牛分枝杆菌Eis对巨噬细胞自噬的调控及机制研究

Eis, a secretory protein of Mycobacterium tuberculosis, can interfere with the Th1 response and enhance the intracellular survival of the pathogen. Similarly, Mycobacterium bovis, the causative agent of bovine tuberculosis which severly threat the bovine industry and human health, also has Eis protein. However, whether the Eis protein of Mycobacterium bovis can regulate the autophagy of host cells and the exact mechanisms remain to be investigated. In this study, we assume that Eis of Mycobacterium bovis can regulate macrophage autophagy via TLR dependent signaling pathway or the IFN-γ/STAT-1 and IFN-γ/Irgm1 mediated signaling pathway. Recombinant BCG with Eis of Mycobacterium bovis will be constructed, and the regulation of Eis to autophagy and to cell survival/death in BCG infected bovine alveolar macrophages will be measured by means of transmission electron microscopy, laser scanning confocal immunofluorescence, Western-blot, etc. Effects of Eis to the survival of BCG in the infected macrophages will also be detected. The regulation of Eis to the key molecules of TLR signaling pathway, IFN-γ/STAT-1 and IFN-γ/Irgm1 signaling pathway in BCG infected bovine alveolar macrophages will be tested as well. C57BL / 6 mice will be used as animal model to explore the regulation of Eis to host autophagy, cell death and lung inflammation in BCG infected mice. This study would shed light on the interaction between Mycobacterium bovis and host cells, and lay a basis for clarifying the mechanism of immunity evasion of Mycobacterium bovis.

结核分枝杆菌Eis增强其在巨噬细胞内存活，可干扰Th1应答，并调控自噬。牛分枝杆菌是牛结核病病原，引起严重危害。牛分枝杆菌也有Eis，但其是否调控宿主细胞自噬及其确切机制仍未知。本研究假设牛分枝杆菌Eis经TLR、IFN-γ/STAT-1和IFN-γ/Irgm1通路调控巨噬细胞自噬，由此拟构建牛分枝杆菌Eis-rBCG，通过激光共聚焦、WB等检测牛分枝杆菌Eis对BCG感染牛肺泡巨噬细胞自噬和细胞存活/死亡的调控，检测Eis对BCG在感染巨噬细胞中存活的影响，检测巨噬细胞中TLR、IFN-γ/STAT-1和IFN-γ/Irgm1通路关键分子表达差异，探讨牛分枝杆菌Eis对宿主细胞自噬调控分子机制。以C57BL/6小鼠为模型，探讨牛分枝杆菌Eis对BCG感染鼠肺泡巨噬细胞自噬、死亡及肺部炎症的调控。有利于从根本上揭示牛分枝杆菌与宿主细胞之间的互作，为阐明牛分枝杆菌免疫逃避机制提供科学依据。

本课题表达了牛分枝杆菌Eis重组蛋白，并发现其对雷帕霉素诱导的RAW264.7巨噬细胞自噬有明显抑制，并有剂量依赖关系。BCG感染可引起RAW264.7细胞发生自噬，采用5μg的Eis蛋白处理即可对BCG引起的RAW264.7细胞自噬产生明显抑制，且具有剂量依赖关系。构建了过表达牛分枝杆菌Eis蛋白的重组耻垢分枝杆菌MS–eis，经SDS-PAGE、Western blot及质谱鉴定，该重组菌能较好表达Eis蛋白，且不影响MS的生长。在GFP-LC3质粒转染细胞上研究发现，与对照MS相比，MS-eis可抑制雷帕霉素引起的LC3点状极化。western blot发现，Eis抑制了LCI向LC3II的转化，从而证实了Eis蛋白抑制了RAW264.7细胞的自噬。进一步研究发现，MS-eis显著降低了Rapamycin诱导引起的LC3I向LC3II的转化，且p62蛋白降解受阻，表明MS-eis有效抑制了Rapamycin引起的自噬。与MS相比，MS-eis组细胞中重组菌存活明显高于MS组，表明Eis有利于细菌在Raw264.7细胞中存活。通过PI检测，发现MS-eis感染细胞存活率显著高于MS感染细胞，表明MS-eis可抑制感染细胞死亡。采用Multi-Pathway 9-plex探讨eis蛋白对RAW264.7细胞自噬调控信号通路，结果显示Eis明显抑制了CREB、JNK、NF-kB/p38/erk1/2活化，推测Eis可能通过CREB、JNK、NF-kB、p38、erk1/2途径调控自噬。通过TLR2/4特异性抑制发现，MS-eis在感染后30min及1h通过TLR2抑制CREB 、JNK、p38、Erk1/2活化，在感染后3h，通过TLR4抑制CREB、NF-kB、Akt、p70s6k活化调控信号通路。Real-time PCR发现MS-eis可降低RAW264.7细胞的IFN-γ转录，Western blot发现MS-eis抑制了STAT1磷酸化，推测Eis可能通过IFN-γ/STAT-1信号通路调控自噬。C57BL/6 小鼠体内研究表明各组IFN-γ的mRNA转录水平差异较小，rapamycin MS-Eis组仅比rapamycin MS对照组低约1.5倍，肺及肾中的CPU无明显差异。表明MS-Eis可能在体内抑制自噬效果不明显，或者反应条件还需进行系统优化。