Vp-PAI Gene Expression Figure 1. Crude bile stimulates the production of TDH and T3SS2-related protein under high osmotic conditions. A. Effect of temperature on the production of TDH and T3SS2-related proteins. Immunoblot analysis of bacterial whole cell pellets (left) and secreted proteins (right) from WT V. parahaemolyticus cultured in LB medium containing 0.5% NaCl at various temperatures (20–42uC). Blots were probed with anti- TDH, anti-VopD2, anti-VopC, anti-VscC2 and anti-RNAP antibodies. B. Effect of osmolarity of the culture medium on the production of TDH and T3SS2- related proteins. Immunoblot analysis of bacteria cultured in LB medium containing various concentrations of NaCl (0.1–0.5 M) at 37uC. C. Effect of crude bile on the production of TDH and T3SS2-related proteins. Immunoblot analysis of bacteria cultured in LB medium containing 0.3 M NaCl at 37uC in the presence of various concentrations of crude bile (0–0.04%). D. Crude bile promoted the T3SS2-dependent cytotoxicity of V. parahaemolyticus. V. parahaemolyticus strains (DvcrD1DvcrD2 [TDH and T3SS1/T3SS2-deficient strain], gray bar; POR-2 [TDH and T3SS1-deficient strain], black bar) were cultured in LB medium containing 0.3 M NaCl at 37uC in the presence of various concentrations of crude bile (0–0.04%) for 3 h. After incubation, bacteria were used for infection of Caco-2 cells for 4.5 h. Cytotoxicity was evaluated according to the amount of LDH released. Error bars represent the SDs of means from triplicate independent experiments. E. Crude bile stimulation overcomes the repression of TDH and T3SS2-related protein production under high osmotic pressure. Immunoblot analysis of V. parahaemolyticus cultured in LB medium containing various concentrations of NaCl (0.1–0.5 M) in the presence (+) or absence (2) of 0.04% crude bile. doi:10.1371/journal.pone.0013365.g001 genes of filtered microarray data into three clusters: genes downregulated by crude bile stimulation in both WT and WTDvtrA (Group1), genes upregulated in both WT and WTDvtrA (Group2) and genes upregulated in WT but not significantly changed in WTDvtrA (Group3) (Fig. 3B). Interestingly, all the genes in Group3 were encoded within the Vp-PAI region. These results indicate that crude bile specifically promotes expression of Vp-PAI genes and that this induction is regulated by vtrA. Identification of a transcription-inducing substance for Vp-PAI genes in crude bile Crude bile is a mixture of organic and inorganic compounds whose major constituents include bile acids, cholesterol, phospho- lipids and the pigment biliverdin [28]. To identify a transcription- inducing substance for Vp-PAI genes in crude bile, we next examined the effect of bile acid depletion. The ability of crude bile to induce vtrB-lacZ transcription disappeared upon treatment with PLoS ONE | www.plosone.org 3 October 2010 | Volume 5 | Issue 10 | e13365