ToxR蛋白调控副溶血弧菌酸耐受反应的分子机制

Vibrio parahaemolyticus is an important food-borne pathogen in mariculture industries, and breakthrough gastric acid barrier is an important way for the infection process. Previous studies had showed that the acid tolerance response play an essential role for the infection of food-borne pathogen. In Vibrio parahaemolyticus, ToxR and CadA proteins participated the regulation of acid tolerance response. In this study, we used the newborn rabbits as animal model to research the acid tolerance response and function in the infection process in vivo and in vitro. Then research the regulation of ToxR and CadC to the cadBA genes in transcript and translate levels in different condition of acids, and study the directly regulation of ToxR and CadC to the cadBA promoter by EMSA, DNase I Footprinting, ChIP and 5’-RACE. Finally, drawn up the model of ToxR protein involved in regulation of acid tolerance response. Clarify the function, molecular mechanism and regulation model of acid tolerance response in the infection process of Vibrio parahaemolyticus, it will be helpful to understanding the significance of the acid tolerance response in the colonization of the gastrointestinal pathogens, and provide a new theoretical support for the prevention of Vibrio parahaemolyticus in seafood.

副溶血弧菌是海水养殖业中重要的食源性病原菌，突破胃酸屏障是其肠道定植的重要途径。研究发现酸耐受反应对食源性病原菌的肠道定植具有重要作用，且副溶血弧菌中ToxR和CadA蛋白参与酸耐受反应的调控。本项目将分别从体外和体内两种条件下考察副溶血弧菌酸耐受反应及其在感染新生兔动物模型中的重要作用。拟从转录水平和翻译水平考察不同酸性条件下ToxR和CadC蛋白对cadBA操纵子的调控情况，并进一步采用EMSA、DNase I Footprinting、5’-RACE和ChIP等分子生物学的实验方法探究ToxR和CadC蛋白调控cadBA启动子的分子机制，最终绘制出ToxR蛋白参与酸耐受反应的调控模型。本项目的主要目标是阐明副溶血弧菌感染过程中酸耐受反应的作用及其调控机制，将有助于认知酸耐受反应对食源性病原菌肠道定植的意义，并为有效的提出海产品中副溶血弧菌的防控手段提供新的分子机制的理论支持。

副溶血弧菌是重要的食源性病原菌，其感染宿主后可以通过宿主胃部酸性环境定植在肠道。本项目鉴定出ToxR、CadC和VPA1701蛋白是副溶血弧菌重要的环境应激和毒力的调控因子。研究证实了副溶血弧菌在经过微酸环境处理后可以增强其在致死性酸环境中存活率。RNA-seq进一步发现ToxR蛋白可以调控ompU介导酸耐受，CadC蛋白调控cadBA的表达介导酸耐受反应，qRT-PCR也证实了ToxR和CadC蛋白对这些基因的调控作用。EMSA实验进一步证实了CadC蛋白可以直接结合到cadBA的启动子上调控其表达。此外，研究也发现在微酸环境中预处理后可以增强副溶血弧菌的运动性和细胞毒性，并绘制出CadC蛋白调控酸耐受反应和毒力的网络图。转录调控因子VPA1701蛋白可以调控副溶血弧菌的运动性和肠道定植。RNA-seq、qRT-PCR和EMSA等实验证实了VPA1701蛋白可以直接结合到侧生鞭毛基因的启动子上调控其表达，以及抑制calR基因的表达来调控副溶血弧菌的运动性。本研究证实了酸耐受反应在副溶血弧菌感染过程中具有重要作用，为有效提出副溶血弧菌的防控手段提供理论支持。