within Vp-PAI, including the genes for TDH and the T3SS2- related proteins. Because mutants of vtrA and vtrB did not exhibit any in vivo fluid-accumulating activity in the rabbit intestine, this demonstrated that these regulators play critical roles in the enterotoxicity of V. parahaemolyticus [23]. Although this shows that expression of Vp-PAI genes is required for induction of fluid accumulation in the intestine, the environmental and/or host- derived factors that trigger the expression of these genes remain unknown. The aim of this study was to identify a host factor that triggers the expression of Vp-PAI genes and to determine its role in the pathogenicity of V. parahaemolyticus. Results Crude bile stimulates the production of TDH and T3SS2- related proteins under intestinal osmotic conditions To identify an environmental factor in the intestine that affects the expression of Vp-PAI genes, we first examined the effect of cultivation temperature on the production of TDH and the T3SS2-related proteins, VopD2 (T3SS2 translocon protein), VopC (T3SS2 effector protein) and VscC2 (T3SS2 apparatus protein), using immunoblotting. Much higher abundances of these proteins were detected when bacteria were cultured at 37 and 42uC, which corresponds to the temperature of the intestine, than at lower temperatures (Fig. 1A). To determine the effect of extracellular osmotic pressure on the production of these proteins, we next grew V. parahaemolyticus at 37uC in LB medium and adjusted the osmotic pressure from 0.1 M to 0.5 M by adding NaCl (Fig. 1B). The greatest production of Vp-PAI proteins was observed for bacteria cultured in medium containing 0.1 M NaCl. Protein production gradually decreased as NaCl concentration increased and was particularly impaired at NaCl concentrations greater than 0.3 M. This result was unexpected because the osmolarity of the intestinal lumen is estimated to exceed that of a 0.3 M NaCl solution [24]. Therefore, we reasoned that a host- derived inducer (or host-derived inducers) that counteracts the inhibitory effect of intestinal osmotic pressure on Vp-PAI protein production must exist in the intestinal tract. After evaluating various candidate factors, we finally identified bile as a potent stimulator of the production of these proteins. As shown in Fig. 1C, crude bile induced the production of TDH and T3SS2-related proteins in a concentration-dependent manner in the presence of 0.3 M NaCl. The inducing activity of crude bile became saturated at a concentration of 0.04%, which corresponds with the concentration of bile in the intestinal tract. A characteristic of pathogenic V. parahaemolyticus is its T3SS2-mediated cytotoxic effects on Caco-2 cells in vitro [25]. Therefore, the effect of crude bile on cytotoxicity was evaluated by preculturing bacteria in the presence or absence of various concentrations of crude bile (Fig. 1D). Although a TDH- and T3SS1-deficient strain (POR-2) precultured without crude bile did not induce any apparent cytotoxic effects, crude bile stimulated the cytotoxicity of this strain in a concentration-dependent manner. No cytotoxic effect was observed in a TDH- and T3SS1/T3SS2-deficient strain (DvcrD1/ DvcrD2) regardless of the presence of bile. To ensure that the stimulatory effect of bile was specific for T3SS2 and TDH, the effect of crude bile on the production of T3SS1-related proteins was also examined (Fig. S1A). In contrast to the aforementioned crude bile-mediated increase in the production of T3SS2-related proteins and TDH, with this strain an increase in the concentration of crude bile was accompanied by a decrease in the production of T3SS1-related proteins. In addition, this repressive effect of crude bile was reflected in decreased T3SS1- dependent cytotoxicity (Fig. S1B). As the osmolarity of the Vp-PAI Gene Expression intestinal lumen is higher than that of a 0.3 M NaCl solution, we next determined whether bile-induced TDH- and T3SS2- protein expression would overcome the repressive effects of high osmotic pressure. Fig. 1E shows that crude bile stimulated the production of these proteins at concentrations of up to 0.5 M NaCl. Together, these results indicate that crude bile is a potent host-derived inducer of TDH and T3SS2-related protein produc- tion under osmotic conditions corresponding to those in the intestinal tract. VtrA and VtrB mediate crude bile-induced production of T3SS2-related proteins and TDH It has been reported that three transcriptional regulators are involved in the expression of TDH and/or T3SS2-related proteins. One of these, Vp-ToxR, shares some identity with the V. cholerae ToxR (Vc-ToxR). The Vc-ToxR is known to regulate expression of multiple genes, including the cholera toxin (CT) and toxin-coregulated pilus (TCP) genes [26], and is also involved in sodium cholate induction of CT [27]. The other candidates are VtrA and VtrB, which were recently identified as master regulators of virulence gene expression in the Vp-PAI [23]. We next examined whether any of these regulators are involved in bile-induced production of TDH and T3SS2-related proteins. No participation of the Vp-ToxR gene in crude bile-induced production of TDH and T3SS2-related proteins was observed (Fig. S2A). A WTDtoxR strain induced fluid accumulation in a rabbit ileal loop to a similar extent as WT bacteria over a range of inoculation doses (Fig. S2B). In contrast to Vp-ToxR, vtrA and vtrB deletion mutants did not induce any fluid accumulation (Fig. S2B), nor did they produce TDH and T3SS2-related proteins, even in the presence of crude bile (Fig. 2A). As a previous report showed that the expression of VtrB is controlled directly by VtrA [23], we next examined whether crude bile affects the expression of these regulators using vtrA-lacZ and vtrB-lacZ transcriptional fusion reporters. As shown in Fig. 2B, crude bile stimulation did not have any influence on vtrA-lacZ transcription. In contrast, substantial induction of vtrB-lacZ transcription by crude bile was observed in both WT and vtrB-deficient V. parahaemolyticus strains (WT and WTDvtrB), whereas deletion of the vtrA gene (WTDvtrA) caused a lack of responsiveness similar to that of a double deletion mutant (WTDvtrADvtrB) (Fig. 2C). Immunoblotting of VtrA and VtrB revealed that the production of VtrA was constant regardless of the presence of crude bile, whereas the production of VtrB protein was induced only when crude bile was present (Fig. 2D). These results indicate that crude bile induces VtrA-mediated VtrB expression and that this transcriptional regulatory cascade is essential for crude bile-induced production of TDH and T3SS2- related proteins. Genome-wide transcriptional analysis of crude bile-responsive genes in V. parahaemolyticus For complete identification of the crude bile-responsive genes of V. parahaemolyticus, genome-wide transcriptional profiles of WT or vtrA-deficient strains grown in the presence of 0.04% crude bile were compared with that of the WT strain grown in the absence of crude bile (Fig. 3). The expression levels of 77 genes displayed significant changes ($ a fourfold difference, P,0.05) in WT cells grown in the presence of crude bile (Table S1). In most cases, upregulation was observed and, interestingly, most of the upregulated genes were located within a distinct region of chromosome 2 (Fig. 3A). This region is included in the Vp-PAI region; in the vtrA-deficient strain, induction of these genes was absent (Table S1). Hierarchical clustering analysis classed the PLoS ONE | www.plosone.org 2 October 2010 | Volume 5 | Issue 10 | e13365