AphB互作蛋白的筛选及其对霍乱弧菌毒力因子调控机制研究

Toxigenic Vibrio cholerae can cause human acute diarrhea – cholera, O139 cholera as an emerging infectious diseases is still epidemic globally. Toxigenic Vibrio cholerae expresses two major virulence factors including cholera toxin CT encoded by ctxAB and pili TCP encoded by tcpA, which are stepwise regulated by more than a regulatory protein. ToxT as a regulatory protein directly activates the expression of tcpA and ctxAB, two membrane proteins ToxR and TcpP coordinately promote the transcription of toxT. In Vibrio cholerae environmental survival and infection process, AphB as a major regulator can activate the transcription of tcpP by binding its promoter and ultimately causes disease. However, it is still unclear which protein can interact with AphB directly to regulate virulence genes. Based on our preliminary studies, in this research, we try to further screen some proteins, which can directly interact with AphB, from the Vibrio cholerae genomic library. We will confirm their interaction and analyze their influence on the transcription and expression of the virulence genes in the virulence regulatory pathway. How these proteins interact and sense micro-environmental signals to regulate the virulence gene expression will be further analyzed. This study will expand our understanding on the regulation mechanism of AphB. The virulence regulatory pathway will be further refined and supplemented. This study will provide us molecular basis for revealing the pathogenesis of Vibrio cholerae.

产毒霍乱弧菌是导致霍乱的病原体，目前仍在全球流行，并引起O139霍乱这一新发传染病。产毒霍乱弧菌主要的致病因子包括霍乱毒素CT和菌毛TCP，这些毒力因子表达受到多个蛋白的级联调控：毒力基因直接被ToxT激活，toxT基因又受到ToxR和TcpP的协同调控，其中tcpP的转录表达又受到AphA和AphB的联合正向调控。在霍乱弧菌环境生存和感染过程中，AphB作为主要调控蛋白，能够感知微环境信号的变化，发挥毒力基因的激活作用。然而哪些蛋白与 AphB直接互作协同发挥毒力调控作用还不清楚。基于我们的前期研究工作,本研究拟通过扩展霍乱弧菌基因文库，利用BACTH进一步筛选与AphB互作的蛋白，通过对初筛蛋白验证及分析其对毒力调控通路下游基因转录、表达的影响，明确蛋白互作以及感受外界信号调节毒力表达的机制。本研究将扩展我们对调控蛋白AphB的认识，完善毒力调控通路，为揭示霍乱弧菌致病性提供分子依据。

产毒霍乱弧菌主要的致病因子包括霍乱毒素CT和菌毛TCP，这些毒力因子表达受到多个蛋白的级联调控：毒力基因直接被ToxT激活，toxT基因又受到ToxR和TcpP的协同调控，其中tcpP的转录表达又受到AphA和AphB的联合正向调控。在霍乱弧菌环境生存和感染过程中，AphB作为主要调控蛋白，能够感知微环境信号的变化，发挥毒力基因的激活作用。然而哪些蛋白与 AphB直接互作协同发挥毒力调控作用还不清楚。本研究建立并扩大了霍乱弧菌基因文库，利用细菌双杂交技术（BACTH）进一步筛选到了与AphB互作的蛋白VC0049、VC0601，Pull Down实验进一步验证了VC0049/AphB、VC0601/AphB互作，明确了AphB的第59、100、134、201和275氨基酸位点在AphB与VC0049互作时起到重要作用，VC0049的第11、18、60和120氨基酸位点在VC0049与AphB互作时起到重要作用，其中任何一个位点的突变均会造成两个蛋白的互作消失。VC0049过表达后，会引起toxR和toxT的转录几乎停止，VC0049过表达后也会引起TcpA表达量大大降低，tcpA的转录水平降低100倍。总之，本研究筛选到VC0049可与霍乱弧菌毒力调控通路的上游调控蛋白AphB互作，明确了VC0049或者VC0049/AphB复合物间接抑制toxR的转录，进而引起toxT转录水平下降，最终导致毒力因子TcpA表达的降低。本课题通过对VC0601/AphB、VC0049/AphB复合物验证及分析VC0049/AphB对毒力调控通路下游基因转录、表达的影响，扩展我们对调控蛋白AphB的认识，为原有的毒力调控通路增添了新“成员”，进一步完善了毒力调控通路，为揭示霍乱弧菌致病性提供分子依据。