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。

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.