鸡伤寒沙门菌双组分信号转导系统CpxAR对MDR和致病性的协调调控机制

The regulative functions of two-component signal transduction system CpxAR on Salmonella gallinarium MDR were identified and confirmed using CpxAR as subject investigated in this study by constructing expression plasmids of CpxAR and site-directed mutagenesis of response regulator cpxR. The expression levels of fusion reporter genes were investigated in various strains by constructing promoter P-lacZ fusion reporter plasmids of the various target genes, and active efflux and/or outer membrane protein genes regulated by CpxAR were made clear. Electrophoretic mobility shift assay(EMSA) and DNA footprinting assay was used to find out if CpxR was directly bound to the promoter of respounding genes to elucidate DNA sequence of target binding sites and its regulative mechanism on MDR. Pathogenicity and mRNA expression levels of virulence- and resistance-associoated genes wer compared and analysed among the representative clinical isolates,cpxR deletion strain and its complementation strain to elucidate the coordinate regulation mechanisim of CpxAR on MDR and pathogenicity of S.gallinarium.Molecular biological methods for the research of TCS regulative mechanisms on MDR in animal pothogens will be set up innovatively to provide techniques and methods for further reserch of regulative network cascades and mutual actions among various TCS,to provide a new idea and theretical basis for finding potential drug effluxs and virulence genes coordinated by CpxAR, novel targets for antimicrobial therapy and controling effectively MDR S.gallinarium infections.

选择对致病性和多重耐药表型有调控作用的双组分信号转导系统CpxAR 为研究对象，构建表达质粒和磷酸化位点特异性突变，确证CpxAR对鸡伤寒沙门菌MDR的调控功能；构建不同目标基因的启动子P-LacZ融合报告质粒并观察其表达，弄清其对何种外排或外膜蛋白基因具有调控作用，经过电泳迁移率变动分析和DNA足迹试验探明cpxR与靶基因启动子的结合位点，阐明其对MDR的调控机制。比较代表性临床流行克隆株、cpxR基因缺失株的致病性，分析MDR及毒力相关基因的mRNA表达变化，阐明CpxAR对鸡伤寒沙门菌MDR和致病性协调调控的分子机制。本试验创新建立适合动物源细菌中TCS对MDR调控机制研究的分子生物学方法，为进一步研究TCS调控网络通路、不同TCS之间的相互作用等奠定基础，发现CpxAR调控的MDR潜在外排泵和同时协调调控的毒力基因，为寻找抗菌作用新靶点、防治鸡MD沙门菌感染提供新的思路和理论依据。

CpxAR是对多种阴性菌致病性有重要调控作用的TCS之一，国内外未见有鸡沙门菌CpxAR 对MDR、致病性协调调控机制的研究报道。本试验成功构建了鸡伤寒沙门菌CpxAR 表达质粒、cpxR磷酸化位点特异性突变株、cpxR缺失株、cpxR和acrB双基因缺失株及互补菌株，证明了CpxAR通过磷酸转移和信号转导实现对MDR的调控，尤其是确证其对氨基糖苷类、内酰胺环类抗生素耐药性的调控作用；成功构建了目标基因的启动子P-LacZ融合报告质粒并观察其表达，再经过RT-PCR初筛，弄清了外排泵acrD和外膜蛋白OmpF、OmpD和STM3031为受控靶外排泵基因、靶外膜蛋白基因。其中，经过电泳迁移率变动分析和DNA足迹试验探明：CpxR蛋白可结合到acrD的启动子区，结合靶位的碱基序列为：GTAAA-gaacg-GCAAA，位于acrD起始密码子上游173 bp，CpxR对acrD具有直接调控作用，对其他受控靶基因外膜蛋白OmpF、OmpD和STM3031则有间接的调控作用。利用噬菌体转导，获得了流行克隆株ST1920（新的ST型）株的cpxR缺失株，LD50结果显示， ST1920型代表株（JS分）的LD50较JS标低约30.31倍，JS标△cpxR突变株的LD50比其亲本株JS标 升高了1.36倍，JS分△cpxR突变株LD50比其亲本株JS分升高了17.54倍。黏附试验和侵袭试验表明：标准菌株、分离菌cpxR缺失后的对PK-15细胞的黏附量分别较亲本菌株分别降低了1.35、2倍，对PK-15细胞的侵袭量分别降低了3.4、3倍。表明：cpxR基因缺失株毒力均明显下降，cpxR对鼠伤寒沙门菌致病性有重要调控作用。. 本试验还应用RT-PCR方法，比较了16株代表性临床分离株、cpxR缺失株的MDR及毒力相关基因的mRNA表达，从反向证明：cpxR可下调OmpF、OmpD的表达量，上调acrD和STM3031的表达量。cpxR基因对hilA、sopB、hilD、 fur、invA、spvC等6个毒力基因的表达有正性调控作用。本课题系统阐明了CpxAR对鸡伤寒沙门菌MDR和致病性协调调控的分子机制，创建了研究细菌TCS对MDR调控机制的分子生物学方法，并为寻找抗菌作用新靶点、防治鸡MDR沙门菌感染提供了新的思路和理论依据。