结核分枝杆菌中一个IV型限制系统的发现与表征

Tuberculosis (TB) is one of the diseases that have the highest adult mortality rate worldwide，and China is one of the most serious global TB epidemic countries. In recent years, in the wake of increasingly serious drug resistance of Mycobacterium tuberculosis (MTB), the phagotherapy on MTB is paid attention again..However, the bacterial restriction-modification (R-M) systems significantly block phage infection. Genomic bioinformatics analysis reveals that MTB possesses multiple R-M systems, the function of which is to restrict the entry of exogenous DNA into the bacterial host. R-M systems severely disturb the curative effect of TB phagotherapy, which also hinders the genetic manipulation of MTB..In previous study we found that Rv2528c is a type IV restriction endonuclease that recognizes and cleaves methylated DNA. On this basis, this project focuses on the identification of methylation pattern recognized by Rv2528c, the characterization of recognition/cleavage sites, and resolving protein crystal for the sake of elucidating the restriction mechanism of Rv2528c. Based on our research, researchers can modify or optimize the MTB phage DNA to obtain higher infection efficiency, and finally promote the development of TB phagotherapy. Furthermore, this project also screens other MTB R-M candidate genes through heterologous expression in Mycobacterium smegmatis(a relative species of MTB), which could contribute to break the MTB R-M system, and further improve genetic manipulation on MTB.

结核病是世界上成人致死率最高的疾病之一，其中我国是全球结核病流行最严重的国家之一。近年来，结核病的耐药性现象日益严重，使针对结核分枝杆菌（MTB）的噬菌体疗法重新得到重视。.细菌的限制修饰系统是噬菌体侵染细菌的重要阻碍。生物信息学分析预测MTB具有多重的限制修饰系统，其功能是限制外源DNA进入宿主，这不但严重影响了噬菌体疗法的效果，同时也阻碍了对MTB的遗传操作。.在前期实验中我们发现Rv2528c是一个切割甲基化DNA的IV型限制酶，本项目拟在此基础上继续鉴定其识别的DNA修饰类型，测定其识别/切割位点，并通过解析其蛋白晶体结构等揭示其限制机理，为优化治疗用噬菌体的DNA序列以获得更高的侵染效率、促进结核病的噬菌体疗法发展打下基础。同时通过在MTB的近缘模式宿主耻垢分枝杆菌中异源表达等方法筛选出更多的MTB限制修饰系统基因，为破解MTB的限制修饰系统，改进MTB的遗传操作体系做出贡献。

结核分枝杆菌存在多个可能的修饰系统且功能未知。前期工作中我们发现了一个可能的IV型限制性内切酶Rv2528c。在本课题研究中，我们首先通过体内实验验证了Rv2528c基因的细胞毒性，并证明Rv2528c通过体内降解基因组DNA来造成大肠杆菌细胞生长迟滞。然后制备多种甲基化修饰的测试质粒，并转化BL21/Rv2528c菌株，以检验Rv2528c识别的甲基化修饰类型。结果发现Rv2528c在体内对M.HhaI、M.MspI、M.BamHI、CpG、M.AluI、M.HaeIII、M.EcoGII、M.TaqI修饰的质粒DNA有明显的限制活性。对于体外活性实验，我们成功表达纯化了带C端6xHis-tag的Rv2528c蛋白，并可以体外降解CpG、GpC、M.EcoGII修饰的质粒DNA。对于蛋白结构分析，我们比较了Rv2528c和来自大肠杆菌K12菌株的同源蛋白EcoKMrr的序列相似性及基于计算机预测的三维结构相似性，比较了两者在三级结构上的异同，为后续研究提供了理论指引。以上发现可为针对结核分枝杆菌的诊疗用分支杆菌噬菌体的DNA甲基化修饰改造提供一定的指导。