鼠伤寒沙门氏菌耐药调控蛋白RamA对自动诱导子AI-2的分子调控机制

项目名称： 鼠伤寒沙门氏菌耐药调控蛋白RamA对自动诱导子AI-2的分子调控机制

项目编号： No.U1504327

项目类型： 联合基金项目

立项/批准年度： 2016

项目学科： 农业科学

项目作者： 孙亚伟

作者单位： 河南科技学院

项目金额： 27万元

中文摘要： RamA是鼠伤寒沙门氏菌产生多重耐药的必要元素，RamA过表达可导致沙门氏菌毒力岛1内侵袭力相关基因（invF, sipA 和prgH）表达水平下降。自动诱导子AI-2调节蛋白LsrR是上述基因表达的必要成分且lsrR基因启动子区含有一个相似的RamA结合盒。本课题通过构建AI-2检测报告菌和基因失活技术，首先确定AI-2在RamA对侵袭力相关基因表达中作用。其次，通过RT-PCR、基因失活、染色体融合、体外EMSA实验，确定RamA调控多重耐药泵（AcrAB-TolC和MdtK）在AI-2外排中作用和RamA对lsrR调控机制。最后，将LsrR在临床多重耐药鼠伤寒沙门氏菌体内失活并检测失活株对细胞侵袭能力。本研究将阐明RamA如何调节AI-2系统从而导致细菌侵袭力改变，将为鼠伤寒沙门氏菌多重耐药产生过程中毒力变化提供首个理论基础，为药物新靶标筛选和临床多重耐药菌防治提供有益指导。

中文关键词： 鼠伤寒沙门氏菌；调控蛋白RamA；自动诱导子AI-2；；分子调控机制

英文摘要： RamA is a required element in the development of multidrug resistance of Salmonella enterica serovar Typhimurium. The overexpression of RamA led to decreased expression level of the invasion-related genes (invF, sipA and prgH) in Salmonella pathogenicity island 1. In the autoinducer AI-2 system the regulator LsrR is a required element for the expression of these invasion-related genes and the promoter region of lsrR contains a similar rambox. In the research, the function of AI-2 was first confirmed in the regulation of RamA to the virulence genes by construcing an AI-2 detection reporter strain and gene knockout. Secondly, the role of multidrug resistance effluxs (AcrAB-TolC and MdtK) in the transport of AI-2 from cytoplasm to niche and the regulation mechanism of RamA to lsrR were decided by RT-PCR, gene knockout, chromosome fusion and EMSA experimen. At last, LsrR was inactivated in clinical isolates of multidrug resistance Salmonella enterica serovar Typhimurium and then, the invasion ability of the mutants to cell were tested. In summary, the research will clarify that RamA lead to the change of invasion ability of Salmonella enterica serovar Typhimurium to cell dependent of AI-2 system. The result will give a theoretical basement in predicting the virulence change in the development of multidrug resistance of Salmonella enterica serovar Typhimurium and provide a good direction in screening new drug target and the prevention and cure of clinical isolates of multidrug resistance bacteria.

英文关键词： Salmonella enterica serovar Typhimurium;Regulator RamA;Autoinducer AI-2;Molecular Regulation Mechanism