with PBS-washed bacteria at a multiplicity of infection of 10. The release of lactate dehydrogenase (LDH) into the medium was quantified using CytoTox96 (Promega). The LDH release (percentage cytotoxicity) was calculated using the following equation: ((OD at 490 nm [OD490] of experimental release – OD490 of spontaneous release)/(OD490 of maximum release – OD490 of spontaneous release)) 6 100. Spontaneous release was defined as the amount of LDH released from the cytoplasm of uninfected cells, whereas maximum release was defined as the amount of LDH released after total lysis of uninfected cells. Microarray experiments Microarray analyses were performed as previously described [23]. Briefly, V. parahaemolyticus strains were grown at 37uC in LB broth containing 0.3 M NaCl to an OD600 of 0.6, and were then incubated with or without 0.04% crude bile for 30 min. Bacteria were harvested by centrifugation and highly purified total RNA was finally isolated using QIAGEN RNeasy mini kits according to the manufacturer’s protocol. RNA was transcribed to DNA and labeled with aminoallyl dUTP using reverse transcriptase (Superscript III; Invitrogen) and random hexamers (TAKARA Bio) as primers. The aminoallyl-labeled DNA was labeled with Cy3 or Cy5 dye. Cy3- or Cy5-labeled probe mixtures were applied to microarray slides, which were then incubated for 16 h at 55uC in a MAUI hybridization chamber. After washing, the microarray slides were scanned using a Scan Array Express Lite instrument (Perkin Elmer Life and Analytical Sciences). Each experiment was repeated in triplicate. Microarray data were analyzed using ScanArray Express software (Perkin Elmer Life and Analytical Sciences). Data were filtered for statistical significance (P,0.05) using a MultiExperiment Viewer t test (MeV, http://www.tm4. org/mev/). The hierarchical clustering of filtered microarray data was performed using the average-linkage method, the euclidean distance metric and MeV. All of gene expression data were MIAME compliant and were deposited in the NCBI Gene Expression Omnibus database (GEO; http://www.ncbi.nlm.nih. gov/geo/) under accession number GSE21666. Reporter gene assays V. parahaemolyticus strains harboring a reporter plasmid were grown for 2 h at 37uC in LB broth containing 0.3 M NaCl with or without 0.04% crude bile or various concentrations of bile salts. The enzymatic activity of cell lysates was measured using Miller’s method with o-nitrophenyl-b-D-galactopyranoside as substrate [52]. Rabbit ileal loop test V. parahaemolyticus strains were grown overnight in LB broth containing 0.3 M NaCl. Cultures were then diluted 1:100 into LB broth with 0.3 M NaCl and grown with shaking for 5.5 h. After incubation, bacteria were harvested by centrifugation and suspended in LB broth containing 0.3 M NaCl. To determine the effect of endogenous bile acid depletion from the rabbit small intestine on fluid accumulation, 5% cholestyramine (Sigma) or Dowex800 400 mesh (Sigma) slurry in PBS was passed through the small intestine. After washing with PBS to remove the remaining resin, the bacterial suspensions (105–109 CFU per loop) were injected into the ileal loops. To determine the effect of coexistence of cholestyramine or Dowex800 400 mesh on fluid accumulation, various concentrations (106–109 CFU/ml) of bacteria were suspended in LB broth containing 1% cholestyramine or Dowex800 400 mesh and then injected into the rabbit ileal loops. The fluid accumulation in each loop was measured 16 h after the challenge. The result was expressed as the amount of accumulated fluid (ml) per cm of ligated rabbit small intestine. All animal Vp-PAI Gene Expression experiments were performed according to an experimental protocol approved by the Ethics Review Committee for Animal Experimentation of the Research Institute for Microbial Diseases (Osaka University, Osaka, Japan). Statistical analysis All data are presented as the mean 6 SD of three determinations per experimental condition. Statistical significance was determined using one-way ANOVA followed by Dunnett’s multiple comparison test. P , 0.05 was considered statistically significant. Supporting Information Figure S1 Crude bile represses production of T3SS1-related proteins. A. Effect of crude bile on the production of T3SS1- related proteins by V. parahaemolyticus. Immunoblot analysis of V. parahaemolyticus cultured in LB broth (0.3 M NaCl) at 37uC in the presence of various concentrations of crude bile (0-0.04%). Blots were probed with anti-VopD1 (T3SS1 translocon protein), anti-VepA (T3SS1 effector protein), anti-VscC1 (T3SS1 apparatus protein) and anti-RNAP antibodies. B. Crude bile represses T3SS1-dependent cytotoxicity of V. parahaemolyticus. V. para- haemolyticus strains (DvcrD1DvcrD2, gray bar; POR-3, 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, the strains were exposed to Caco-2 cells for 4.5 h. Cytotoxicity was evaluated according to the amount of LDH released. Error bars represent SDs for triplicate independent experiments. Found at: doi:10.1371/journal.pone.0013365.s001 (8.73 MB TIF) Figure S2 ToxR is not necessary for crude bile-induced TDH and T3SS2-related protein production or for V. parahaemolyti- cus-induced fluid accumulation. A. Production of TDH and T3SS2-related proteins by the toxR mutant strain in the presence of crude bile. Immunoblot analysis of V. parahaemolyticus strains cultured in LB broth containing 0.3 M NaCl at 37uC with (+) or without (-) 0.04% crude bile. Blots were probed with anti-TDH, anti-VopD2, anti-VopC, anti-VscC2, anti-VtrB, anti-VtrA, and anti-RNAP (RNA polymerase) antibodies. B. Fluid accumulation induced by the toxR mutant strain. Fluid accumulation induced by various doses (106-109 CFU per loop) of the toxR mutant (gray bars) and a high dose (109 CFU per loop) of WTDvtrA (light blue bar) or WTDvtrB (orange bar) were compared with that of fluid accumulation in the presence of the WT (black bars). Data are expressed as the amount of accumulated fluid (ml) per cm of ligated rabbit small intestine. Error bars represent SDs for experiments conducted in sextuplicate. Found at: doi:10.1371/journal.pone.0013365.s002 (9.39 MB TIF) Figure S3 Production of TDH and T3SS2-related proteins in various concentrations of bile salts. Immunoblot analysis of bacterial whole cell pellets from V. parahaemolyticus cultured in LB medium containing 0.3 M NaCl at 37uC in the presence of various concentrations (0.1-1000 mM) of bile salts (taurodeox- ycholate, TDC; glycodeoxycholate, GDC; deoxycholate, DC; taurochenodeoxycholate, TCDC; glycochenodeoxycholate, GCDC; taurocholate, TCA; glycocholate, GCA). Blots were probed with anti-TDH (A), anti-VopD2 (B), anti-VopC (C), anti- VscC2 (D) or anti-RNAP (E) antibodies. F: Immunoblot analysis of bacterial whole cell pellets from V. parahaemolyticus cultured with or without 1000 mM CA, CDC or 0.04% crude bile. Blots were probed with anti-TDH, anti-VopD2, anti-VopC, anti-VscC2 or anti-RNAP antibodies. PLoS ONE | www.plosone.org 10 October 2010 | Volume 5 | Issue 10 | e13365