LuxS/AI-2群体感应系统对副溶血性弧菌耐药性及其传递的调控作用研究

Vibrio parahaemolyticus is the foodborne pathogen commonly found in seafood. It is a challenge for clinical therapy and threat to the human public health if the pathogenic antibiotic-resistant V. parahaemolyticus strains enter into the human body via the food chain. It has been known that the quorum sensing system LuxS/AI-2 affects many physiological processes, including antibiotic resistance. In order to provide a new idea and breakthrough for the study on antibiotic resistance mechanism of pathogens and for prevention and control of antibiotic-resistant pathogens, the multiple antibiotic-resistant V. parahaemolyticus strains from seafood were chosen as the research object in this project and were studied based on quorum sensing. The relationship between the key genes luxS, pfs and the activities of AI-2 were analyzed, based on which, the mutant strains with luxS deletion was constructed and the AI-2 was synthesized in vitro. The relationship between antibiotic resistance, transcription level of resistance genes, conjugation frequency of resistance plasmid, transcription level of regulatory genes of plasmid and AI-2 were carried out by blocking the synthesis of AI-2 in vivo and adding the synthetic AI-2. From the above results, the regulation effect and its molecular mechanism on the antibiotic resistance and its dissemination of Vibrio parahaemolyticus by quorum sensing system LuxS/AI-2 were revealed from DNA-mRNA-protein level, which provide the basis for explaining the resistance dissemination mechanism of Vibrio parahaemolyticus and developing a theory of inhibiting the antibiotic resistance dissemination of Vibrio parahaemolyticus based on quorum sensing.

副溶血性弧菌是海产品中广泛存在的食源性致病菌，其致病性耐药菌通过食物链进入人体，会给临床治疗带来挑战，威胁人类健康。已知细菌种间交流的LuxS/AI-2群体感应系统能调控细菌包括耐药性在内的多种生理过程。为了给致病菌耐药机制研究及耐药致病菌的防控提供新思路和突破口，本项目从群体感应入手，以海产品中分离的副溶血性弧菌多重耐药菌为研究对象，在分析合成关键基因luxS和pfs与AI-2信号分子活性相关性的基础上，构建luxS基因缺失株和体外合成AI-2分子，从内源阻断合成及外源添加两个角度分析AI-2与菌株耐药性、耐药基因转录水平、耐药质粒接合频率、质粒调控基因转录水平的相关性，从DNA-mRAN-蛋白质三个水平揭示LuxS/AI-2对副溶血性弧菌耐药性及其传递的调控作用与分子机制，为解析副溶血性弧菌的耐药性扩散机制提供依据，为建立一种基于群体感应策略抑制副溶血性弧菌耐药性传播的理论奠定基础。

副溶血性弧菌是海产品中广泛存在的食源性致病菌，其致病性耐药菌通过食物链进入人体，会给临床治疗带来挑战，威胁人类健康。已知细菌种间交流的LuxS/AI-2群体感应系统能调控细菌包括耐药性在内的多种生理过程。本项目从群体感应入手，以海产品中分离的副溶血性弧菌耐药菌为研究对象，在分析信号分子AI-2活性与合成关键基因luxS和pfs相关性的基础上，体外合成信号分子AI-2和构建luxS基因缺失株，并以四环素耐药性为例，从外源添加及内源阻断合成两个角度分析LuxS/AI-2群体感应系统对副溶血性弧菌耐药性及其传递的影响及机制。研究结果发现，所有副溶血性弧菌均含有LuxS/AI-2群体感应系统，大部分菌株高产信号分子AI-2，培养条件能调控副溶血性弧菌AI-2的活性和合成关键基因luxS和pfs的表达，部分条件下AI-2的活性与luxS和pfs的表达量具有明显相关性。通过对副溶血性弧菌的luxS和pfs进行基因克隆和原核表达，获得可溶性的蛋白LuxS和Pfs，并在体外合成具有生物活性且活性较高的AI-2。AI-2能显著提高副溶血性弧菌对四环素的耐受性，同时在一定程度上提高菌株四环素耐药基因的表达水平。通过融合PCR和自杀质粒基因敲除技术成功构建了副溶血性弧菌luxS缺失突变株，luxS的缺失造成菌株四环素耐药性显著降低，同时耐药基因的表达量也显著降低。从外源添加AI-2和内源阻断AI-2合成两方面都证明了LuxS/AI-2群体感应系统能促进副溶血性弧菌四环素耐药性的增强。AI-2能增强副溶血性弧菌接合频率，同时提高部分接合转移调控基因的表达水平，表明AI-2能促进副溶血性弧菌四环素耐药基因的传递扩散。项目的研究结果为解析副溶血性弧菌的耐药性扩散机制提供依据，为建立一种基于群体感应策略抑制副溶血性弧菌耐药性传播的控制技术奠定基础。