Ⅵ型分泌系统在迟缓爱德华氏菌侵染鱼类过程中的作用机制研究

Type Ⅵ secretion system (T6SS) is a new bacterial secretion system found in recent years. It secrets effectors to exert various functions, that include infection. T6SS is one of the most important virulence determinants of Edwardsiella tarda, and is indispensable for the organism to invade target host cells. By now, the only known T6SS proteins are coded by genes of evp (Edwardsiella tarda virulent protein) cluster. While the present evidence indicates that the EVP proteins are structural components of the secretion apparatus, instead of effectors. The evp cluster was proved to possess two promoters in all. In our previous work, we constructed a mutant strain with a deletion of both the promoters. And the T6SS secretion apparatus of this mutant strain is completely inactivated. In the present research, we aim to obtain the entire extracellular and intracellular proteins of E. tarda wild type strain and T6SS deficient strain under infection conditions, and then identify the T6SS-related proteins (including the effectors) with comparative proteomics analysis. The functional roles of the T6SS-related proteins vital for E. tarda infection will be investigated. And the finding will be combined and integrated to illustrate the effect and mechanism of T6SS in the process of E. tarda infection of fish.

Ⅵ型分泌系统（T6SS）是近几年发现的一种新型细菌分泌系统，它通过分泌效应蛋白(effectors)在细菌的致病及其它过程中发挥功能。T6SS是迟缓爱德华氏菌（Edwardsiella tarda）的重要毒力因子，为该菌入侵宿主细胞所必需。目前对E. tarda T6SS的了解仅限于evp基因簇，已有的证据表明该基因簇主要编码T6SS输送器蛋白，而非效应蛋白。研究证实evp基因簇共含有两个启动子，申请人在前期研究中通过缺失突变这两个启动子而获得了T6SS输送器功能完全丧失的E. tarda突变株。本研究拟提取E. tarda野生株及其T6SS突变株在侵染时表达的全部胞外和胞内蛋白，以比较蛋白质组学研究获取包括效应蛋白在内的T6SS相关蛋白，通过研究在侵染过程中起关键作用的T6SS相关蛋白的功能，揭示T6SS在E. tarda侵染鱼类过程中的作用机制。

迟缓爱德华氏菌是一种宿主范围广泛的胞内病原菌。它导致海水养殖鱼类的迟缓爱德华氏菌病，造成严重的经济损失。迟缓爱德华氏菌依赖其VI型分泌系统（Type VI secretion system, T6SS）感染宿主。目前对该菌T6SS效应蛋白所知甚少。本项目旨在确定迟缓爱德华氏菌T6SS效应蛋白和其它相关蛋白，探索T6SS在该菌侵染宿主过程中的作用机制。为实现这一目标，我们建立了一套基于CRISPR (clustered regularly interspaced palindromic repeats)的转录控制技术，该方法具有高效、简便、可同时靶向多个位点的特点，可广泛应用于迟缓爱德华氏菌和其它水产病原菌的研究。应用上述技术，我们通过双位点干扰，对编码迟缓爱德华氏菌T6SS分泌装置的EVP基因簇进行了高效转录抑制，并以分泌组定量质谱比较分析确定了数十种T6SS相关蛋白，为迟缓爱德华氏菌T6SS效应蛋白和致病机制研究打下了坚实基础。此外，我们还发现迟缓爱德华氏菌可借助其T6SS抑制巨噬细胞的死亡以干预宿主免疫反应，并针对可能在这一过程中起主要作用的T6SS效应蛋白开展了研究。