AcrAB-TolC功能失活与CpxAR激活协同增强沙门菌对黏菌素敏感性的分子机制

黏菌素耐药性是全球关注的焦点。前期原创性发现：沙门菌AcrAB-TolC失活（△acrB）与CpxAR激活（cpxR过表达，△cpxR/pcpxR）协同作用时，菌株（JS△acrB△cpxR/pcpxR）对黏菌素敏感性显著增强，但其分子机制尚未阐明。本课题通过P-lacZ转录融合构建，探究AcrAB-TolC失活（基因缺失、EPIs应用）和CpxAR激活间的协同作用；基于转录组学研究，探明差异化表达基因及其信号通路；基于代谢组学分析，挖掘差异化的细胞内代谢物和代谢途径；最后在分离菌中，应用EPIs及其配合CpxAR激活剂分析验证。从源头揭示AcrAB-TolC失活与CpxAR激活协同，增强沙门菌对黏菌素敏感性的分子机制。本研究不仅对开拓细菌代谢、外排泵、双组份调控系统及其与耐药关系研究的新领域有重要学术价值，而且可为多途径寻找抗菌作用新靶点和防控耐药菌感染的新思路提供科学依据。

Colistin resistance in bacterial has been the focus of attention in the world in recent years. Our previous studies have originally shown that the susceptibility of the constructed strain (JS∆acrB∆cpxR/pcpxR) to colistin was greatly enhanced when the active efflux pump AcrAB-TolC functional inactivation derived from∆acrB mutation in Salmonella enterica serovar Typhimurium acted synergistically with CpxAR activation (the overexpression of the cpxR gene in TCS CpxAR of JS∆cpxR/pcpxR mutant strain) . However, molecular mechanism of this phenomenon has still remained unclear to date. In this project, the synergistical relationship between AcrAB-TolC functional inactivation and CpxAR activation in Salmonella isolates will be elucidated through deleting tolC gene (∆tolC mutant), phage transduction, the reconstruction of promoter P-lacZ transcription fusion and using efflux pump inhibitors. Based on transcriptomic studies, the differentially expressed genes among reconstructed deletion mutants will be found out. Overmore, the differentially metabolic molecules and metabolic paths will be unearthed based on metabolomic analysis. In the end, analysis and confirmation were performed in the clinic isolates by using efflux pump inhibitors (EPIs) and the combination of EPIs+CpxAR activator (EDTA). From the multi-perspectctives and multilevels of phenotype, gene, mRNA expression and metabolomics, the metabolites, metabolic paths and their signal pathway of AcrAB-TolC functional inactivation by using various methods and CpxAR activation synergistically increasing the expression of the cpxR and mgrB genes and changing the express of other genes relative to colitin resistance will deep be uncovered, and the molecular mechanism of AcrAB-TolC inactivation’synergistically enhanging the susceptibility of the tested strains to colistin will systematically be elucidated. This project has not only important academic value for setting up innovatively new research area of mutual relationship among bacterial metabolism, Efflux pump, two component signal transduction system (TCS ) and bacterial resistance but also can provide scientific foundation for finding novel targets for antimicrobial therapy and a new way to control of colistin-resistant bacteria infection.