Vp-PAI Gene Expression Figure 5. Endogenous bile acids in the intestinal tract are necessary for efficient induction of fluid accumulation by V. parahaemolyticus. A. Absorptive removal of endogenous bile salts from the intestinal tract using cholestyramine attenuates V. parahaemolyticus- induced fluid accumulation. Rabbit ileal loops were treated with PBS (white bars), 5% cholestyramine (black bars) or 5% Dowex800 400 mesh (gray bars) before washing with PBS and infection with various doses of bacteria (105–109 CFU). Fluid accumulation in each loop was measured 16 h after the challenge. Values are expressed in terms of the amount of accumulated fluid (ml) per cm of ligated rabbit small intestine. Error bars represent SDs for experiments conducted in sextuplicate. Asterisks indicate significant differences from the PBS-treated ileal loops (P,0.001). B. Addition of TDC or GDC restores the attenuated fluid accumulation of cholestyramine-treated ileal loops. V. parahaemolyticus (107 CFU) were suspended in LB medium containing 0.04% crude bile or 100 mM TDC, GDC, CA or CDC before injection into cholestyramine-treated ileal loops. The fluid accumulation in each loop was measured 16 h after infection. Error bars represent SDs for means from experiments conducted in sextuplicate. Asterisks indicate significant differences from nonsupplemented control loops (P,0.05). C. Prevention of V. parahaemolyticus-induced fluid accumulation by coadministration of cholestyramine. Various doses of V. parahaemolyticus (106–109 CFU) were suspended in LB medium alone (white bars), LB medium containing 1% cholestyramine (black bars) or LB medium containing 1% Dowex800 400 mesh (gray bars) before injection into rabbit ileal loops. Fluid accumulation in each loop was measured 16 h after infection. Error bars represent SDs for experiments conducted in sextuplicate. Asterisks indicate significant differences from the means for resin-free ileal loops (*P,0.001, **P,0.05). D. Prevention of trh-positive V. parahaemolyticus- and non-O1/non-O139 V. cholerae-induced fluid accumulation by coadministration of cholestyramine. TH3996 (trh-positive V. parahaemolyticus) or RIMD2214243 (non-O1/non- O139 V. cholerae) (109 CFU) were suspended in LB medium alone (white bars), LB medium containing 1% cholestyramine (black bars) or LB medium containing 1% Dowex800 400 mesh (gray bars) before injection into rabbit ileal loops. Fluid accumulation in each loop was measured 16 h after infection. Error bars represent SDs for experiments conducted in sextuplicate. Asterisks indicate significant differences from resin-free ileal loops (*P,0.05). doi:10.1371/journal.pone.0013365.g005 enhanced by several bile acids [35,36]. It was also reported that bile and DC increased adherence of both KP-positive and KP- negative V. parahaemolyticus to Int-407 cells in vitro [37]. Recently, it was reported that bile modulated T3SS genes expression of non- O1/non-O139 V. cholerae [38]. As described above, bile is considered closely associated with the production of virulence factors of enteric pathogens. However, the importance and role of bile during the in vivo infection process has remained largely unknown. Here, we demonstrated that bile acids are important not only for the production of T3SS2-related proteins and TDH in PLoS ONE | www.plosone.org 8 October 2010 | Volume 5 | Issue 10 | e13365