胆汁酸介导的霍乱弧菌毒素基因表达的分子调控机制研究

项目名称： 胆汁酸介导的霍乱弧菌毒素基因表达的分子调控机制研究

项目编号： No.31470244

项目类型： 面上项目

立项/批准年度： 2015

项目学科： 生物科学

项目作者： 杨梦华

作者单位： 浙江农林大学

项目金额： 85万元

中文摘要： 霍乱弧菌是人类重要的肠道病原菌，胆汁酸可通过增强毒素基因调控蛋白TcpP形成二聚体，从而诱导霍乱弧菌毒素基因的表达。但是胆汁酸如何增强TcpP二聚体形成的分子机制尚不清楚，而且肠道中除了胆汁酸分子之外，霍乱弧菌毒素基因的表达是否受其它信号因子的调控，也不明晰。因此，本项目将通过Octet分子互作、等温滴定量热和体外共沉淀技术，从研究胆汁酸分子与膜蛋白TcpP的相互作用入手，揭示胆汁酸分子对细胞膜蛋白功能与结构的影响，进而通过RNA-Seq等手段构建胆汁酸影响霍乱弧菌毒素基因表达和群体感应信号的分子调控网络，阐明霍乱弧菌中毒素基因的分子调控机制。同时，通过文库构建、同位素标记等方法筛选影响毒素基因表达的胆汁酸分子竞争性抑制剂，为筛选干预肠道细菌毒素性腹泻疾病的非抗生素类靶向药物提供重要的理论依据。

中文关键词： 霍乱弧菌；病原微生物；耐药性；胆汁酸；毒力因子

英文摘要： Vibrio cholerae is the causative agent of the diarrheal disease cholera. For V. cholerae to successfully infect a host, it must readily alter its phenotypic properties to allow for positive adaptations to the host environment. However, which host factors V. cholerae sense to and how V. cholerae initiate transcriptional regulatory cascades to induce virulence genes in the small intestines arelargely unknown. Using a newly developed ex vivo tissue model, we found that taurocholic acid, a bile acid present in mammalian small intestines, induces key V. choleraevirulence regulatory pathways. Although bile salts activated virulence genes expression by affecting the transmembrane transcription factor TcpP, yet, the exact mechanism is unclear. Whether bile salts directly interact with TcpP protein, or bile salts enhance TcpP activity by interacting with other regultory factors would be further studied in this application. We will apply in vitro pull-down assay, luminescence assay and other biochemical and genetic methods to elucidate the mechanism how bile salts enhance TcoP activity so as to activate virulence genes expression in V.cholerae. These studies will thus allow us to gain a fuller understanding of the bacteria-host interactions that have allowed V. choleraeto become such a successful human pathogen. On the other hand, we will screen for those competitive inhibitors for the taurocholate acid, which can interfere the activation of of dimerization of TcpP by taurocholate acid. These inhibitors will be able to attenuate the induction effect of virulence genes of V.cholerae by the bile salt molecules in the small intestine, so they would be the very good candidates for developing the non-antibiotic drugs for the treatement of diarrheal in the future.

英文关键词： vibrio cholerae;pathogen;drug resistant;bile salt;virulence factor