Vp-PAI Gene Expression Figure 4. Identification of a transcriptional inducer for Vp-PAI genes in crude bile. A. VtrB expression in bile acid-sequestered crude bile. V. parahaemolyticus carrying the vtrB-lacZ transcription fusion vector were incubated in LB medium (0.3 M NaCl) with no crude bile (2), crude bile, cholestyramine-treated crude bile (Cho-Bile), or Dowex-treated crude bile (Dow-Bile). b-Galactosidase activity is expressed in Miller units. Data are expressed as the average 6 SD of three separate experiments. B. Bile acid depletion from crude bile diminishes induction of TDH and T3SS2-related protein production. Immunoblot analysis of V. parahaemolyticus cultured in LB broth containing 0.3 M NaCl at 37uC with no crude bile (2), crude bile, cholestyramine-treated crude bile (Cho-Bile) or Dowex-treated crude bile (Dow-Bile). Blots were probed with anti-TDH, anti-VopD2, anti-VopC, anti- VscC2, anti-VtrB and anti-RNAP (RNA polymerase) antibodies. C. VtrB expression in the presence of various bile salts. vtrB-lacZ transcriptional activity was measured in the presence of various concentrations (0.1–1000 mM) of taurodeoxycholate (TDC; black line), glycodeoxycholate (GDC; red line), deoxycholate (DC; blue line), taurochenodeoxycholate (TCDC; green line), glycochenodeoxycholate (GCDC; light blue line), taurocholate (TCA; purple line), glycocholate (GCA; pink line), chenodeoxycholate (CDC; gray line) or cholate (CA; orange line). The data are expressed as the average 6 SD of the ratio of LacZ activity in cells grown with bile acids relative to LacZ activity in cells grown without bile acids for three separate experiments. D. TDC and GDC independently induce the production of VtrB protein. Immunoblot analysis of V. parahaemolyticus cultured in LB medium containing 0.3 M NaCl at 37uC with various concentrations (0.1–1000 mM) of TDC (left panel) or GDC (middle panel) or with 1000 mM of CA, CDC or with 0.04% crude bile (right panel). Blots were probed with anti-VtrA and anti-VtrB antibodies. E. The addition of TDC or GDC restores the diminished activity of cholestyramine-treated crude bile to induce vtrB expression. Cholestyramine-treated crude bile (Cho-bile) was supplied with 0.04% crude bile or 100 mM of TDC, GDC, CA or CDC. Effects on vtrB expression were assessed according to a b-galactosidase assay. b-Galactosidase activity is expressed in Miller units. Data are expressed as the average 6 SD of three separate experiments. F. Effect of the addition of bile acids to cholestyramine-treated crude bile on the production of VrtB protein. Cholestyramine-treated crude bile (Cho-bile) was supplied with 100 mM of TDC, GDC, CA or CDC. Effects on VtrB production were assessed according to immunoblotting using anti-VtrB and anti-RNAP antibodies. doi:10.1371/journal.pone.0013365.g004 Endogenous bile acids in the intestinal tract of rabbits are necessary for induction of fluid accumulation To determine whether the presence of endogenous bile acids in the intestine is necessary for in vivo enterotoxicity, fluid accumu- lation induced by various doses of V. parahaemolyticus in cholestyramine-treated rabbit ileal loops were compared with fluid accumulation in untreated loops (Fig. 5A). Cholestyramine- treated rabbit ileal loops were prepared by inoculating the resin into the loops. The loops were then washed with PBS to remove residual resin before infection (see Material and methods). Although fluid accumulation in cholestyramine-treated loops inoculated with 109 CFU of V. parahaemolyticus was similar to that in untreated loops, cholestyramine significantly reduced fluid accumulation at inoculation doses ranging from 106 to 108 CFU. In contrast, fluid accumulation in control resin (Dowex800 400 mesh)-treated loops was similar to that in untreated loops for every inoculation dose. To determine whether the attenuating effect of cholestyramine treatment on fluid accumulation was caused by absorptive removal of endogenous bile acids from the intestinal tract, we examined the effect of adding crude bile or individual bile salts to cholestyramine-treated loops (Fig. 5B). Fluid accumulation in cholestyramine-treated loops was significantly increased by the addition of crude