结核分枝杆菌ESAT6蛋白抑制巨噬细胞自噬体-溶酶体融合及促进结核分枝杆菌存活的调控机制

Autophagosome-lysosome fusion is crucial in autophagic function, and usually inhibited by intracellular pathogens for survival. Our previous study demonstrates that virulent proteins of mycobacterium tuberculosis can induce a significant increase of autophagosome in murine macrophage, and recent finding shows that ESAT6 inhibits autophagy flux and contribute to the survival of mycobacterium tuberculosis. We deduce that ESAT6 may promote mycobacterium tuberculosis survival by blocking autophagosome-lysosome fusion, but the mechanism is rarely known. The critical molecules involved in autophagosome-lysosme fusion contain mTOR, BECN1and Rab7, and our preliminary experiment shows that ESAT6 activates mTOR significantly. Hence, we speculate that ESAT6 may block autophagosome-lysosome fusion through mTOR/Rab7 pathway in macrophage. This study is to observe the role of ESAT6 in autophagosome-lysosome fusion and bacterium survival in macrophage from mice or tuberculosis patients; and the effect of mTOR/Rab7 pathway regulated by ESAT6 on autophagosome-lysosome fusion and bacterium survival. This study aims to clarify the mechanism underlying autophagy function and mycobacterium tuberculosis survival in macrophage, and exploring new clues of regulating autophagy and protecting tuberculosis infection.

自噬体-溶酶体融合是自噬功能的关键环节，常被胞内病原体抑制以促进其存活。我们以往研究证实结核分枝杆菌毒力蛋白可诱导巨噬细胞自噬体增多，最近发现ESAT6抑制自噬流量并促进胞内结核分枝杆菌存活。推测ESAT6可能通过抑制自噬体-溶酶体融合促进结核分枝杆菌存活，但ESAT6抑制自噬体-溶酶体融合的作用机制尚无报道。mTOR、BECN1及Rab7是介导自噬体成熟及融合的关键分子，预实验表明ESAT6显著上调mTOR活性。因此，我们分析ESAT6可能通过mTOR/Rab7途径抑制巨噬细胞自噬体-溶酶体融合。本项目拟观察ESAT6对小鼠及结核患者巨噬细胞的自噬体-溶酶体融合及胞内结核杆菌存活作用；ESAT6调控mTOR/Rab7通路对自噬体-溶酶体融合及结核分枝杆菌存活的影响。旨在阐明ESAT6调控巨噬细胞自噬功能及结核杆菌生存的作用机制，探讨自噬调控及抗结核感染的新途径。

我们研究发现结核分枝杆菌毒力蛋白ESAT6可抑制巨噬细胞自噬溶酶体活性，ESAT6诱导PI3K/mTOR通路活化，并通过介导UVRAG磷酸化活化RAB7、干扰RAB7参与自噬体溶酶体融合。ESAT6诱导mTOR磷酸化的功能结构域为67-95aa区域，因靠近羧基末端，称之为羧基末端结构域（CTD）。将具有跨膜转导性的TAT肽段与CTD肽段柔性连接构成TAT-ESAT6(CTD)多肽，体外实验证实具有抑制巨噬细胞mTOR磷酸化功能。自噬抑制促进结核分枝杆菌的增殖，ESAT6抑制溶酶体降解功能依赖于mTOR的活化，并促进结核分枝杆菌细胞内增殖。阻断PI3K/mTOR通路增强巨噬细胞对结核分枝杆菌RD1基因缺陷株BCG及临床多重耐药株MDR的杀伤作用。体内实验证实mTOR抑制剂联合异烟肼可增强小鼠肺部BCG感染抑制率。因此，本研究揭示了ESAT6调控巨噬细胞自噬功能及结核杆菌生存的作用机制，为自噬调节用于抗结核感染治疗提供了新思路。