基于“群体感应-肠道菌群-肠上皮细胞”途径探讨产淬灭酶芽孢菌干预嗜水气单胞菌感染鲫鱼作用机制

Based on the “quorum sensing” in microbial community ecology and the “microbial stimulation theory” of intestinal mucosal immune response, this project will use Crucian carp as the animal model to investigate the preventive effect of quorum quenching enzyme producing Bacillus sp. against Aeromonas hydrophila infection through oral administration. The structure of the intestinal microbiota will be analyzed by using pyrosequencing the 16S rRNA genes, metabolite profiles in the intestinal contents will be analyzed by GC-MS. The immune-histochemical method will be used for the detection of intraepithelial lymphocytes, eosinophile granulocyte and other immune related cells. ELISA will be used to determine the cytokines and antibodies in serum. Inhibition of A. hydrophilia infect the intestinal epithelial cells by quorum quenching enzyme or QSI-1 will be conducted by in vitro cell culture. The project aims to explore the effect of quorum quenching enzyme producing Bacillus sp. on the structure of intestinal microflora and the mucosal immune system in Crucian carp, and clarify the mechanism of quorum quenching enzyme producing Bacillus sp. against A. hydrphila infection in fish. This project will provide new ideas for healthy rearing of aquatic animals through regulating gut microflora and enhancing mucosal immune system.

本项目基于微生物间的“群体感应理论”和肠道黏膜免疫应答形成的“微生物刺激理论”，以鲫鱼为模型动物，通过口服方式接种能产生群体感应淬灭酶的芽胞杆菌QSI-1,对鲫鱼抗嗜水气单胞菌感染进行干预，应用16S rRNA基因高通量序列测定技术分析各肠道菌群组成，通过GC/MS代谢组学平台对肠内容物代谢组谱进行分析，组织学和免疫组织与化学方法检测各肠段组织上皮内淋巴细胞、嗜酸性粒细胞等免疫相关细胞数量；定量PCR检测免疫相关基因的表达水平；酶联免疫方法测定血清中抗体及细胞因子的含量；通过体外细胞培养对淬灭酶以及淬灭酶产生菌抑制嗜水气单胞菌感染上皮细胞进行分析。探究群体感应淬灭酶产生菌对鱼肠道微生物区系、代谢以及黏膜固有免疫的影响，阐明群体感应淬灭酶产生菌干预嗜水气单胞菌感染的作用机制，从菌群调控及增强黏膜免疫方面为水产动物健康养殖提供新思路。

抗生素的使用导致细菌耐药性的产生，群体感应淬灭是抗感染的有效途径。本研究基于微生物间的“群体感应理论”和肠道黏膜免疫应答形成的“微生物刺激理论”，以鲫鱼为模型动物，通过口服方式接种能产生群体感应淬灭酶的芽胞杆菌QSI-1,对鲫鱼抗嗜水气单胞菌感染进行干预，应用16S rRNA基因高通量序列测定技术分析各肠道菌群组成，通过GC/MS代谢组学平台对肠内容物代谢组谱进行分析；定量PCR检测免疫相关基因的表达水平；酶联免疫方法测定血清中抗体及细胞因子的含量；通过体外细胞培养对淬灭酶以及淬灭酶产生菌抑制嗜水气单胞菌感染上皮细胞进行分析，对产群体感应淬灭酶的芽孢菌提高鲫鱼抗嗜水气单胞菌感染的机制。结果表明群体感应淬灭酶产生菌QSI-1以及淬灭酶能抑制嗜水气单胞菌毒力因子产生以及生物膜的形成，并能抑制嗜水气单胞菌内化；体内研究发现：群体感应淬灭酶产生菌QSI-1能有效降低鲫鱼肠道内信号分子含量，调节肠道菌群，促进有益菌的生长，抑制病原菌的生长，并提高肠道内短链脂肪酸的含量；淬灭酶产生菌QSI-1能增强鲫鱼免疫应答反应，能显著增强肠道黏膜屏障紧密性，促进免疫相关细胞因子的产生，刺激鲫鱼粘液中与免疫相关蛋白的表达，并能提高血清和粘液中抗体水平。该研究揭示了群体感应淬灭酶产生益生菌提高鲫鱼抗感染机制，为益生菌作为抗生素替代品在水产养殖中的应用提供了理论基础。