副溶血性弧菌CalR调控vopB2的分子机制  

The transcriptional regulation role of CalR on vopB2 in Vibrio parahaemolyticus

作　　者：张凌宇 唐浩 仇越 张义全 王丽[3] ZHANG Ling-Yu;TANG Hao;QIU Yue;ZHANG Yi-Quan;WANG Li(Nanjing University Hospital,Nanjing,Jiangsu 210000,China;School of Medicine,Jiangsu University,Zhenjiang,Jiangsu 212013,China;The First Hospital Affiliated to Henan University,Kaifeng,Henan 475000,China)

机构地区：[1]南京大学医院,江苏南京210000 [2]江苏大学医学院,江苏南京210000 [3]河南大学第一附属医院,河南开封475000

出　　处：《微生物学通报》2019年第7期1672-1677,共6页Microbiology China

基　　金：国家自然科学基金青年项目(81501779);河南省高等学校重点科研项目(18A320024);江苏省自然科学基金青年基金(BK20160505)~~

摘　　要：【背景】副溶血性弧菌是一种非常重要的食源性致病菌,CalR蛋白是一种全局转录调节因子。III型分泌系统2 (Type 3 secretion systems 2 T3SS2)是副溶血性弧菌主要的毒力因子,vopB2是T3SS2中的一个关键效应蛋白。【目的】研究副溶血弧菌CalR对vopB2的转录调控机制。【方法】利用引物延伸实验鉴定vopB2及vtrA的转录起始位点,并根据产物的丰度判断CalR对靶基因的调控关系;采用实时定量RT-PCR研究靶基因mRNA在WT和ΔcalR中转录丰度,验证CalR对靶基因的转录调控关系,进一步利用LacZ实验通过比较β-半乳糖苷酶活性的差异来判定CalR对靶基因的调控关系;利用凝胶阻滞实验分析His-CalR对靶基因启动子区是否具有直接的结合作用。【结果】vopB2有两个转录起始位点A(-130和-28)且其活性受CalR的直接抑制;引物延伸和LacZ结果表明CalR对vtrA的转录并无调控作用。【结论】CalR直接抑制vopB2的转录,该抑制作用与vtrA无关联。[Background] Vibrio parahaemolyticus is a seafood-borne pathogen.CalR is a global transcriptional regulator in V.parahaemolyticus.Type 3 secretion systems 2(T3 SS2) is one of the major virulence determinants of V.parahaemolyticus.VopB2 is a key effector protein of T3 SS2.[Objective] To study the transcriptional regulation of vopB2 by CalR in Vibrio parahaemolyticus.[Methods] Primer extension assay was employed to detect the transcription start sites and the amount of primer extension porroducts of target genes in ΔcalR and WT.Quantitative RT-PCR was then carried out to calculate the transcriptional variation of target genes between ΔcalR and WT.LacZ assay was used to verify regulation relationship by measuring the β-galactosidase activities in cellular extracts using a β-Galactosidase Enzyme Assay System(Promega).Finally the electrophoretic mobility shift assay(EMSA) was applied to analyze the DNA-binding activity of His-CalR to target promoters in vitro.[Results] We detected one transcription start site for vopB2,which was located at 130 bp and 28 bp upstream of vopB2 and its transcribed activity was under the negative control of the CalR.We also found that His-CalR was bind to the promoter region of vopB2 directly.Besides,our data showed that CalR had no regulatory effect on vtrA transcription,a known regulator for vopB2.[Conclusion] V.parahaemolyticus CalR represses the transcription of vopB2 directly,which is not associated to vtrA.

关 键 词：副溶血弧菌 转录调控 CalR vopB2 vtrA 

分 类 号：R378[医药卫生—病原生物学]