyticus, RIMD2210633, revealed that this bacterium possesses two sets of genes for T3SS (Makino et al., 2003). One set was located on chromosome 1 (T3SS1), and the genes displayed a high similarity with those of T3SS from Yersinia spp., except for the genes encoding effector proteins. In contrast, T3SS2, located on chromosome 2, was not similar to any particular T3SS of any other bacteria reported to date. The distribution of those genes among V. parahaemolyticus strains and the G+C content of the DNA regions containing T3SS1 and T3SS2 suggest that T3SS1 is intrinsic to the species, while T3SS2 has a feature of laterally transferred DNA (Makino et al., 2003). Functional analysis of both T3SSs from V. parahaemolyticus revealed that T3SS1 was involved in the cytotoxic activity of the organism against HeLa cells, while, in a rabbit model, T3SS2 was demonstrated to play a role in enterotoxicity (Park et al., 2004b). Despite that, the role of T3SS2 remains elusive, mainly because assay systems to analyse biological activities of T3SS2 are lacking, apart from the ileal loop test. Nevertheless, as T3SS2 is found exclusively among KP-positive strains of V. parahaemolyticus, it is very likely that it is involved in its pathogenicity to humans. In this study, we demonstrated that T3SS2 was involved in the cytotoxic effect against two intestinal cell lines, Caco-2 and HCT-8. Moreover, we identified vopT (VPA1327) as one of the genes that contribute to this cytotoxicity. Furthermore, we demonstrated that VopT is not only secreted by T3SS2, but is also injected into the cytoplasm of the host cells. To our knowledge, this is the first report on an effector translocated by the T3SS2 of V. parahaemolyticus. VopT is similar to the ADPRT domain of ExoT and ExoS (45% and 44% identity respectively), two effector proteins secreted by T3SS of P. aeruginosa. ExoS and ExoT are bifunctional cytotoxins, and both contain an N-terminal RhoGAP domain and a C-terminal ADP ribosylation domain (Barbieri and Sun, 2004). Protein modification catalysed by ADPRT occurs as a result of the transfer of ADP-ribose from NAD+ to specific amino acid residues on substrate proteins. As a result, the biological activities of the substrate proteins are usually severely changed (Ziegler, 2000). Two glutamic acid residues within the C-terminal ADPRT domain of ExoS and ExpT (E379, 381 of ExoS and E383, 385 of ExoT respectively) are necessary for the ADP ribosylation reaction (Liu et al., 1997; Radke et al., 1999). Binding to the mammalian protein 14-3-3, also known as FAS, activates both ExoS and ExoT (Coburn and Gill, 1991; Fu et al., 1993). Comparison between the amino acids in the ADPRT catalytic residues and the FAS-binding residues of both ExoS and ExoT with that of VopT revealed that these amino acids were conserved among these three proteins. Mutation in these conserved amino acid residues on VopT resulted in reduced yeast growth inhibition activity (Fig. 4), strongly suggesting that they are essential for the activity of VopT. Accordingly, these mutations also resulted in the loss of its cytotoxic effect (Fig. 4) and its ability to ADP-ribosylate Ras (Fig. 5). These findings confirm that VopT is an ADPRT cytotoxin. While sharing high amino acid identity (72%) within the C-terminal ADPRT domain, there were some differences between the cytotoxic and the ADP ribosylation activity of ExoS and ExoT. While ExoS was reported to induce cell death that was dependent on its ADP ribosylation activity, ExoT exhibited no effect on cell viability (Frithz-Lindsten et al., 1997; Sundin et al., 2001). Recent studies have shown that ExoS and ExoT ADP-ribosylate different substrates. ExoS has a broad substrate specificity and is able to ADP-ribosylate a large number of LMWG proteins, including Ras (McGuffie et al., 1998; Fraylick et al., 2002a,b). In contrast, ExoT did not efficiently ADP-ribosylate Ras or other LMWG proteins that are normally ADP-ribosylated by ExoS (Liu et al., 1997; Sundin et al., 2001). However, ExoT is able to ADP-ribosylate other mammalian cell proteins, such as Crk-I and Crk-II, an activity confirmed both in vitro and in vivo (Sun and Barbieli, 2003). The biological activity of VopT can be considered similar to that of ExoS in the point that VopT demonstrated both cytotoxic and Ras modification activities. Interestingly, in as far as it has been investigated, Ras was the only LMWG protein modified by VopT. How VopT and other ADP-ribosylating toxins recognize their protein substrates is an interesting issue. It has been reported that ADP ribosylation of Arg41 by ExoS disrupted Ras signalling by inhibiting the guanine nucleotide exchange factor (GEF)-catalysed nucleotide exchange, which uncoupled Ras signal transduction (Ganesan et al., 1998; 1999). However, it is not yet clear whether the cytotoxic effect by VopT is a consequence of Ras signalling disruption. Nevertheless, it can be expected that making this pathway clear would provide us with new insights on the difference in the cytotoxic sensitivity obtained with the different cell types as shown in Fig. 1. In this study, we found that T3SS2-dependent cytotoxicity was significantly decreased, but not completely abolished, by deleting the vopT gene, suggesting that other unknown effector(s) are involved in this cytotoxicity. There are a number of hypothetical genes in the T3SS2 region. It is possible that additional effector genes responsible for this cytotoxicity are encoded in this region, and we plan to explore this issue in our future research. As practically all clinical isolates of V. parahaemolyticus produced TDH, research into the pathogenicity of this bacterium had bee
Referência(s)