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

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EC100lpir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EC100lpir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。

Abstract:

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 (△toxR) 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 and △toxR 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 and △toxR strains to detect the β-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 in △toxR 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 vpa0198 significantly increased in △toxR strain relative to those in WT strain, suggesting that ToxR repressed the transcription of scrA, scrG and vpa0198. lacZ fusion assay showed that ToxR was able to repress the promoter activities of scrA, scrG and vpa0198 in both V.parahaemolyticus and E.coli 100λpir. EMSA results showed that His-ToxR was able to bind to the regulatory DNA regions of scrA and scrG, but not to the regulatory DNA region of vpa0198. In conclusion, ToxR inhibited the production of c-di-GMP in V.parahaemolyticus via directly regulating the transcription of enzyme genes associated with c-di-GMP metabolism, which would be beneficial for V.parahaemolyticus to precisely control bacterial behaviors including biofilm formation.