结核分枝杆菌的拟核结合蛋白Rv0047c参与细菌胁迫应答调控的分子机制研究

Stress response is essential for infection and survival of Mycobacterium tuberculosis in the host. However, our understanding on underlying molecular mechnisms is still largely incomplete. Nucleoid-associated proteins,a global gene regulation proteins, regulate gene expression by organizing and condensing bacterial chromosome. In previous studies, we found a hypothesis protein Rv0047c of Mycobacterium tuberculosis exhibited several characteristics similar to nucleoid-associated protein. In addition, over-expression of Rv0047c significantly increased resistance of Mycobacterium tuberculosis to antibiotics and H2O2. This project will focus on systematically identifying DNA-binding characteristics, physiological roles and regulation mechanisms of Rv0047c in Mycobacterium tuberculosis through integrating genetics and molecular biology experimental methods. First, DNA-binding characteristics of Rv0047c will be studied by EMSA assay and atomic force microscope observation. Second, the physiological roles of Rv0047c in Mycobacterium tuberculosis will be analyzed by detecting bacterial-growth curve and morphology of rv0047c knockout and over-expression recombinant strains under various environmental stimuli. Third, target genes regulated by Rv0047c will be identified by microarray analysis, RT-PCR and ChIP experiment to explore the regulation pathways. The results will elucidate the roles of a new nucleoid-associated protein Rv0047c and provide insights into the nucleoid-associated protein mediated stress response mechanisms of mycobacteria.

胁迫应答对于结核分枝杆菌的感染和在宿主体内的生存十分关键，然而相关的分子机制尚不清楚。拟核结合蛋白是一类通过结合并改变染色体结构调控基因表达的全局调控蛋白。申请人团队前期研究发现结核分枝杆菌未知功能蛋白Rv0047c具有拟核结合蛋白特征，并且表达该蛋白能显著增加细菌的药物抗性和抗氧化能力。本项目将结合遗传学和分子生物学技术手段系统研究Rv0047c与核酸的结合特征及其对结核分枝杆菌生理调控的分子机制。首先采用EMSA和原子力显微镜等技术手段研究Rv0047c与核酸的相互作用机制；进而通过监测不同环境胁迫下rv0047c基因异常表达菌株的生长变化情况，鉴定其生理功能；最后，利用全基因组芯片分析、RT-PCR及ChIP技术鉴定Rv0047c所调控的靶基因。研究结果将阐明拟核结合蛋白Rv0047c的生理功能及其调控结核分枝杆菌胁迫应答的分子机制。

拟核结合蛋白( nucleoid-associated proteins，NAPs)是细菌中一类分子量较小的碱性蛋白，类似于真核生物中的组蛋白，能包装和浓缩细菌染色体DNA。本研究以结核分枝杆菌未知功能蛋白Rv0047c（命名为NapM）为研究对象，综合运用基因操作技术、蛋白质功能研究技术以及细胞生物学技术研究其特征和功能，重点针对NapM影响结核分枝杆菌潜伏感染和药物抗性进行研究。结果显示：（1）发现NapM为结核分枝杆菌中一个新的保守的拟核结合蛋白，能非特异性结合DNA，并具有浓缩和桥连DNA的功能，与细菌形成拟核的结构相关；（2）NapM能广泛调控超过150个分枝杆菌基因的表达，为一个广泛调控子；（3）NapM影响分枝杆菌对利福平和乙胺丁醇等多种药物的抗性；（4）NapM通过调控ABC转运系统ms4656-ms4659的表达，从而达到调控耻垢分枝杆菌的乙胺丁醇抗性；（5）NapM通过与复制起始蛋白DnaA相互作用，调控DnaA的DNA结合活性以及ATP水解活性，并进而影响结核分枝杆菌的潜伏感染。当前研究的科学意义在于，鉴定了分枝杆菌中一个未知蛋白的功能，并确定其为拟核结合蛋白，为分枝杆菌广泛调控的基因库添加新成员；其次本研究鉴定的NapM能调控复制起始过程，为阐明结核分枝杆菌潜伏感染机制提供了重要理论基础。