vitro but also for induction of fluid accumulation in vivo. To our knowledge, this is the first report showing that bile acid stimulation is actually responsible for the virulence of enteric pathogens in vivo. It has been reported that bile acids induce CT expression in V. cholerae strain O395 in a ToxRS-dependent and ToxT-indepen- dent manner [27]. Vp-ToxR of V. parahaemolyticus, a homolog of the V. cholerae ToxR, has been reported to be essential for TDH production and for the enterotoxic activities of the AQ3815 strain [39]. In our investigation, bile-induced production of TDH and T3SS2-related proteins and V. parahaemolyticus-induced fluid accumulation were dependent on VtrA and VtrB and no participation of Vp-ToxR in these processes was observed (Fig. S2A and B). Therefore, we conclude that Vp-ToxR is not responsible for bile-mediated expression of these virulence factors or for V. parahaemolyticus-induced fluid accumulation. However, this conclusion should be validated by studies on other strains of V. parahaemolyticus. It has been demonstrated that host adrenergic agonists such as norepinephrine activate transcription not only of genes encoded by the T3SS gene cluster, but also of flagellar- and shiga-toxin- producing genes [40]. There is an intriguing report that shows that the pathogenicity of V. parahaemolyticus is also augmented by norepinephrine [41]. Therefore, we considered the possibility that the suppressive effect of the cholestyramine-treated ileal loop on fluid accumulation was due to absorptive removal of norepineph- rine. However, as shown in Fig. S4, exogenous norepinephrine administration did not result in recovery of fluid accumulation in the cholestyramine-treated ileal loop. Norepinephrine does not appear to elevate transcription of T3SS2 genes [41]. Thus, we conclude that norepinephrine removal does not contribute to the attenuating effect of cholestyramine treatment on fluid accumu- lation and that the effect of norepinephrine on V. parahaemolyticus- induced fluid accumulation reported by Nakano et al. occurs via a pathway other than that through which the effects of bile acids are exerted. In contrast to the effect of crude bile on the production of T3SS2-related proteins and TDH, expression of T3SS1-related proteins was suppressed by bile (Fig. S1A), which was also illustrated by decreased cytotoxic activity against Caco-2 cells (Fig. S1B). However, bile did not significantly affect the level of transcription of these genes according to microarray analysis (Fig. 3A and Table S2), suggesting that a compound (or compounds) in crude bile is involved in posttranscriptional regulation of T3SS1-related protein expression. This study revealed that 1) V. parahaemolyticus recognizes its arrival in the intestine by sensing bile acids, 2) bile acids induce the transcription of Vp-PAI genes via VtrA and VtrB and 3) transduction of this signal facilitates fluid accumulation in the host. This mechanism of bile acid-induced expression of virulence genes in the human intestinal tract appears to be common to T3SS2-positive pathogens because fluid accumulation induced by trh-positive V. parahaemolyticus and non-O1/non-O139 V. cholerae was also inhibited by cholestyramine (Fig. 5). These prevention effects of cholestyramine treatment on fluid accumulation in vivo were less effective compared with that of in vitro assays. We predict two possible causes. One possibility is that cholestyramine treatment could not remove endogenous bile salts from the intestinal tract completely. As shown in Fig. 1 and Fig. 4, bile and bile acids can induce expression of Vp-PAI genes at low concentration. Therefore, residual bile acids in intestine might remain to stimulate Vp-PAI genes expression. Another possibility is that a host-derived inducer(s) other than bile acids exists in the intestinal tract. We will plan to explore this issue in our future research. Despite of this, it is amazing that these pathogens, which Vp-PAI Gene Expression occur naturally in marine, estuarine or aquatic environments, are equipped with an extremely specialized sensing system for adapting to the human body and causing disease. Cholestyramine is a bile acid sequestrant that binds bile acids in the gastrointestinal tract, forming an insoluble complex [42,43]. Such sequestration stimulates the conversion of plasma cholesterol into bile acids to normalize intestinal bile acid levels. As it has very few side effects, cholestyramine is frequently used to treat hypercholesterolemia [43]. Given the increase in numbers of antibiotic-resistant pathogenic bacteria and the emergence of multidrug-resistant strains of vibrios from patients and environ- mental sources worldwide [44–48], drugs targeted at suppressing bacterial virulence mechanisms instead of killing bacteria or inhibiting their growth (the aims of most conventional antibiotics) constitute an alternative approach to treating infections [49–51]. Inactivation of bile acids using sequestrants such as cholestyramine exploits a weakness of T3SS2-positive pathogens and represents a potential novel antivirulence therapy that may attenuate the development of drug-resistant bacteria. This new approach warrants further validation. Materials and Methods Bacterial strains and plasmids V. parahaemolyticus strain RIMD2210633 (KP positive, serotype O3:K6) [17] was used for constructing deletion mutants and for functional analysis. E. coli DH5a and SM10lpir were used for general manipulation of plasmids and mobilization of plasmids into V. parahaemolyticus. The strains and plasmids used in this study are listed in Table S2. Immunoblot analysis V. parahaemolyticus was grown overnight in LB broth containing 3% NaCl at 37uC. For measurement of protein production under various temperature conditions, an overnight culture was diluted 1:100 into LB with 0.5% NaCl and grown to an OD600 of 1.0. For measurement of protein production under various osmotic conditions, an overnight culture was diluted 1:100 into LB medium containing various concentrations of NaCl and grown to an OD600 of 1.0. For crude bile and bile salt induction experiments, cells were grown for 3 h in LB medium containing 0.3 M NaCl with or without crude bile (OX Gall powder, Sigma) or bile salts (Sigma). After incubation, bacterial cultures were centrifuged and bacterial pellets were solubilized using Laemmli buffer. Secreted proteins were harvested by precipitation with trichloroacetic acid (10% v/v) on ice for 60 min, followed by centrifugation at 48,000 g for 60 min. The pellets were rinsed in cold acetone and solubilized in Laemmli buffer. Samples for western blot analysis were separated using SDS PAGE (12.5% polyacrylamide; COSMO BIO). The transferred membranes were probed with anti-VscC1, anti-VopD1, anti- VepA, anti-VscC2, anti-VopD2, anti-VopC, anti-TDH, anti-VtrA or anti-VtrB polyclonal antibodies or with anti-RNA polymerase (RNAP) b-subunit monoclonal antibody (SANTA CRUZ BIO- TECHNOLOGY) and then probed with horseradish peroxidase- conjugated goat anti-rabbit antibody (ZYMED). The blots were developed using enhanced chemiluminescence western blotting kits (GE HEALTHCARE). Cytotoxicity assay Cytotoxicity assays were performed as previously described [23]. Briefly, V. parahaemolyticus strains were grown in LB medium containing 0.3 M NaCl with or without 0.04% crude bile for 3 h and were washed with PBS. Caco-2 cells were cocultured for 4.5 h PLoS ONE | www.plosone.org 9 October 2010 | Volume 5 | Issue 10 | e13365