基于群体感应通路解析腐败希瓦氏菌对副溶血弧菌TDH和被膜形成的作用机制

Thermostable hemolysin (TDH) and biofilm produced by pathogenic VP (Vibrio parahaemolyticus) in aquatic products and environment are considered to be the most important hazard factors of food poison and food contamination. SP (Shewanella putrefaciens), coexisting with VP, can affect the generation of hazard factors, and bring new challenges for food security assessment and control. At present, the studies focusing on the effects of actions and mechanism of SP on VP THD/biofilm formation are scarce. Based on the theory of quorum sensing, this study will build a series of quorum sensing signal system deletion mutant of VP with gene knockout technology, co-culture SP with VP and VP mutants respectively, ascertain the effects of actions of SP on the TDH production and biofilm formation of VP in coexisting system by detecting the expression differences of gene TDH with FQ-PCR (fluorescence quantitative polymerase chain reaction) and detecting the micromorphology and composition characteristics of biofilm with FISH (fluorescence in situ hybridization) and LSCM (laser scanning confocal microscopy). By contrasting the effects of actions of SP on VP before and after gene knock-out the function pathway of SP to VP is ascertained. The key signal molecules related to the function pathway is detected by signal molecular reporter bacteria detection and LC-MS-MS. The regular pattern of dynamic cascade of signal molecules and the effects of actions are clarified, and the action law of SP to VP can be well researched. This study will provide theoretical basis for the food safety hazard assessment and the control in co-existing condition of VP and SP.

水产品及其环境中致病性副溶血弧菌(VP)产生的耐热溶血素（TDH）和生物被膜(BF)是引起食物中毒的重要危害因子，腐败希瓦氏菌(SP)与VP共存可对其危害因子生成产生影响，为食品安全评估和控制带来新的挑战。但目前缺乏SP对VP TDH/BF形成作用效应及机制的研究。基于群体感应理论，本研究首先采用基因敲除技术构建VP群体感应信号系统缺失系列突变株，然后将SP分别与VP、VP系列突变株共培养，以荧光定量PCR检测tdh基因表达，荧光原位杂交和共聚焦显微技术观察BF微观形貌及组成特征，明确共存体系下SP对VP TDH/BF形成的作用效应；通过对比基因敲除前后作用效应的改变，探明SP对VP的作用途径；采用信号分子报告菌和LC-MS-MS法对与作用途径相关的关键信号分子进行测定，并明确其与作用效应的动态级联规律，探明SP对VP的作用规律。本项目将为SP共存条件下VP危害因子评估及控制提供理论依据。

腐败希瓦氏菌作为水产品的特定腐败菌常常和副溶血弧菌共存于海产品及其加工环境中，两菌共存对副溶血弧菌毒力因子的产生或有促进作用，可能带来比预期更大的食品安全问题，其共存作用效应需要评估，作用机制需要揭示。由于群体感应系统对细菌毒力因子的生成起到关键调控作用，故基于该系统开展研究可以更加高效地揭示腐败希瓦氏菌对副溶血弧菌毒力因子形成具有促进作用的机制。可为两菌共存的食品安全风险评估和寻找控制靶点提供理论依据。研究考察了共培养体系中对虾腐败过程优势菌群分离株对副溶血性弧菌毒力因子生成的影响，发现某些腐败菌与致病性副溶血弧菌共存时其毒力因子的表达会显著增强，其中腐败希瓦氏菌camtF菌株对tdh基因表达上调作用最为明显。采用基因敲除技术构建了副溶血性弧菌AI-1/AI-2信号分子合成酶基因LuxM/LuxS缺失菌株，探讨了LuxM和LuxS在毒力因子调控中的作用。发现AI-1信号分子合成酶luxM基因对副溶血性弧菌的溶血活性、tdh基因表达和生物被膜的形成起主要的负调控作用，AI-2信号分子合成酶luxS基因对副溶血弧菌溶血活性、tdh基因表达起次要的正调控作用，对生物被膜形成起次要的负调控作用。比较了腐败希瓦氏菌与副溶血弧菌间的AHLs信号分子谱异同；对比了腐败希瓦氏菌对副溶血弧菌亲本株、突变株的毒力因子生成的作用效应差异；探讨了腐败希瓦氏菌-信号分子-副溶血弧菌毒力因子间的级联作用规律。综合分析后发现，腐败希瓦氏菌能够降解副溶血性弧菌的AHLs信号分子，使共存体系中的AHLs信号分子浓度降低，从而解除了对副溶血弧菌的毒力因子表达的负调控作用，使副溶血弧菌的毒力因子的表达增强。通过分离纯化和鉴定，证明降解AHLs信号分子的物质属于酰胺酶类。研究结果最终从分子水平上揭示了腐败希瓦氏菌对副溶血弧菌的tdh基因表达和生物被膜形成的促进作用机制。