cpxR调控沙门菌对黏菌素敏感性的分子机制

Colistin is last-line therapeutic option of multidrug-resistant (MDR) gram-negative pathogen infections, but the colistin-resistant (Colr) strains are recently increasing in the word. Our previous studies have shown that the complementation of the cpxR gene in Salmonella enterica serovar Typhimurium JS∆acrB∆cpxR mutant strain with the cpxR gene greatly enhanced the susceptibility of the complemented strain to colistin and this effect was found to be associated with decreases of the pmrB mRNA expression level of TCS PmrAB. Signal pathway of cpxR regulation on susceptibility of strain to colistin was further identified and confirmed through the construction of pmrB, pmrD and phoQ complementary strains and mgrB deleted strains in this project. Electrophoretic mobility shift assay (EMSA) and DNA footprinting assay was used to uncover regulative target gene and sequence point. From the perspectctives of the regulative network cascades and the regulative target point , the molecular mechanism of cpxR’ enhanging the susceptibility of the tested strains to colistin, when complementated in cpxR and acrB double-deleted strain, was systematically studied and deep revealed. This project has not only important practical significance for finding novel targets for the antimicrobial therapy and a new way to reduce resistance to colistin but also has the important academic value for setting up innovatively molecular biological methods for the research of TCS regulative mechanisms on colistin susceptibility and mutual action relations among various TCS.

黏菌素是治疗MDR阴性菌感染的最后手段，但其耐药菌株正全球性日益增长。前期证明：cpxR在伤寒沙门菌cpxR和acrB双基因缺失株中互补，对黏菌素敏感性增强与pmrB的表达量降低密切相关。本课题进一步构建pmrB、pmrD、phoQ基因互补菌株和mgrB基因缺失株，分析鉴定cpxR基因调控沙门菌对黏菌素敏感性的信号通路；通过EMSA和DNA足迹试验，揭示其调控的靶基因和靶序列。从调控通路、调控靶点等多视角系统开展研究，深层次揭示cpxR调控沙门菌对黏菌素敏感性的分子机制。本课题不仅对寻找抗菌作用新靶点、寻找降低细菌对黏菌素耐药的新途径具有重要的现实意义，而且对创新建立TCS对黏菌素敏感性调控机制的分子生物学方法、深入研究不同TCS之间的相互作用关系，具有重要学术价值。

前期研究表明：当双组分系统CpxAR应答调节子cpxR在JS△acrB△cpxR中回补时，沙门菌对黏菌素的敏感性显著升高。本课题研究cpxR调控沙门菌对黏菌素敏感性的信号通路、调控靶点。前者包括：pmrB、pmrD、phoQ互补菌株构建，mgrB缺失株构建及互补试验和RT-PCR；后者包括：EMSA和DNase I footprinting 分析、ESI/MS分析和竞争试验。. 2018年成功构建pmrB、pmrD、phoQ互补菌株、mgrB缺失株及其回补株。MIC测定表明：与JS相比，黏菌素对各菌株MIC值分别上升了4-16倍或下降了2-4倍。RT-PCR发现：与JS相比，JS∆∆/pR中黏菌素耐药相关基因phoP、phoQ、pmrB、pmrC、pmrH 和 pmrD的mRNA表达量均显著下降（> 1/2），而mgrB的表达量显著上升（3.5倍），表明CpxR通过调控黏菌素耐药相关的MgrB-PhoPQ-PmrD-PmrAB-PmrC/H通路，使JS∆∆/pR的黏菌素敏感性显著上升。EMSA发现，CpxR能与黏菌素耐药相关基因phoPQ、pmrC、 pmrH 和 pmrD的启动子结合，而不能与pmrAB和mgrB的启动子结合。. 2019年成功构建含有pmrAB、phoPQ、mgrB、pmrC、pmrD 和pmrH启动子区的融合报告质粒，完成了β-半乳糖苷酶报告质粒相关试验；DNA印迹发现，CpxR能结合到phoPQ、pmrC、pmrH 和 pmrD启动子区中的CpxR-box 序列5’-GTAAA-(N)4−8-GTAAA-3’。ESI/MS分析表明：CpxR通过下调黏菌素耐药相关基因的表达水平，减少类脂A的L-Ara4N和pEtN修饰，使JS∆∆/pR的黏菌素敏感性上升。竞争试验表明：JS∆∆/pR在对黏菌素敏感性显著上升的同时产生了适应性代价。. 上述研究揭示了cpxR增强沙门菌黏菌素敏感性的分子机制：沙门菌cpxR超临界表达可直接调控phoPQ、pmrC、pmrH和pmrD的表达，间接调控pmrA和mgrB的表达，使类脂A的L-Ara4N和pEtN修饰减少，而△acrB显著增强cpxR和mgrB表达，即与cpxR超临界表达协同使JS∆∆/pR对对黏菌素的敏感性显著升高，其增强CpxR和mgrB表达的分子机制值得深入研究。