baeR基因对沙门菌耐药相关蛋白表达及耐药基因的调控作用

Effects of baeR gene of Salmonella on expression of drug resistance-related proteins and regulation of drug resistance genes

【目的】探究baeR对鼠伤寒沙门菌耐药相关蛋白表达水平的影响以及对耐药基因的调控作用,为鼠伤寒沙门菌耐药性的深入研究提供理论依据。【方法】采用非标记定量蛋白质组学技术,以差异倍数(FC)≥1.5且P<0.05为标准,筛选鼠伤寒沙门菌baeR和acrB双缺失CR株(鼠伤寒沙门菌ATCC13311体外诱导环丙沙星耐药株)(C1)、baeR过表达C1株(CA)和baeR回补株(CM)的差异表达蛋白,对筛选的差异蛋白进行GO富集和KEGG通路富集分析,探究差异蛋白的主要功能和涉及的生物过程;采用平行反应监测(PRM)蛋白质组学技术对筛选到的抗药相关差异蛋白CysP、GltI、ArgT、MdtA、OmpF、ArcA、FrdD、DcuB、TorR、UhpA、NarZ、PgtA、MraY、CheM、CheA、Trg进行验证;采用LacZ报告基因融合实验研究baeR对靶基因pmrF和mdtC的调控作用。【结果】CA/C1比较组中共筛选到393个差异蛋白,其中上调182个,下调211个;CM/C1比较组中共筛选到391个差异蛋白,其中上调202个,下调189个。差异表达蛋白主要集中在外排泵、外膜蛋白、生物膜、生物合成、双组分系统等通路。PRM验证结果定量到CysP、GltI、ArgT、OmpF、ArcA、DcuB、TorR、PgtA、CheM、CheA、Trg等11个蛋白,其表达水平变化趋势与非标记定量结果一致,证实了蛋白质组结果的可靠性。baeR可以正向调控pmrF和mdtC的表达。【结论】明确了baeR过表达对鼠伤寒沙门菌耐药相关蛋白水平的影响及对相关耐药基因的调控作用。

【Objective】This study investigated the effects of baeR on expression levels of drug resistance-related proteins in Salmonella typhimurium and the regulation of drug resistance genes to provide basis for further research on drug resistance of Salmonella typhimurium.【Method】Using non-label quantitative proteomics technology,differentially expressed proteins of Salmonella typhimurium baeR and acrB double deletion CR(ATCC13311 induced ciprofloxacin-resistant strains in vitro)strain(C1),baeR overexpressing C1 strain(CA)and baeR complementing strain(CM)were screened with the standards of difference fold≥1.5 and P<0.05.The differentially expressed proteins were subjected to GO enrichment and KEGG pathway enrichment analysis to explore the main functions and related biological processes.The differential proteins related to drug resistance including CysP,GltI,ArgT,MdtA,OmpF,ArcA,FrdD,DcuB,TorR,UhpA,NarZ,PgtA,MraY,CheM,CheA and Trg were verified using parallel reaction monitoring(PRM)proteomic technology.The LacZ reporter gene fusion experiment was used to study the regulation of baeR protein on target genes of pmrF and mdtC.【Result】A total of 393 differential proteins were screened in the CA/C1 comparison group,of which 182 were up-regulated and 211 were down-regulated.A total of 391 differential proteins were screened in the CM/C1 comparison group,of which 202 were up-regulated and 189 were down-regulated.The differentially expressed proteins mainly concentrated in the pathways of efflux pump,outer membrane protein,biofilm,biosynthesis,metabolism and virulence.The PRM verification obtained 11 proteins of CysP,GltI,ArgT,OmpF,ArcA,DcuB,TorR,PgtA,CheM,CheA and Trg.The change trend of their expression levels was consistent with unlabeled quantitative results,confirming the reliability of the proteomic results.The baeR gene positively regulated the expressions of pmrF and mdtC.【Conclusion】The effect of baeR overexpression on the levels of drug resistance-related proteins in Salmonella typhimurium and the regulation of related drug resistance genes were clarified.