沙门菌双组分系统BaeSR的耐药调控机制

Salmonella are serious zoonotic pathogens. The study of resistance mechanism to increasingly serious multidrug is of great significance. Studies have shown that BaeSR is a two-component signal transduction system (TCS) that is closely related to Salmonella multidrug resistance. Our previous results of the transcriptome sequencing of Salmonella parental strain and two selected resistant strains in vivo and in vitro showed that baeSR expression of selected resistant strains were both up-regulated compared with parental strain, and correlated with the expression of the efflux pump gene, but the regulatory mechanism of BaeSR on multidrug resistance is not clear. Therefore, we constructed baeSR gene deletion strains and recovery strains of Salmonella parental strain, selected resistant strains and clinical resistant strain, and analyzed its effect on drug resistance and biological characteristics. Then the transcriptomics and proteomics analysis of these strains were carried out to screen differential expression genes regulated by baeSR gene, especially the efflux pump genes and its associated proteins. Finally, knocked out the baeSR gene indirectly regulated efflux pump genes, to determine its function with baeSR gene by drug sensitivity test, and use gel blocking and LacZ reporter gene detection technology to explore its regulatory role in drug resistance related pathways. The study will provide an important basis for exploring the mechanism of bacterial resistance formation, finding drug targets and developing new antibacterial drugs.

沙门菌是危害严重的人兽共患病原菌，其多重耐药现象日趋严重，探究其耐药机制具有重要意义。研究表明BaeSR是一种与沙门菌多重耐药密切相关的双组分信号转导系统。项目组前期对沙门菌标准株与诱导耐药株进行转录组测序发现，诱导耐药株baeSR基因表达均上调，且与外排泵基因表达相关，但BaeSR对多重耐药的调控机制尚不明确。本项目拟构建沙门菌标准株、诱导耐药株和临床耐药株baeSR基因缺失株与回复株，分析其对耐药性和生物学特性的影响。然后开展菌株转录组学和蛋白质组学分析，筛选baeSR基因调控的差异表达耐药基因，尤其是外排泵基因及其蛋白。最后敲除baeSR基因间接调控的外排泵基因，测定MIC值，验证其与baeSR基因的功能关联性，进而利用凝胶阻滞及LacZ报告基因酶活检测技术探明其在耐药相关通路中的调控作用。研究结果将为探索细菌耐药性形成机制、寻找药物靶标和开发新型抗菌药物提供重要依据。

沙门菌（Salmonella）是重要的人兽共患病原菌，可引起动物和人的胃肠炎、败血症等，随着抗菌药物的广泛与不合理使用，沙门菌多重耐药现象日趋严重。研究沙门菌的耐药机制，寻找防治耐药沙门菌的潜在药物靶点，对控制耐药沙门菌感染具有重要意义。研究表明BaeSR是一种与沙门菌多重耐药密切相关的双组分信号转导系统。项目组前期对沙门菌标准株与诱导耐药株进行转录组测序发现，诱导耐药株baeSR基因表达均上调，且与外排泵基因表达相关，但BaeSR对多重耐药的调控机制尚不明确。本项目使用环丙沙星、多粘菌素和替加环素对鼠伤寒沙门菌标准菌株（ATCC 13311）进行体内（秀丽隐杆线虫）和体外耐药性诱导，构建了诱导耐药株baeSR，acrB基因的缺失株、回复株和过表达株，发现baeSR，acrB基因影响菌株的运动性及生物膜形成，协同调控菌株的耐药性。转录组和蛋白组测序发现，基因缺失、过表达前后，多个基因和蛋白质的表达水平发生显著改变，差异基因包括耐药和毒力基因，主要富集在代谢途径、ABC转运系统、双组份信号转导系统、鞭毛组装、β-内酰胺抗性等通路。对基因缺失前后，蛋白质的磷酸化水平进行比较发现，BaeSR双组分系统和AcrB外排泵影响鼠伤寒沙门菌中蛋白质的磷酸化，并可能通过影响蛋白质的磷酸化来影响鼠伤寒沙门菌的耐药性和毒力。成功表达BaeR蛋白，LacZ报告基因融合实验结果表明，BaeR过表达后可以正向调控ompW，sodB，tolC，mdtD，spy和marR的表达，凝胶阻滞实验（EMSA）结果表明BaeR蛋白可以直接结合到marR的启动子区，调控marR基因的表达。综上所述，本项目探明了沙门菌 BaeSR 的耐药调控作用和机制，为深入探索沙门菌耐药性形成机制、寻找潜在药物靶标和开发新型抗菌药物提供了重要依据。