迟钝爱德华氏菌T6SS参与抑制宿主炎症小体活化的机制研究

Edwardsiella tarda is a leading pathogen in China's aquaculture industry that causes severe diseases in board host range in fish. The disease properties of E. tarda are due in large part to their type VI secretion system (T6SS). Inflammasomes are cytosolic multiprotein complexes that assemble in response to the invading infectious microbes. They play a critical role in host innate immunity to defense against the infection of pathogenic organisms. In our previous work, we demonstrated that E. tarda could activate inflammasomes such as NLRP3 and NLRC4. Surprisingly, low activation of NLRP3 inflammasome was found and this was due to the suppression by a T6SS in E. tarda. The objective of this proposal is to systematically identify and characterize T6SS effectors that interfere with NLRP3 inflammasome. The studies will include identifying host targets, examining their underlying biochemical and molecular mechanisms, and elucidating the role of T6SS in escaping innate defense. The knowledge generated from these studies will lead to biotechnological and aquaculture-relevant applications, such as rapid diagnostics, new targets for novel vaccines, and effective therapeutics.

迟钝爱德华氏菌是一种重要的广宿主病原，严重威胁我国水产养殖业的健康发展。VI型分泌系统（T6SS）是迟钝爱德华氏菌重要的毒力因子，在该菌感染过程中发挥重要作用。炎症小体是宿主固有免疫系统的组成部分，是宿主抵抗病原微生物感染的重要免疫防御手段之一。本项目前期研究结果表明：迟钝爱德华氏菌侵染宿主后，能激活NLRP3和NLRC4两种类型的炎症小体，但NLRP3炎症小体的激活程度很低，而该菌的T6SS可能参与了NLRP3炎症小体的抑制过程。从前述重要的实验现象出发，本项目拟系统鉴定迟钝爱德华氏菌T6SS效应物，深入解析T6SS效应物参与抑制宿主炎症小体活性的的分子机制，从而揭示出全新的细菌T6SS生物学功能和细菌免疫逃逸机制。本项目的成功实施将有助于深入理解迟钝爱德华氏菌的致病机制，为该菌病害的早期预警、诊断以及疫苗和药物靶点的设计提供理论支持。

本项目发现海洋鱼类病原菌迟钝爱德华氏菌(Edwardsiella tarda)能通过其三型分泌系统(T3SS)在小鼠巨噬细胞内激活NLRC4和NLRP3两种炎症小体，而其六型分泌系统(T6SS)却能够抑制NLRP3炎症小体的激活。进一步的研究证明，迟缓爱德华氏菌在感染过程中，能够通过其T6SS将一个效应蛋白EvpP注射到小鼠巨噬细胞内，正式由该蛋白介导了T6SS对NLRP3炎症小体的抑制作用。此外，EvpP也能抑制由三磷酸腺苷(ATP)、尼日利亚菌素(Nigericin)以及单钠尿酸盐晶体(Monosodium urate crystals)所诱导的NLRP3炎症小体激活。进一步的分子机制研究发现，EvpP能够抑制小鼠巨噬细胞内钙离子浓度的升高，导致下游Jnk信号通路受阻，后续ASC接头蛋白无法寡聚化，从而阻断NLRP3炎症小体的激活。体内感染实验表明，EvpP对NLRP3炎症小体的抑制作用能帮助迟缓爱德华氏菌在动物体内的定植和感染。本研究首次报道一个全新的细菌T6SS效应物EvpP抑制宿主NLRP3炎症小体通路而促进细菌免疫逃逸的机制，上述工作对于深入理解病原菌与宿主的互作机制、指导抗感染药物的理性设计有着重要意义。