ToxR负调控副溶血弧菌中c-di-GMP的合成  

ToxR represses the synthesis of c-di-GMP in Vibrio parahaemolyticus

作　　者：张苗苗[1] 薛星帆 孙君芳 吴齐敏 李雪[2] 周冬生 倪斌[1] 陆仁飞[2] 张义全[1,2] ZHANG Miaomiao;XUE Xingfan;SUN Junfang;WU Qimin;LI Xue;ZHOU Dongsheng;NI Bin;LU Renfei;ZHANG Yiquan(School of Medicine,Jiangsu University,Zhenjiang 212013,Jiangsu,China;Department of Clinical Laboratory,Affiliated Nantong Hospital 3 of Nantong University,Nantong 226006,Jiangsu,China;State Key Laboratory of Pathogen and Biosecurity,Beijing Institute of Microbiology and Epidemiology,Beijing 100071,China)

机构地区：[1]江苏大学医学院,江苏镇江212013 [2]南通大学附属南通第三医院检验科,江苏南通226006 [3]北京微生物流行病研究所病原微生物生物安全重点实验室,北京100071

出　　处：《生物工程学报》2022年第12期4719-4730,共12页Chinese Journal of Biotechnology

基　　金：国家自然科学基金(82072239);中央高校基本科研业务费专项资金(JUSRP121061)。

摘　　要：副溶血弧菌(Vibrio parahaemolyticus)是世界范围内引起海产品相关食物中毒的主要致病菌,具有很强的生物膜形成能力。ToxR是一种膜结合调控蛋白,对副溶血弧菌生物膜形成具有一定的调控作用,但具体机制尚未见报道。c-di-GMP是一种普遍存在于细菌中重要的第二信使,参与调控细菌的多种生物学行为包括生物膜的形成。本文探究ToxR对副溶血弧菌中c-di-GMP代谢的调控作用。利用酶联免疫吸附法(enzyme linked immunosorbent assay,ELISA)测定副溶血弧菌野生株(wild-type,WT)和toxR突变株(ΔtoxR)中c-di-GMP水平的差异。挑选c-di-GMP代谢相关基因scrA、scrG和vpa0198为进一步研究的靶标,采用实时定量qPCR实验检测靶基因在WT和ΔtoxR中的转录水平差异;将靶基因调控区DNA序列克隆入pHRP309质粒中无启动子的β半乳糖苷酶基因上游,采用lacZ报告基因融合实验进一步研究ToxR对靶基因的转录调控关系;将重组质粒分别导入含有pBAD33或pBAD33-toxR的EC100λpir中,采用lacZ报告基因融合实验研究ToxR是否能在异体宿主中调控靶基因的表达;PCR扩增靶基因上游调控区DNA序列,并纯化His-ToxR蛋白,用凝胶阻滞实验(electrophoresis mobility shift assay,EMSA)研究His-ToxR与靶基因启动子区DNA序列是否具有结合作用。ELISA结果显示ΔtoxR中c-di-GMP含量显著性高于WT中的,说明ToxR抑制c-di-GMP的产生;实时定量qPCR结果表明WT中scrA、scrG和vpa0198的转录水平显著性高于ΔtoxR中的,表明ToxR抑制它们的转录;lacZ报告基因融合实验结果表明ToxR可抑制副溶血弧菌和EC100λpir中scrA、scrG和vpa0198的启动子区活性;EMSA实验显示His-ToxR能特异性地结合到scrA和scrG的上游调控区DNA序列上,而对vpa0198的上游调控区DNA序列无结合作用。综上所述,ToxR通过直接调控相关酶蛋白基因的转录来抑制副溶血弧菌内c-di-GMP的合成,从而有助于精确调控生物膜形成等细菌行为。Vibrio parahaemolyticus,the main pathogen causing seafood related food poisoning worldwide,has strong biofilm formation ability.ToxR is a membrane binding regulatory protein,which has regulatory effect on biofilm formation of V.parahaemolyticus,but the specific mechanism has not been reported.c-di-GMP is an important second messenger in bacteria and is involved in regulating a variety of bacterial behaviors including biofilm formation.In this study,we investigated the regulation of ToxR on c-di-GMP metabolism in V.parahaemolyticus.Intracellular c-di-GMP in the wild type(WT)and toxR mutant(ΔtoxR)strains were extracted by ultrasonication,and the concentrations of c-di-GMP were then determined by enzyme linked immunosorbent assay(ELISA).Three c-di-GMP metabolism-related genes scrA,scrG and vpa0198 were selected as the target genes.Quantitative real-time PCR(q-PCR)was employed to calculate the transcriptional variation of each target gene between WT andΔtoxR strains.The regulatory DNA region of each target gene was cloned into the pHR309 plasmid harboring a promoterless lacZ gene.The recombinant plasmid was subsequently transferred into WT andΔtoxR strains to detect theβ-galactosidase activity in the cellular extracts.The recombinant lacZ plasmid containing each of the target gene was also transferred into E.coli 100λpir strain harboring the pBAD33 plasmid or the recombinant pBAD33-toxR to test whether ToxR could regulate the expression of the target gene in a heterologous host.The regulatory DNA region of each target gene was amplified by PCR,and the over-expressed His-ToxR was purified.The electrophoretic mobility shift assay(EMSA)was applied to verify whether His-ToxR directly bound to the target promoter region.ELISA results showed that the intracellular c-di-GMP level significantly enhanced inΔtoxR strain relative to that in WT strain,suggesting that ToxR inhibited the production of c-di-GMP in V.parahaemolyticus.qPCR results showed that the mRNA levels of scrA,scrG and vpa0198significantly increased inΔt

关 键 词：副溶血弧菌 转录调控 ToxR C-DI-GMP 

分 类 号：TS201.3[轻工技术与工程—食品科学]