酰基高丝氨酸内酯介导的群体感应调控副溶血性弧菌乙醇耐受机制研究

Vibrio parahaemolyticus (VP) is an important seafood-associated pathogen. Quorum sensing (QS) mechanism a hot spot of VP researches that microorganisms coordinate their physical behaviors by their populations. It was found that VP secreted two N-aclhomoserine lactone (AHL) molecules as QS signals and had a complex QS regulation network. But it is rarely reported that VP uses QS system regulating its adversity stress behaviors. We found that VP was not susceptible to ethanol of low concentration. A scientific hypothesis is put forward in this study that QS controls adversity stress behavior like ethanol-tolerance in VP. And a series of assays have been designed to proof the hypothesis by exploring the roles of AHL-introduced QS in VP ethanol-tolerance. First, a vanM-knockout strain which could not product AHL signals would be constructed to test VP AHL signals’ roles in ethanol-tolerance. Second, QS core regulator opaR gene expression levels in different AHL pathways would be analyzed and opaR-knockout strain would be constructed to analyze ethanol-tolerance ability, so that it would be revealed whether AHLs play roles in ethanol-tolerance through a OpaR-dependent pathway. Third, proteomic assays iTRAQ method would be performed to find QS-regulated ethanol-tolerance factors by comparing QS-regulated protein profile (prepared from iTRAQ with vanM-/ opaR-/ wildtype VP strains) and ethanol-tolerance-associated protein profile (prepared from iTRAQ with ethanol treated/untreated VP strains). At last, the functions of QS-regulated ethanol-tolerance factors would be validated by realtime-PCR and gene knockout methods. This study would hierarchically reveal the mechanism of QS regulating VP ethanol-tolerance from three levels of QS signal (AHL), QS regulator (OpaR) and QS effector (QS-regulated ethanol-tolerance factor). It would shed the light on research of VP survival strategies and control of VP-associated foodsafety risk.

副溶血性弧菌(VP)是一种重要的水产品相关病原菌，群体感应( QS)是它一大研究热点。研究发现VP利用两种酰基高丝氨酸内酯（AHL）为QS信号，具有强大QS调控网络，但鲜见QS调控逆境胁迫的研究。我们发现VP耐受乙醇，提出QS调控VP逆境胁迫的假设，研究AHL介导的QS在乙醇耐受中的作用机制：构建不产AHL的vanM缺失突变株，分析AHL缺陷及补充不同AHL对乙醇耐受能力的影响；realtime-PCR研究AHL对QS调控子OpaR表达的影响，构建opaR缺失株探索OpaR在QS调控乙醇耐受中的作用；利用iTRAQ技术研究QS调控蛋白谱和乙醇耐受蛋白谱，比较两组蛋白发掘QS调控的乙醇耐受因子；利用realtime-PCR和基因敲除等手段对它们进行功能验证。本研究将从"信号分子-调控通路-效应因子"三个层次揭示QS调控VP乙醇耐受的机制，为研究VP逆境生存策略、防控VP食品安全危害提供思路。

副溶血性弧菌是一种重要的食源性致病菌，群体感应系统被认为在它的致病性和环境适应性调节中发挥重要作用。本项目主要围绕酰基高丝氨酸內酯群体感应信号调节副溶血性弧菌乙醇耐受能力，研究副溶血性弧菌群体感应系统调控其逆境胁迫的机制，同时利用蛋白质组学等手段，拓展对副溶血性弧菌其他群体感应通路的认识。主要研究内容包括：分析酰基高丝氨酸内酯信号及其合成酶VanM对副溶血性弧菌乙醇耐受能力的影响；探索群体感应核心调控子OpaR对乙醇耐受能力影响以及其在酰基高丝氨酸内酯调控乙醇耐受中的作用；通过蛋白质组学手段发掘受酰基高丝氨酸内酯调控的乙醇耐受因子，并验证这些乙醇耐受因子的功能和表达调控机制；拓展研究了另一副溶血性弧菌群体感应通路及功能。我们发现：酰基高丝氨酸内酯群体感应信号及其合成酶基因vanM的存在促进了副溶血性弧菌的乙醇耐受；OpaR同样能够影响副溶血性弧菌的乙醇耐受能力，并且其表达收到群体感应信号分子的影响；副溶血性弧菌的cqsA群体感应通路以3-羟基-4-十一烷酮为信号分子，蛋白质组学分析显示其调控众多靶标基因的表达。本研究证实了群体感应在群体感应系统在副溶血性弧菌乙醇胁迫条件下的生存调节作用，拓展了副溶血性弧菌生存调节机制的研究，为副溶血性弧菌防控策略的研究提供了理论基础。在本项目的资助下，项目组在国际学术期刊Frontiers in Microbioloy、Journal of Microbiology和Microbial Pathogenesis各发表论文1篇,，取得了预期成果；项目组的3名主要成员均获得高级职称晋升，人才队伍水平得到大幅提高，为进一步开展相关研究打下了基础。