副溶血弧菌T3SS2效应蛋白VPA1324操控宿主cdG免疫信号通路的肠毒性机制

Vibrio parahaemolyticus type III secretion system 2 (T3SS2) injecting effector proteins into host cells to subvert the host normal functions has been suggested to be the intestinal toxicity mechanism for the bacterium. However, there are only a small number of effector proteins have been revealed for their biological functions and mechanisms of manipulation of host cells. Our previous work found that VPA1324 is a T3SS2-secreted protein, and the protein was testified to be an EAL-type phosphodiesterase, which could hydrolyze cdG, a second messenger widely found in bacteria. Whereas, the cdG is also an essential signaling molecule that can activate host STING and NLRP3-dependent innate immune signaling pathways. Therefore, based on these results, we proposed a hypothesis that VPA1324 could be a new T3SS2 effector protein, and it could inhibit host innate immune defense through hydrolysis of cdG. In this proposal, we plan firstly to verify that VPA1324 could be injected directly into the host cells post-introduction by bile salts. Then we will confirm that the protein could hydrolyze intracellular cdG through determination of the changes of cdG after the protein and cdG both transfection into host cells. After that, system analysis of the changes of the key proteins in host STING and NLRP3-dependent signaling pathways resulting by VPA1324 to verify the protein could block the immune signaling pathways. Lastly, we confirm the impact of VPA1324 on Vibrio parahaemolyticus intestinal toxicity, thus illustrates the protein manipulation of the host innate immune defense as a novel intestinal pathogenic mechanism. The results of this research will not only promote to completely reveal the mechanism of T3SS2-dependent intestinal toxicity, but also provide a new perspective for the investigation of host signal transduction and innate immune defense.

副溶血弧菌T3SS2向宿主注入效应蛋白来破坏宿主细胞的正常功能为其肠毒性机制，但目前仅有少数几个效应蛋白得到研究，需要鉴定新的效应蛋白并揭示其操控宿主的作用机制。申请人前期研究发现VPA1324为T3SS2分泌蛋白，并明确其为磷酸二酯酶，可水解细菌第二信使cdG；因cdG可激活宿主STING及NLRP3依赖的天然免疫信号通路，为此提出VPA1324为T3SS2效应蛋白，通过水解cdG来阻止免疫信号通路的激活。本课题首先验证VPA1324被直接注入宿主细胞，明确其为效应蛋白；然后将其及cdG转染至宿主细胞，确定其在宿主细胞内水解cdG；再系统分析其对宿主免疫信号通路中关键成分的影响，证实其阻止免疫信号通路的激活；最后明确其在副溶血弧菌肠毒性中的作用，进而揭示其通过水解cdG抑制宿主天然免疫防御的全新操控机制，不仅促进全面揭示T3SS2的肠毒性机制，也为宿主的信号转导及免疫防御研究提供新视角。

副溶血弧菌不仅是水生动物主要病原菌，也是世界范围内食用海产品导致肠胃炎等疾病的关键原因，其Ⅲ型分泌系统2(T3SS2)向宿主注入效应蛋白来操纵宿主正常功能已被揭示为其导致肠毒性的关键机制，但目前仅揭示了少数几个效应蛋白的功能及其操控宿主机制。申请人在前期基础上，提出副溶血弧菌VPA1324为T3SS2效应蛋白，通过水解信号分子c-di-GMP来抑制宿主依赖c-di-GMP激活的免疫防御通路的抗免疫机制。为证实这一观点，主要开展了：⑴确定了VPA1324为T3SS2分泌蛋白，进一步通过构建含vpa1324-cyaA融合基因的菌株，证实VPA1324被T3SS2注入宿主细胞，明确了VPA1324为T3SS2效应蛋白；⑵体外实验证实了VPA1324具有磷酸二酯酶活性，能够水解c-di-GMP分子；⑶构建了表达VPA1324的宿主细胞，通过c-di-GMP激活免疫通路，明确了VPA1324抑制宿主细胞STING二聚体的形成、TBK1及IRF3的磷酸化修饰、IRF3入核及IFNβ的分泌，从而阐明了VPA1324抑制c-di-GMP-STING免疫信号通路的作用机制；⑷构建了含穿膜肽的PEP-1-VPA1324融合蛋白，证实其能引导VPA1324进入宿主细胞，利用体外NLRP3炎症小体激活模型，明确了VPA1324显著抑制c-di-GMP/NLRP3炎症信号通路；⑸构建了各种相应基因缺陷菌株，利用细胞、组织和动物感染模型分析了各菌株的毒性，明确了VPA1324在副溶血弧菌T3SS2依赖的毒性作用及致病性。本研究基于病菌与宿主之间相互作用，从病菌抗宿主免疫这一新的视角，发现并阐明了病菌的全新致病机制。本研究成果不仅促进全面及深入阐明副溶血弧菌的致病性机制，也为真核细胞的信号转导及免疫防御机制研究提供新视角，同时为新靶点药物研究和临床治疗提供新策略，在此基础上深入开展研究，有望获得突破。