应激条件下结核分枝杆菌Pup-蛋白酶体系统靶蛋白发现及致病机制研究

Proteasome system and the prokaryotic ubiquitin-like protein (Pup) had been found within Mycobacterium tuberculosis (M.tb), which is the pathogenic bacterium of tuberculosis. Pub, like ubiquitin, targets proteins for degradation，　thus the specific　cellular processes or　signal transduction pathways were affected to regulate the M.tb growth situation as well as its pathogenicity. M.tb encounters and adapts to the stress conditions, such as hypoxia and nutrient limitation，during persistent infection in host lesions. However, current studies of M.tb pup-proteasome system are mostly performed under the in vitro normal growth condition, which cannot reflect the pup-proteasome system how to regulate M.tb growth and its pathogenicity. In this study, the pupylome under the stress conditions(hypoxia and nutrient starvation) will be characterized and compared with the normal culture condition using technologis of proteomics, gene knock-out and establishment of in vitro and in vivo infection model. We will also identify the specific substrates of pup-proteasome dynamically according to the growth conditions. Molecular mechanisms of the growth condition-dependent recognition and pupylation of substrates in M.tb will be clarified. Furthermore, the role of pup-proteasome system in the stress condition adaption, persistence and virulence of M.tb will be explored in this study. It will provide new evidences for development of new anti-tuberculosis drugs and vaccines.

结核病病原菌-结核分枝杆菌中存在蛋白酶体和原核类泛素蛋白（prokaryotic ubiquitin-like protein，Pup），Pup可标记多种靶蛋白介导蛋白降解，调节结核分枝杆菌的生长或影响其致病性。目前对于结核分枝杆菌Pup-蛋白酶体系统的研究多集中在正常培养状态下相关蛋白的功能分析。而结核分枝杆菌感染宿主后处于缺氧和营养缺乏等应激生长环境，目前的研究成果不能全面反映和揭示Pup-蛋白酶系统对结核分枝杆菌生长调控及致病性的影响。本项目拟通过蛋白质组学、基因敲除、体内外感染模型构建等技术鉴定和比较正常生长条件和应激生长条件（低氧和营养缺乏）下结核分枝杆菌Pup化蛋白谱，研究该系统对靶蛋白的"动态调控"，揭示不同生长条件下该系统特异性识别并催化靶蛋白Pup化的分子机制，研究Pup-蛋白酶体系统在结核分枝杆菌适应应激环境及在持留和致病性中的作用，为抗结核新药和疫苗发现提供理论依据。

结核病是由结核分枝杆菌感染所致，结核病已成为世界范围内的重大公共卫生问题。结核分枝杆菌持留感染占全球人口的 1/3,其中的 1/10 将最终罹患结核病。结核分枝杆菌的持留感染也 是结核病学防治工作中亟需解决的重大问题。结核分枝杆菌中存在蛋白酶体和原核类泛素蛋白（prokaryotic ubiquitin-like protein，Pup），Pup可标记多种靶蛋白介导蛋白降解，调节结核分枝杆菌的生长或影响其致病性。目前对于结核分枝杆菌Pup-蛋白酶体系统的研究多集中在正常培养状态下相关蛋白的功能分析。而结核分枝杆菌感染宿主后处于缺氧和营养缺乏等应激生长环境，目前的研究成果不能全面反映和揭示Pup-蛋白酶系统对结核分枝杆菌生长调控及致病性的影响。本课题旨在研究体外正常培养和应激培养条件下结核分枝杆菌中 Pup 化蛋白谱（ PUPylome） 的差异。本课题成功筛选出基因pup敲出菌株和回补菌株，建立结核分枝杆菌快速厌氧培养模型和探索摸索出全蛋白的提取条件，蛋白的纯化和质谱的鉴定正在进行中。阐明与正常培养相比较，在应激培养条件下结核分枝杆菌中 Pup 化蛋白种类的差异，探讨 Pup-蛋白酶体系统对靶蛋白的降解是否受到生长环境的调控。为抗结核新药和疫苗发现提供理论依据。