中国罗非鱼源无乳链球菌溯源及跨宿主感染分子机制研究

Streptococcus agalactiae has significantly impacted the sustainable development of tilapia industry around these five years in China. Depending on our previous system study on the epidemiology of streptococcal disease in Tilapia during a period of 2006 to 2013, making together with studies by domestic and foreign scholars, our research group has proposed an academic hypothesis that human-to-fish transmission of Streptococcus agalactiae. Based on the above consideration, this project is proposed to study the evolution and relationships between tilapia-derived and human-derived wild-type S.agalactiae speices through comparison analyses of their clinical experimental informations, serotypes and genotypes. Meanwhile, in order to explain evolution relationship between tilapia source and human source S.agalactiae and the molecular mechanism of Tilapaia infected S.agalactiae from human, sequencing and comparing four strains of different resources of wild-type S.agalactiae isolated from fish and human (include the stains can and not can infected Tilapia) whole genome DNA. In addition, functional mechanism of virulent facors, transfer elements, or some other active key genes in the processing of such pathogenic human-derived S.agalactiae to cross species barriers to infect tilapia meningeal cells, will be studied by gene mutation and deletion, transcriptome analysis, RNA interferene, and immunohistochemical technologies. In sum, this project aim to discusses the evolution and relationships between tilapia-derived and human-derived S.agalactiae speices in China, as well as the molecular mechanism of S.agalactiae cross-species host transmission, provide experimental and theoretical basis for scientific prevention and control of S.agalactiae disease.

近5年，无乳链球菌病已严重影响我国罗非鱼产业可持续发展。项目组在2006-2013年我国罗非鱼链球菌病流行病学系统研究的基础上，结合国内外学者研究结果，提出"我国罗非鱼源无乳链球菌可能来源于人"的学术主张。为此，项目拟通过菌株临床信息、血清型及基因型比较研究，初步理清我国罗非鱼源与人源无乳链球菌间进化亲缘关系；同时，通过感染试验、4株罗非鱼源和人源菌株（能/不能感染罗非鱼）全基因组测序及比较分析，从基因组水平进一步阐明我国罗非鱼源与人源无乳链球菌的亲缘关系及人源无乳链球菌跨宿主感染罗非鱼的分子机制；另外，利用基因突变和缺失、转录组分析、RNAi及免疫组化等技术对毒力因子、转移元件及其它关键基因在人源无乳链球菌跨宿主感染罗非鱼脑膜细胞的作用机制进行研究。项目探讨中国罗非鱼源与人源无乳链球菌的亲缘关系及人源无乳链球菌跨宿主感染的分子机制，为科学防控无乳链球菌病提供实验和理论依据。

无乳链球菌对包括人、鱼等在内的广泛宿主具有致病性，近10年来，无乳链球菌病已严重影响我国罗非鱼产业的可持续发展。项目组在2006-2013 年我国罗非鱼链球菌病流行病学系统研究的基础上，结合国内外学者研究结果，提出“我国罗非鱼源无乳链球菌可能来源于人”的学术主张。项目通过菌株临床信息、血清型及基因型比较研究，梳理我国罗非鱼源与人源无乳链球菌间进化亲缘关系；同时，通过全基因组、转录组及蛋白组测序等研究手段进一步对我国罗非鱼源与人源无乳链球菌的亲缘关系及人源无乳链球菌跨宿主感染罗非鱼的分子机制进行研究。项目共获取308株罗非鱼源和92株人源无乳链球菌试验菌株并对其进行相关荚膜血清型、MLST、PFGE鉴定，初步理清我国罗非鱼源无乳链球菌与人源无乳链球菌进化上的亲缘关系，并筛选获得了对罗非鱼强致病力/无致病力的人源菌株NNA048/NNA038（V，ST1）及鱼源菌株HN016/YM001（Ia，ST7）。多组学分析结果显示，NNA048基因组相较于NNA038特异具有一个长度为49.8 kb编码68个蛋白的完整噬菌体序列；显著差异蛋白主要与代谢及代谢物运输和生物合成有关，噬菌体可能是造成菌株毒力差异的主要原因；YM001基因组相较于HN016有D1（5,832 bp）和D2（11,115 bp）两处显著的大片段缺失；显著差异蛋白与细胞壁结构功能、耐酸性、耐低氧能力等相关，可能是导致YM001对罗非鱼失去致病力的主要原因。此外，人源无乳链球菌与血脑屏障的相关作用机制研究表明，超毒力粘附因子HvgA基因的F9片段可极显著促进对人脑膜血管内皮细胞的粘附作用（Ming Chen et al ,2019, IF=5.39）。.本项目基本理清了我国罗非鱼源无乳链球菌与人源无乳链球菌进化上的亲缘关系，并深入探究了人源无乳链球菌跨宿主感染的分子机制，为科学防控无乳链球菌病提供了实验和理论依据。本项目已发表14篇学术论文，其中SCI论文共10篇（一区3篇，二区6篇；5分以上论文1篇），项目团队在罗非鱼链球菌病领域发表SCI论文总数居世界前列，已成为世界上研究鱼类链球菌病的重要力量。