bile or the vtrB-inducing bile salts, TDC and GDC, whereas addition of CA and CDC, which did not induce vtrB expression, did not increase fluid accumulation. As it was reported that norepinephrine—a hormone that exerts a modula- tory effect in the gastrointestinal tract—enhances T3SS2-depen- dent fluid accumulation in a rat ileal loop model (Nakano et al., 2007), we examined whether the attenuating effect of cholestyr- amine-treatment on fluid accumulation was due to absorptive removal of norepinephrine. However, norepinephrine addition did not restore the reduced fluid accumulation of cholestyramine- treated loops (Fig. S4). We next examined the effect of coadministration of cholestyramine resin and the bacterial suspension on the induction of fluid accumulation (Fig. 5C). The preventive effect of cholestyramine resin on fluid accumulation was stronger when added together with the bacterial suspension than when the loops were pretreated with cholestyramine and then inoculated (cf. Fig. 5C with Fig. 5A) and a significant decrease in fluid accumulation was observed even with the highest dose of V. parahaemolyticus (Fig. 5C). In contrast, coadministration of Dowex800 400 mesh resin had no effect on fluid accumulation. As a T3SS gene cluster similar to the T3SS2 gene cluster of KP- positive V. parahaemolyticus is present in trh-positive (KP-negative) V. parahaemolyticus and non-O1/non-O139 V. cholerae [21,22], we next examined whether cholestyramine would also prevent the induction of fluid accumulation by these species. For the non- O1/non-O139 V. cholerae, we first performed PCR-based geno- typing to confirm the presence of T3SS2-related genes (Fig. S5). Fluid accumulation induced by both trh-positive V. parahaemolyticus and the V. cholerae strain was significantly decreased when the bacteria were injected simultaneously with cholestyramine (Fig. 5D). These results indicate that endogenous bile acids in the intestinal tract are at least one of a host-derived inducer for induction of fluid accumulation by T3SS2-positive pathogens and sequestration of bile acids by cholestyramine resin may be useful for preventing diarrhea caused by these bacteria. Discussion T3SS2-related genes encoded in the Vp-PAI region are considered to be involved in the pathogenicity of V. parahaemolyticus to humans [18,20]. We previously showed that two positive regulators, VtrA and VtrB, are essential for expression of these genes and that vtrA- and vtrB-null mutants lack the capacity to induce fluid accumulation in vivo [23]. However, nothing has been known about environmental factors that affect expression of Vp- PAI genes or the host-derived factors that trigger the production of these virulence genes. In this study, we demonstrated that several bile acids in crude bile strongly elevate transcription of Vp-PAI- encoded genes under intestinal osmotic conditions. The Vp-PAI gene-inducing activity of crude bile and bile acids was saturated at concentrations of 0.04% and 100 mM, respectively. Although the bile acid concentration in the intestine varies, it usually ranges from 0.2% to 4% [28]. Therefore, the concentrations of crude bile and bile acids used in this study are representative of concentra- tions in vivo. The presence of bile in the lumen of the human intestine is necessary for the digestive process [28]. Bile acids, a major component of crude bile, affect the production of the virulence factors of several enteric pathogens [28,30]. For instance, bile has been shown to reduce the invasion of Salmonella typhimurium into eukaryotic cells through transcriptional repression of T3SS genes located in the Salmonella pathogenicity island (SPI-1) [31]. In contrast to Salmonella, bile induces Shigella spp. invasiveness in that bile salts DC and CDC induce secretion of the T3SS protein, Ipa, which promotes the invasion of epithelial cells [32]. In V. cholerae, production of the major virulence factors, CT and TCP, was substantially reduced by bile in a ToxT-dependent manner (ToxT is a transcriptional activator of these genes) [33,34]. Osawa et al. demonstrated that production of TDH by V. parahaemolyticus was PLoS ONE | www.plosone.org 7 October 2010 | Volume 5 | Issue 10 | e13365