迟缓爱德华氏菌操控宿主cAMP信号通路的作用机制研究

Edwardsiella tarda is a serious pathogen for farmed fish and can cause edwardsiellosis which is a major threat in mariculture. It is known that E. tarda utilizes its type VI secretion system (T6SS) to manipulate host signaling and blunt its immune defense. However, the underlying mechanisms are yet to be explored. cAMP is an important second messenger, which suppresses immune system in normal physiological conditions. In a previous work, we discovered that E. tarda is able to regulate the cAMP levels of infected host cells and the transcription of an isoform of phosphodiesterase; the latter is responsible for cAMP degradation. The effector and its mode of action involved in this observation require further investigation. In this proposal, with the help of a group of identified potential T6SS effectors and a CRISPR genome editing tool from a previous study, we are aimed to conduct an in-site research to give insight to the subversion of host cAMP signaling by E. tarda.

迟缓爱德华氏菌（Edwardsiella tarda）世界范围内海水养殖鱼类的重要病原菌，它所导致的鱼类迟缓爱德华氏菌病（edwardsiellosis）给我国养殖产业造成严重经济损失。已有研究表明，迟缓爱德华氏菌能以T6SS效应蛋白劫持宿主胞内信号转导，并钝化其免疫防卫机能，但具体机制目前所知尚浅。环化腺苷单磷酸（cAMP）是一种主要的胞内第二信使，对免疫应答有普遍抑制作用。本项目的前期研究发现，迟缓爱德华氏菌可借助其T6SS调控宿主细胞cAMP含量和一种磷酸二酯酶亚型的转录水平，但其中涉及的效应蛋白及其作用机制尚不清楚。本项目拟基于前期研究发现以及申请人已建立的迟缓爱德华氏菌CRISPR基因组编辑平台和已筛选出的T6SS潜在效应蛋白，开展感染状态下的原位研究，以求深入解析迟缓爱德华氏菌操控宿主cAMP信号通路的作用机制。

迟缓爱德华氏菌可通过干预宿主免疫反应促进自身感染。环腺苷单磷酸cAMP是一种二级信使，在动物中具有免疫调节功能。迟缓爱德华氏菌可通过其VI型分泌系统（T6SS）改变宿主细胞cAMP水平。本项目围绕迟缓爱德华氏菌操控宿主cAMP信号通路的作用机制展开研究，厘清了迟缓爱德华氏菌T6SS体外诱导条件，通过敲除T6SS分泌装置鞘蛋白编码基因获得了迟缓爱德华氏菌T6SS功能缺陷株，通过针对野生株和T6SS功能缺陷株分泌蛋白的比较蛋白质组学分析获得了潜在的T6SS分泌蛋白，通过报告基因检测确定了T6SS分泌蛋白，通过分析野生株和T6SS分泌蛋白突变株感染引起的宿主cAMP水平变化情况，鉴定得到了迟缓爱德华氏菌操控宿主细胞cAMP代谢的T6SS效应蛋白，解析了迟缓爱德华氏菌操控宿主cAMP水平的作用机制及其免疫学意义。本项目促进了对迟缓爱德华氏菌与其宿主免疫系统相互作用机制的深入理解。