结核杆菌与宿主吞噬/自噬通路精细互作网络及其机制研究

Tuberculosis is one of the top challenge to animal husbandry and public health in our country. During the process of evasion and parasitism, many proteins of Mycobacterium interact with host cells proteins to block the host phagocytosis/autophagy system, interfere with antigen presentation, and escape the host immune clearance. However, little is known about the fine interaction network between Mycobacterium tuberculosis and host phagocytosis/autophagy pathways, which greatly impedes the understanding of the survival mechanism of Mycobacterium in host cells. Moreover, this also hinder the development of vaccine against tuberculosis. Based on the previously developed new high-throughput yeast two-hybrid system (RLL-Y2H), the current project aim to delineate the fine protein-protein interaction map between nmycobacterium and host phagocytosis/autophagy signaling pathway proteins. Next, we will explore the molecular mechanism of Mycobacterium interfering the host phagocytosis/autophagy signaling pathway based on the key protein-protein interaction pair. After knocking out/over-expression of Mycobacterium and host cell interacting genes, we will combine live cell imaging, super-resolution imaging and other molecular methods to analyze the dynamic processes of the maturation of phagocytosis and autophagosomes, lysosome fusion and other molecular details. Finally, by using antigen-presenting assay, we will try to reveal the key secreted proteins of Mycobacterium which interfere with the antigen presentation of the host cell to elucidate the molecular mechanism of Mycobacterium escaping the host clearance and promote the development of anti-tuberculosis vaccine.

结核病是我国畜牧业和公共卫生业的极大挑战。结核杆菌在寄生过程中与宿主细胞大量蛋白相互作用,以抵抗宿主吞噬/自噬系统，干扰抗原提呈、达到逃逸机体免疫清除。然而目前对结核杆菌与宿主吞噬/自噬通路的精细互作网络仍知之甚少，极大的限制了对结核杆菌逃逸免疫机制的认识,也使得结核病疫苗研发难以精准、有靶向性的开展。本项目将基于前期研发的新型高通量酵母双杂交系统（RLL-Y2H)，解析结核杆菌与宿主吞噬/自噬信号通路蛋白相互作用精细网络；以结核杆菌干扰宿主吞噬/自噬信号通路的关键相互蛋白关键节点为突破口，通过敲出/过表达结核和宿主细胞相关基因，结合活细胞成像、超分辨成像等手段解析这些蛋白干扰宿主吞噬\自噬体的形成、成熟及其与与溶酶体融合的动态过程及分子细节；最后还将利用抗原提呈assay，揭示结核杆菌关键分泌蛋白干扰抗原提呈的分子途径，为阐明结核杆菌逃避宿主清除分子机制与抗结核疫苗开发奠定基础。

结核病是我国畜牧业和公共卫生业的极大挑战。目前对结核杆菌与宿主吞噬/自噬通路的精细互作网络仍知之甚少，极大的限制了对结核杆菌逃逸免疫机制的认识,也使得结核病疫苗研发难以精准、有靶向性的开展。本项目将基于前期研究基础，通过交叉学科手段，取得了以下进展：构建得到结核杆菌蛋白与宿主吞噬/自噬通路蛋白互作的子网络图谱。解析了结核杆菌Rv3722c与宿主TRAF3互作、促进M.tb细胞增殖、诱导的NF-κB、IFN-β产生的分子机制。利用结核分枝杆菌的内源性III-A型CRISPR-Cas10系统，构建了结核杆菌全基因组基因编辑、RNA干扰和基因组规模的RNA干扰筛选体系。通过对约5000种化合物库的高通量筛选最后鉴定出了orbifloxacin化合物良好的抗结核杆菌活性、发现其作用靶标蛋白Gyr ase的D94G点突变会引起结核杆菌对该药物产生耐药。解析了结核杆菌在不同细胞中的存活差异机制。解析了OASL与胞内DNA感受cGAS-STING通路调控结核杆菌胞内存活机制。系统的描绘了结核杆菌感染宿主后表观遗传变化全景图，为阐明结核杆菌逃避宿主清除分子机制与抗结核疫苗开发奠定基础。相关成果发表在Nature Communications、mSystem、Frontiers in Microbiology杂志。此外一篇文章审稿中。获批发明专利一项。