灿烂弧菌VsEprK蛋白靶向刺参Akt/mTOR通路抑制体腔细胞自噬的分子机制

细胞能够以异源自噬的方式消除入侵的病原体，同时，致病性细菌也进化出免疫逃逸机制，通过分泌蛋白干扰宿主自噬通路。申请人前期从刺参病原菌灿烂弧菌中鉴定到一种具有磷脂酰肌醇3-激酶（PI3K）结构域的效应子VsEprk，该蛋白能显著抑制刺参体腔细胞自噬活性。在高等生物中PI3K能够激活Akt/mTOR通路进而抑制自噬，但该蛋白是否通过靶向刺参Akt/mTOR调控灿烂弧菌感染过程中的体腔细胞自噬及其相应分子机制尚不明确。本项目拟采用基因敲除、过表达等技术，明确在灿烂弧菌感染过程中VsEprk负调控体腔细胞自噬；进而利用Akt抑制剂、磷酸化蛋白检测等技术，确定VsEprk靶向调控刺参Akt/mTOR通路抑制自噬；最后通过酶活检测、细胞内共定位等技术探究VsEprk激活Akt/mTOR通路的分子机制。研究结果将为认识灿烂弧菌靶向宿主免疫防御途径的侵染机制提供基础，为灿烂弧菌的特异性防控提供理论支持。

Consistent with xenophagy being an important host defense perse, pathogenic bacteria has evolved strategies to escape from or paralyze the autophagy system. Our previous studies identified an effector VsEprk from Vibrio splendidus with a phosphatidylinositol 3-kinase (PI3K) domain. Purified rVsEprk can significantly inhibit the coelomocyte autophagy activity of sea cucumber Apostichopus japonicus. In higher organisms, PI3K can activate the Akt/mTOR signal pathway to inhibit autophagy. However, it remains unclear that whether VsEprk regulates coelomocyte autophagy during V. splendens infection by targeting Akt/mTOR and what is the molecular mechanism. Through VsEprk gene knockout, overexpression and rescue techniques, we will demonstrate that VsEprk negatively regulates coelomocyte autophagy during V. splendidus infection. Through Akt inhibitors and analysis phosphorylation level, we will certify VsEprk targets Akt/mTOR signal pathway to inhibit autophagy. The molecular mechanism of VsEprk activation of Akt/mTOR pathway is explored through enzyme activity detection and intracellular co-localization. The results of this project will provide a basis for understanding the infection mechanism of V. splendens targeting host immune defense pathway, and provide theoretical support for the specific prevention and control of V. splendens.