Vp-PAI Gene Expression Figure 2. VtrA and VtrB are required for the production of crude bile-induced T3SS2-related proteins and TDH. A. Loss of vtrA and/or vtrB diminishes crude bile-induced TDH and T3SS2-related protein production. Western blot analysis of bacterial pellets (left panel) and secreted proteins (right panel) from isogenic mutants of WT V. parahaemolyticus cultured in the presence (+) or absence (2) of 0.04% crude bile. Blots were probed with RNAP, TDH and T3SS2-related antibodies. B. Crude bile stimulation does not affect the transcription of vtrA. V. parahaemolyticus strains carrying a control vector (left) or a vtrA-lacZ transcription fusion vector (right) were cultured in the absence (gray bars) or presence (black bars) of 0.04% crude bile. b-Galactosidase activity is expressed in Miller units. Data are expressed as the average 6 SD of three separate experiments. C. Crude bile stimulates vtrA-mediated vrtB transcription. V. parahaemolyticus strains carrying a control vector or a vtrB-lacZ transcription fusion vector were cultured in the absence (gray bars) or presence (black bars) of 0.04% crude bile. b-Galactosidase activity is expressed in Miller units. Data are expressed as the average 6 SD of three separate experiments. D. Crude bile induces the production of VtrB protein. Immunoblots of WT V. parahaemolyticus cultured in the presence (+) or absence (2) of 0.04% crude bile. Blots were probed with anti-VtrA (upper panel), anti-VtrB (middle panel) and anti-RNAP (lower panel) antibodies. doi:10.1371/journal.pone.0013365.g002 bile acid sequestered with cholestyramine resin (Cho-bile), whereas no such effect was observed after treatment with the control resin, Dowex800 400 mesh (Dow-bile), which does not bind bile acids [29] (Fig. 4A). Similar results were observed for immunoblotting of TDH and T3SS2-related proteins, as the cholestyramine-treated crude bile lacked the capacity to induce them (Fig. 4B). These PLoS ONE | www.plosone.org 4 October 2010 | Volume 5 | Issue 10 | e13365