迟钝爱德华氏菌T3SS效应蛋白的系统鉴定及其与鱼类宿主互作机制研究

The disease properties of fish pathogen Edwardsiella tarda are due in large part to their type III secretion system (T3SS), which they use to inject harmful effectors into host cells and to escape from the host immune system. T3SS-deficient mutants are attenuated by 1-2 logs in their LD50 values and they are excellent candidates for the development of fish vaccines. However, little of Edwardsiella tarda T3SS effectors have been understood in respective of their types, structures and interactions with host. We will use the next generation sequencing technology (RNA-seq) and bioinformatics analysis to systematically identify all T3SS effector genes in the genome of E. tarda EIB202. The putative T3SS effectors will be validated by translocation assays and characterized further in order to determine their roles in pathogen-host interaction. We will examine how T3SS effectors mimic or disrupt host cellular functions during bacterial infections and immune escape using animal hosts as well as tissue culture models. Functional characterization will include elucidating host signal transduction pathways and the underlying biochemical and molecular mechanisms in controlling and regulating the host immune response, identifying host targets and effector-host protein interactions, examining the effector functional domains and their structure. This work will provide a platform for studies on effector function and host response, and explain how the bacteria escape the host innate defense. Economic results will include identifying new targets for novel vaccines and effective therapeutics targeting bacterial fish pathogens.

迟钝爱德华氏菌基因组编码的III型分泌系统(T3SS)及注射到宿主细胞内的效应蛋白是该菌逃逸宿主免疫系统并在鱼类中致病所必需的关键毒力因子。可是，对该菌T3SS效应蛋白的种类、结构和作用机制还知之甚少。本项目拟利用基于新一代高通量测序技术，结合生物信息学方法，在全基因组范围内系统筛查该菌T3SS效应蛋白；在动物及细胞模型上系统评价各候选T3SS效应蛋白基因缺失株在感染中的差异化表型，鉴定在细菌-宿主相互作用中具有重要功能的T3SS效应蛋白；在鱼类模型上研究重要T3SS效应蛋白在感染和免疫逃逸过程中的作用机制，在细胞模型上探索其作用于宿主细胞的信号通路、相关受体或作用靶点；通过蛋白质晶体学手段，深入阐释T3SS效应蛋白结构与其酶学功能或分子间结合功能的相互关系。本研究有助于深入理解迟钝爱德华氏菌通过T3SS效应蛋白操控宿主细胞通路、逃避宿主免疫防御的感染机制，为该菌病害的控制提供理论支持。

本项目主要围绕主要围绕迟钝爱德华氏菌效应蛋白的种类、结构和作用机制开展研究，利用新一代高通量测序技术，结合生物信息学方法，在全基因组范围内系统筛查获得迟钝爱德华氏菌菌效应蛋白200多个；在动物及细胞模型上系统评价各候选T3SS效应蛋白基因缺失株在感染中的差异化表型，鉴定在细菌-宿主相互作用中具有重要功能的T3SS效应蛋白EseG、EseK和EseH；在鱼类模型上研究了毒力因子溶血素蛋白等在感染和免疫逃逸过程中的作用机制，在细胞模型上探索其作用于宿主细胞的信号通路、相关受体或作用靶点；通过蛋白质晶体学手段，深入阐释效应蛋白Trxlp的结构与其酶学功能或分子间结合功能的相互关系。在Cell Host & Microbe，Nature Communications，iScience, PLoS Pathogens (2篇)，mBio，Cellular Microbiology （3篇），Virulence，Frontiers in Cellular and Infection Microbiology等SCI期刊发表高水平研究论文21篇。该研究为深入理解迟钝爱德华氏菌通过效应蛋白操控宿主细胞通路、逃避宿主免疫防御的感染机制提供有效证据，为该菌病害的控制提供理论支持。