Bile Acid-Induced Virulence Gene Expression of Vibrio parahaemolyticus Reveals a Novel Therapeutic Potential for Bile Acid Sequestrants Kazuyoshi Gotoh1, Toshio Kodama2*, Hirotaka Hiyoshi1, Kaori Izutsu1, Kwon-Sam Park2,3, Rikard Dryselius1,4, Yukihiro Akeda2,5, Takeshi Honda2, Tetsuya Iida1 1 Laboratory of Genomic Research on Pathogenic Bacteria, International Research Center for Infectious Diseases, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan, 2 Department of Bacterial Infections, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan, 3 Department of Food Science and Bio Technology, College of Ocean Science and Technology, Kunsan National University, Kunsan, Korea, 4 Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden, 5 Laboratory of Clinical Research on Infectious Disease, International Research Center for Infectious Diseases, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan Abstract Vibrio parahaemolyticus, a bacterial pathogen, causes human gastroenteritis. A type III secretion system (T3SS2) encoded in pathogenicity island (Vp-PAI) is the main contributor to enterotoxicity and expression of Vp-PAI encoded genes is regulated by two transcriptional regulators, VtrA and VtrB. However, a host-derived inducer for the Vp-PAI genes has not been identified. Here, we demonstrate that bile induces production of T3SS2-related proteins under osmotic conditions equivalent to those in the intestinal lumen. We also show that bile induces vtrA-mediated vtrB transcription. Transcriptome analysis of bile-responsive genes revealed that bile strongly induces expression of Vp-PAI genes in a vtrA-dependent manner. The inducing activity of bile was diminished by treatment with bile acid sequestrant cholestyramine. Finally, we demonstrate an in vivo protective effect of cholestyramine on enterotoxicity and show that similar protection is observed in infection with a different type of V. parahaemolyticus or with non-O1/non-O139 V. cholerae strains of vibrios carrying the same kind of T3SS. In summary, these results provide an insight into how bacteria, through the ingenious action of Vp-PAI genes, can take advantage of an otherwise hostile host environment. The results also reveal a new therapeutic potential for widely used bile acid sequestrants in enteric bacterial infections. Citation: Gotoh K, Kodama T, Hiyoshi H, Izutsu K, Park K-S, et al. (2010) Bile Acid-Induced Virulence Gene Expression of Vibrio parahaemolyticus Reveals a Novel Therapeutic Potential for Bile Acid Sequestrants. PLoS ONE 5(10): e13365. doi:10.1371/journal.pone.0013365 Editor: Adam J. Ratner, Columbia University, United States of America Received July 7, 2010; Accepted September 7, 2010; Published October 13, 2010 Copyright: 2010 Gotoh et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by Grants-in-Aid for Young Scientists (21790419, http://kaken.nii.ac.jp/ja/p/21790419) and Scientific Research on Priority Areas, Matrix of Infection Phenomena (18073003, http://kaken.nii.ac.jp/ja/p/18073003), a Grant-in-Aid for Scientific Research (B) (20390128, http://kaken.nii.ac.jp/ ja/p/20390128) and Special Coordination Funds for Promoting Science and Technology (17019058, http://kaken.nii.ac.jp/ja/p/17019058) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: kodama@biken.osaka-u.ac.jp Introduction Vibrio parahaemolyticus is a Gram-negative marine bacterium responsible for acute gastroenteritis associated with the consump- tion of raw or undercooked contaminated seafood [1]. V. parahaemolyticus infection is a growing public health concern because of the emergence of pandemic strains that have caused severe outbreaks worldwide [2,3]. Most V. parahaemolyticus strains isolated from clinical cases exhibit hemolytic activity on a special blood agar called Wagatsuma agar [4]. This hemolysis, termed the Kanagawa phenomenon (KP), has been considered a good virulence marker of pathogenic V. parahaemolyticus [5]. The KP is caused by thermostable direct hemolysin (TDH) produced by this bacterium [6]. As purified TDH has multiple biological activities, including induction of fluid accumulation in the rabbit intestine, it has been considered a major virulence factor of V. parahaemolyticus [6–16]. Since these studies, whole genome sequencing of a KP-positive V. parahaemolyticus strain revealed the presence of two sets of type III secretion systems: T3SS1 and T3SS2 [17]. Comparative genome analysis using microarrays showed that an 80 kb pathogenicity island (Vp-PAI) on chromosome II, which encodes two tdh genes and the T3SS2 gene cluster, is unique to KP-positive pathogenic strains [18,19]. According to a recent report that evaluated the fluid-accumulating activity of virulence gene deletion mutants in a rabbit ileal loop model, T3SS2, not TDH or T3SS1, contributes to V. parahaemolyticus-induced enterotoxicity [20]. T3SS gene clusters similar to the T3SS2 of KP-positive V. parahaemolyticus have been detected in both TDH-related hemoly- sin (trh)-positive (KP-negative) V. parahaemolyticus and non-O1/non- O139 V. cholerae, which are also pathogenic to humans [21,22]. The T3SS2-related genes of trh-positive V. parahaemolyticus are involved in enterotoxicity [22], whereas those of non-O1/non- O139 V. cholerae appear to be required for intestinal colonization [21]. Consequently, the T3SSs are believed to be pivotal for the pathogenicity of these bacteria. Recently, we reported that two ToxR-like proteins, VtrA and VtrB, specifically regulate the transcription of genes encoded PLoS ONE | www.plosone.org 1 October 2010 | Volume 5 | Issue 10 | e13365