基于群体感应途径的荧光假单胞菌对大菱鲆的致腐机制

The spoilage of seafood is mainly caused by the bacterial growth, in which the special spoilage bacteria play a key role. Quorum sensing (QS), an important regulatory system in microorganism which controlled by bacterial communication signals, acylated homoserine lactones (AHLs), has become a hot topic in life science field. However, most studies of QS focused on the virulence factors regulations in pathogenic bacteria. The roles of QS in seafood spoilage and the regulation mechanisms of QS in spoilage bacteria were still poorly elucidated. In this project, the special spoilage bacteria, Pseudomonas flurosecens (PF), separated from the turbot (Scophthalmus maximus L.) during cold storage will be firstly identified, and the AHLs-producing bacteria will be screened from the separated strains. The types and structures of AHLs from the AHLs-producing PF will be analyzed by biosensors combining with HPLC-MS. Secondly, an AHL-deficient mutant of PF will be constructed by the mini-Tn5 transposon delivery system. The difference in spoilage properties of the AHL-deficient mutant, separated strains and type culture strains of PF will be investigated to obtain the direct evidences of QS involved in the bacterial spoilage of fish. Finally, the difference in protein expression of the AHL-deficient mutant, separated strain and type culture strain of PF will be analyzed by proteomic techniques to reveal turbot spoilage mechanism caused by PF under QS regulation. This study aims to clarify the spoilage mechanisms of fish caused by microorganism deeply, to provide theoretical basis for the development of new seafood preservation techniques base on the targeted inhibition of the spoilage bacteria communication underlying the QS systems. The study will contribute to the innovation and enrichment of the seafood storage and preservation theories.

水产品腐败主要由微生物活动引起，其中特定腐败菌起关键作用。AHLs等信号分子介导的群体感应（QS）作为微生物重要的生理调控系统成为生命科学研究热点之一，但研究多集中在病原菌致病因子的调控方面，对腐败菌的QS调控机制及其在水产品腐败中的作用仍不明晰。鉴于此，本项目将根据前期研究，详实鉴定大菱鲆冷藏时的特定腐败菌-荧光假单胞菌（PF），筛选AHLs活性菌株，采用生物感应器结合HPLC-MS等方法对AHLs进行测定；利用转座子突变技术构建AHLs缺失菌株，比较缺失菌株、分离菌株及标准菌株体内外培养时致腐性能的差异，获得QS参与鱼类腐败的直接证据；采用蛋白质组学技术研究不同PF菌株蛋白质表达差异与致腐性能的相关性，阐明QS对PF致腐性能的调控机制。研究旨在深层次揭示微生物引起的鱼类腐败机制，为开发靶向控制腐败菌QS系统的水产品保鲜新技术提供理论依据，对创新和丰富水产品贮藏保鲜理论具重要意义。

微生物的生长代谢是引起水产品等高蛋白食品腐败的重要因素，其中特定腐败菌起到了关键作用。由信号分子介导的群体感应系统能够有效调控微生物的生理行为，因此有望成为控制微生物致腐能力的关键靶点。但之前研究多集中在病原菌致病因子的调控方面，对腐败菌的群体感应调控机制及其在水产品腐败中的作用仍不明晰。鉴于此，本项目围绕“群体感应对大菱鲆荧光假单胞菌的致腐机制”这一重大科学问题，深入开展了大菱鲆冷藏过程中的菌群演替规律、关键致腐菌群体感应及信号分子腐败调控机制、环境共存菌对优势腐败菌影响以及基于群体感应系统的抑制剂筛选及作用机制探究等相关工作，旨在丰富我国水产品保鲜理论及手段。研究结果表明大菱鲆优势腐败菌荧光假单胞菌能够借由rhlI/R系统调控自身运动性、刺激反应、粘附性以及生物膜合成等相关基因的表达，从而调控自身的致腐行为。且温度、pH、NaCl浓度等环境因素均能通过影响rhlI/R系统来影响荧光假单胞菌致腐因子的产生，其中温度（35°C）、高pH以及高NaCl浓度均有利于控制菌株相关系统的表达。同时，研究发现荧光假单胞菌可以通过受体蛋白RhlR感受外界共存菌分泌的信号分子来增强自身的致腐性，这进一步明晰了腐败菌在自然条件下的协同致腐机制。此外，研究结果证明肉桂醛、香兰素等天然成分能够通过竞争性结合受体蛋白的方式有效抑制菌株的群体感应，从而起到延缓大菱鲆腐败的作用。综上，本课题为从微生物的群体感应入手解决水产品腐败问题提供了确切的实验依据以及应用经验。