副溶血弧菌VP0057介导蛋白质磷酸化修饰调控毒力的分子机制

Vibrio parahaemolyticus is the leading worldwide cause of food-borne illnesses associated with the consumption of raw sea-food. Although hemolysins, adhesion, type III and type VI secretion systems have been revealed as the major virulence factors of the bacterium, their regulatory mechanisms are still ill-defined. Recently, the phospho-modification of proteins triggered by eukaryotic-like Ser/Thr protein kinase (eSTPK) has been found to play central roles in bacterial cellular regulation and closely associated with bacterial virulence. Our previous work on Vibrio parahaemolyticus indicated that VP0057, an uncharacterized protein is most likely to be an eSTPK and it is involved in the activation of the type III secretion system. Therefore, based on these observations, we suggest VP0057 regulates the virulence factors through phosphorylation of proteins. In this proposal, we plan firstly to construct VP0057 gene deletion and complement strains and further confirm the regulatory roles of VP0057 by comparative analyzing the variations of virulence factors and pathogenicity of these strains. Then we will systematic characterize the differentiations of protein expression and protein phosphorylation caused by the deletion of VP0057, and follow by the determination of the phosphorylation target proteins which involved in virulence factors. After that, we will further confirm that the protein phosphorylation is catalyzed by VP0057 and the expression or secretion of virulence factors are regulated by phosphorylation modification. The results of this research will not only reveal a novel regulatory mechanism of virulence from a perspective of protein phosphorylation mediated by VP0057, but also provide a new strategy and a new target for the development of new drugs or vaccines based on phosphorylation sites and regulatory enzymes.

副溶血性弧菌是全球范围内由海产品引起食源性疾病的首要致病菌。虽然溶血素、粘附素、Ⅲ型及Ⅵ型分泌系统已被揭示为主要毒力因子，但它们的调控机制仍不明确。近年发现由Ser/Thr蛋白激酶引起的蛋白质磷酸化修饰是细菌功能调控的核心，且与毒力密切相关。申请人前期研究显示副溶血弧菌未知蛋白VP0057为Ser/Thr蛋白激酶，且与Ⅲ型分泌系统的激活有关，为此申请人提出VP0057通过磷酸化修饰调控毒力。本项目拟构建VP0057缺陷菌株，分析其对毒性及毒力因子的表达、分泌的影响，进一步明确其对毒力的调控作用；系统分析缺失VP0057所引起的蛋白质表达、磷酸化修饰的变化，进而确定与毒力因子相关的磷酸化靶蛋白及其磷酸化位点，证实VP0057催化这些位点的磷酸化，并调控毒力因子的表达、组装或分泌。本项目深入揭示VP0057调控毒力的分子机制，为基于磷酸化修饰位点及调控酶的新药或疫苗研制提供新思路和新靶点。

副溶血弧菌是引起食源性疾病的主要致病菌之一。虽然Ⅲ型及Ⅵ型分泌系统、粘附素等已被揭示为主要毒力因子，但它们的调控机制仍不明确。近年发现由Ser/Thr蛋白激酶引起的蛋白质磷酸化修饰是细菌功能调控的核心，且与毒力密切相关。申请人在前期研究基础上，提出副溶血弧菌Ser/Thr蛋白激酶VP0057通过磷酸化修饰来调控毒力这一观点。为此，本项目采用基于生物化学、分子生物学、生物信息学、微生物学、定量蛋白质组学等的技术手段开展了一系列研究。首先，成功构建了重组载体，获得vp0057基因敲除菌（Δvp0057）和回补菌株（+ vp0057），并通过对VP、Δvp0057和+ vp0057三菌株进行综合比较分析，发现缺失vp0057基因，导致生物膜产量减少，细菌的运动能力、对细胞的黏附能力、肠毒性均下降（但在细菌低密度下，对细胞毒性却有所增加）。这些结果明确了vp0057对副溶血弧菌的毒力具有调控作用。其次，对副溶血弧菌的主要毒力因子T3SS1、T3SS2、T6SS和铁离子摄取相关等一系列基因的表达差异进行分析，从分子水平上确定了缺失vp0057导致这些毒力因子相关基因的表达变化是该菌毒力发生改变的主要原因。再次，完成了缺失vp0057引起的细菌蛋白质表达（蛋白质组）和蛋白质磷酸化修饰（磷酸化蛋白质组）的变化，并对差异蛋白质近了系统的生物信息学分析；发现了由VP0057催化的磷酸化靶蛋白及磷酸化位点，初步揭示了VP0057介导磷酸化修饰调控副溶血弧菌毒力的分子机制。本项目较为系统地研究了副溶血弧菌VP0057（Ser/Thr蛋白激酶）对其毒力的调控作用及其作用机制，本项目的研究成果不仅促进全面、深入地阐明副溶血弧菌的毒力和致病机制，也将有助于基于磷酸化调控的药物靶点的发现，为新药研究和临床治疗提供全新的策略和新思路，具有巨大的应用前景。