与鳗爱德华氏菌ET080813细胞内生活方式密切相关的新型III型分泌系统效应蛋白的鉴定与表征

Edwardsiella species, namely E. piscicida and E. ictaluri, are common and important pathogens of marine fish. They all process a type III and a type VI secretion systems (T3SS and T6SS) and these are important virulence mechanisms of these organisms. E. anguillarum is a recent addition as the third Edwardsiella species that is important in fish disease; it possesses two T3SSs and three T6SSs and is very virulent in fish. However, not much is known about the T3SS effectors and functions of E. anguillarum. In this proposal, I plan to use our well-established platform to study the T3SS effectors of E. anguillarum ET080813. Our first objective is to characterize ET080813 further by looking at its abilities to adhere, invade and replicate intracellularly, as well as study the extracellular protein profile, LD50 values, and formation of the Edwardsiella-containing vacuoles (ECVs). Our second objective will be to identify novel T3SS effectors of ET080813 using bioinformatics and transcriptome approaches; genuine effectors will be confirmed by translocation and secretion assays. We will apply our established platform for the study of novel T3SS Edwardsiella effectors and relate their functions to the intracellular lifestyle of the bacterium. The knowledge generated will provide new targets for vaccines and effective therapeutics that can be applied to prevent Edwardsiella and other fish diseases to benefit the aquaculture industry in China.

爱德华氏菌属的杀鱼爱德华氏菌和鲶爱德华氏菌，是鱼类普遍且主要的病原菌。它们均具有1套III型和1套VI型分泌系统，是该菌种中重要的毒力机制。鳗爱德华氏菌是第三种引起鱼类疾病的关键病原菌，它具有2套T3SS和3套T6SS系统，对鱼类具有很强的毒性。但是，人们对于鳗爱德华氏菌的T3SS分泌系统及其功能知之甚少。本课题拟利用我们已经建成的平台研究鳗爱德华氏菌ET080813中的T3SS效应蛋白。首先，我们将观察该菌进行细胞内黏附、侵入及自我复制的能力，测定其半数致死量LD50值，以及观察携带爱德华氏菌的胞内囊泡的形成规律。在此基础上，利用生物信息学和转录组学的方法，鉴别和确定该菌的新型T3SS效应蛋白。此项研究将为预防爱德华氏菌和其他鱼类疾病的疫苗及其他治疗和诊断方法提供新的靶标，从而有利于中国水产业的发展。

爱德华氏菌是国内外鱼类普遍且主要的致病菌，比如杀鱼爱德华氏菌，鲶爱德华氏菌以及鳗爱德华氏菌。其中杀鱼爱德华和鲶爱德华氏菌均含有一套III型和一套VI分泌系统，而鳗爱德华氏菌含有两套III型和三套VI分泌系统。这两套分泌系统是爱德华氏菌最重要的毒力机制。由于鳗爱德华氏菌是一个新发现的菌种，其T3SS和T6SS的研究报道并不多。因此，本课题拟利用已经建成的平台，通过生物信息学、转录组学以及蛋白组学的方法鉴定鳗爱德华氏菌新型的III型分泌效应物。对新型分泌效应物的认识将增加人们对爱德华氏菌的效应物及其对病原-宿主互作所起作用的了解。此外，该研究为未来开发爱德华氏菌的疫苗及鱼类疾病的疗法打下基础。经本课题经费资助，目前本课题发表了两篇高影响力的论文，一篇论文正在准备投稿。项目具体研究分为以下二部分：1. 表征鳗爱德华氏菌标准菌株ET080813的毒力及其对宿主（细胞）的感染规律 。2. 通过生物信息学、转录组学以及蛋白组学的方法鉴定新型III型分泌效应物。还鉴定了至少13个效应物，未来也将对它们进行功能分析。

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