宿主冠蛋白介导结核分枝杆菌逃避溶酶体杀伤的分子机制

Coronin is a family of highly conserved actin-bindng proteins, participating in a variety of important cell biology processes. Since 2003, we systematically analyzed the biochemical activities of Coronin. We demonstrated its unique ability to remodel branched actin filaments, and revealed the underlying mechanisms of Coronin in modulating cell migration. Tuberculosis is an infectious disease caused by Mycobacterium tuberculosis. With the rise of drug resistent strains, it becomes a serious threat to human health. There is a pressing need to develop new strategy to treat tuberculosis. Previous studies have shown that after entering the cell by phagocytosis, mycobacterial packed in phagosomes can recruit a host protein, Coronin, to delay and/or prevent the fusion of phagosomes to lysosome, which results in the survival of mycobacterial. However, the molecular mechanism underlies this observation is still not clear. Lately, we found that mycobacterial not only recruites Coronin, but also actin, to survive inside host cells. We speculate that Coronin employs its actin remodeling activity to prevent the fusion of phagosomes to lysosomes. In this proposed research, we will use live-cell imaging, combined with modern molecular techniques, to study how mycobacterial uses Coronin to survive in host cells. We will discuss the possibility of increasing the efficiency of phagosome-lysosome fusion to suppress, or even eliminate mycobacterial infection. We hope our study can provide a new drug targeting strategy not only for tuberculosis, but also other infectious diseases.

冠蛋白是高度保守的微丝细胞骨架的结合蛋白，参与细胞分裂、迁移和吞噬等重要生理过程。课题申请人系统分析了冠蛋白的生化活性，证实其可调控微丝聚合和解聚合，是重建分叉微丝的关键蛋白【J Biol Chem；J Cell Sci；Cell两篇】。有研究显示，结核分枝杆菌可富集冠蛋白于吞噬体表面，逃避细胞内溶酶体的杀伤，但相关分子机制尚无定论。我们近期发现，结核分枝杆菌在逃避杀伤时，不仅富集宿主的冠蛋白，也富集宿主的微丝；因此推测，结核分枝杆菌进入宿主细胞后，过量富集的宿主冠蛋白通过调控微丝重建，抑制了吞噬体与溶酶体的有效融合，使病原菌逃避杀伤，得以存活并扩增。本课题将采用活细胞成像和各类分子生物学技术手段，研究病原菌利用宿主冠蛋白逃避溶酶体杀伤的分子机制，探讨利用宿主溶酶体清除病原菌的可能性，研究成果将为冠蛋白作为结核分枝杆菌逃避杀伤的关键蛋白提供有力依据，并为结核治疗提供全新思路。

在本项目的四年资助下，我们采用细胞生物学的实验手段，探索了结核分枝杆菌进入巨噬细胞后逃避溶酶体杀伤的相关分子机制：(1) 结核分枝杆菌进入细胞与传统内吞途径相关，可以被M.smeg菌或乳胶颗粒模拟；(2) 含有结核分枝杆菌的吞噬泡和溶酶体在融合中，招募了冠蛋白1A，其周围富集的微丝信号不强；(3) RNAi干扰冠蛋白1A并没有显著增加细胞内结核分枝杆菌周围的微丝、没有增加含有溶酶体信号的含菌的吞噬泡；(4) 药物诱导增加细胞内自噬反应没有增加含有溶酶体信号的含菌的吞噬泡。目前实验结果否决了通过干扰冠蛋白来促进细胞对结核分枝杆菌的杀伤的抗结核药物开发思路。