TOR通路介导的细胞自噬在仿刺参抗灿烂弧菌感染中的作用研究

Autophagy mediated by mTOR pathway is a particularly important immune defense mechanism in the pathogens infection of mammals, has become hotspot in the international. Our previous studies showed that Vibrio splendidus could induce Apostichopus japonicus coelomocytes autophagy during skin ulcer syndrome (SUS) disease burst, and this process is closely linking with the expression level of gene from TOR pathway. However, it remains unclear that the induced autophagy whether directly regulated by TOR pathway, TOR pathway how to modulate autophagy, and what is the role of autophagy in bacterial infection. Based on this background, we intend to illustrate the molecular mechanism of autophagy under V. splendidus exposure. Through TOR activator, RNA interference and rescue techniques, we will demonstrate that the induced autophagy by V. splendidus is directly regulated by TOR signaling pathway. Through site-specific mutagenesis and analysis phosphorylation level, we will certify the molecular mechanism of autophagy regulation of TOR is determined by the specific sites phosphorylation of ULK. We will also to reveal the role of autophagy in the innate immune defense mechanism of A. japonicus by the co-localization of the V. splendidus, autophagosome and lysosome, and analysis the survival rate of V. splendidus. The results of this project will greatly enrich the research contents of marine invertebrate innate immunology, and provide new ideas for understanding and directing control of SUS disease.

mTOR通路介导的细胞自噬是哺乳动物抗病原感染中极为重要的免疫防御机制，已经成为国际上的研究热点。申请人前期研究发现在刺参腐皮综合症发生过程中，灿烂弧菌诱导的宿主细胞自噬与TOR通路基因的表达水平息息相关。但对于该病原诱导的自噬是否直接经由TOR通路调控、TOR通路如何调控以及自噬发生在抗细菌感染中的作用等关键科学问题尚不清楚。为此，本项目拟采用分子生物学、细胞生物学等技术手段，通过TOR激活剂、RNAi及蛋白拯救等技术，明确TOR通路直接参与灿烂弧菌介导的自噬发生；通过定点突变及磷酸化western blot检测，解析TOR依赖ULK特定位点的磷酸化实现自噬调控的分子途径；通过对灿烂弧菌、自噬体、溶酶体的共定位及灿烂弧菌存活率等指标的测定，揭示自噬发生在刺参先天免疫防御机制中的作用。研究成果将极大的丰富海洋无脊椎动物先天免疫学的研究内容，并为认识和定向防治刺参腐皮综合症提供新思路。

自噬作为免疫系统、尤其是先天免疫系统的重要组成部分，是激活免疫应答、控制感染性疾病的重要途径。本项目率先瞄准自噬调控在刺参细菌性疾病发生中的作用这一关键科学问题，系统探讨了灿烂弧菌是如何通过调控自噬以逃逸宿主监控导致腐皮综合症发生的分子机制。为此，本项目采用分子生物学、细胞生物学等技术手段，通过电镜观察、TOR激活剂/抑制剂、RNA干扰等技术，明确了TOR通路直接参与灿烂弧菌介导的自噬发生；通过western blot检测等技术，解析了TOR通路依赖于ULK途径促进自噬体形成；通过双荧光素酶系统、miRNA-137过表达或抑制表达、靶基因干扰等技术，系统探讨了灿烂弧菌通过miRNA-137负调控靶基因Atg13表达，抑制刺参体腔细胞自噬以逃逸宿主监控导致腐皮综合症发生的分子机制。研究成果极大的丰富了海洋无脊椎动物先天免疫学的基础研究，并引领自噬这一国际热点进入海洋；同时为刺参细菌性疾病的免疫防治提供理论依据和新途径，并将有助于开发作用于miRNA或其靶标的新的治疗手段，促进我国刺参养殖业健康、可持续发展。