肺炎克雷伯菌质粒携带的新型双组分调控系统BaeS/CopR对多黏菌素耐药基因mcr-8的调控机制

多黏菌素耐药肺炎克雷伯菌的频现，已成为一个突出的公共卫生事件。虽已证实染色体上双组分调控系统突变和质粒携带的mcr基因是多黏菌素耐药的主要原因，但目前仍有部分高水平多黏菌素耐药菌的产生机制不明。前期研究发现，肺炎克雷伯菌mcr-8基因质粒上含有一组新型的双组分调控系统BaeS/CopR，但具体功能尚待证实。本项目拟通过baeS/copR的遗传溯源，阐明该系统的遗传演化规律；利用凝胶阻滞、差异二维蛋白电泳等试验明确其结合位点和靶向调控蛋白，并通过比较不同因素对baeS/copR转录水平的影响，揭示BaeS/CopR系统对环境刺激的应答反应，佐证其为双组份调控系统；然后通过构建缺失株和互补株，确证其对mcr-8基因表达的调控作用及其对多黏菌素高水平耐药的贡献。该项目的完成有助于我们明确部分多黏菌素高水平耐药肺炎克雷伯菌株的产生机制，也可为该耐药菌的防控提供新靶点。

The frequency of colistin resistant Klebsiella pneumoniae has become a prominent public health event. Although it has been confirmed that mutations in the two-component regulatory system (TCS) on chromosomes and the mcr genes loacted on plasmids are the main causes of colistin resistance, the mechanism of some high level colistin resistant bacteria is still unknown. Previous studies showed that mcr-8-carrying plasmids contained a novel TCS BaeS/CopR, which are related to the high expression of mcr-8 in K. pneumoniae. Based on the domain analysis of BaeS/CopR, we speculated that BaeS/CopR was a novel two-component regulation system, which was located on plasmid, but its specific function has yet to be confirmed. This project intends to elucidate the genetic evolution law of the BaeS/CopR system through the genetic tracing of baeS/copR. The binding sites and targeted regulatory proteins were identified by EMSA and two-dimensional gel electrophoresis. Through comparing the effects of different factors on the transcription level of baeS/copR revealed the response of baeS/copR system to environmental stimuli, which can be used to prove that it was a two-component regulatory system. Then, the regulation of mcr-8 gene expression and its contribution to high level of colistin resistance were confirmed by constructing the deletion and complementary strains. The completion of this project will help us to clarify the mechanism of some high level colistin resistance K. pneumoniae strains, and also provide new targets for the prevention and control of this resistant bacteria.