结核分枝杆菌耐多药/广泛耐药机制研究及新药靶基因的筛选

The transmission of multi-drug resistant and extensively-drug resistant.Mycobacterium tuberculosis (MDR/XDR-TB) posed severe challenges to the global prevention and treatment of tuberculosis. Currently, the mechanism of MDR/XDR in Mycobacterium tuberculosis and its regulation are not well understood. We have previously performed comprehensive analyses of whole genome sequences from two clinical XDR-TB isolates, and identified 284 new mutated genes which were never reported in the scientific literature. 17 of these genes were selected according to biological functions of their encoded proteins which are predicted to be most likely associated with MDR/XDR-TB and characteristics of mutated amino acids, as well as software analysis techniques, in which 3 new mutated genes of hemK、glyA and MT3842 are predicted to be most likely associated with MDR/XDR-TB and characteristics of mutated amino acids. Based on our preliminary studies, we plan to select these three mutated genes and verify their mutation frequencies in 31 Mycobacterium tuberculosis isolates (1 standard strain H37Rv, 10 drug sensitive-TB, 10 MDR-TB and 10 XDR-TB isolates, respectively) through PCR amplification and sequence analysis. One of these three genes which is frequently mutated in MDR/XDR-TB and encode protein with enzymatic functions will be subsequently chosen for gene cloning, protein expression and purification. Purified protein will be analyzed by Western blot, ELISA and enzyme activity assays to determine the effects of mutations on protein functions. In addition, known inhibitors and/or activators of these enzymes will be added to drug susceptibility tests against different anti-TB drugs to determine how changed enzymatic activities will affect drug susceptibility of XDR-TB strains in order to explore the mechanism of extensive-drug resistance in Mycobacterium tuberculosis as well as its regulations and to provide new drug targets for the treatment of MDR/XDR-TB.

耐多药及广泛耐药结核分枝杆菌的传播给全球结核病防治带来了严峻挑战。目前关于结核分枝杆菌耐多药/广泛耐药机制及其调控尚未阐明。本课题组前期对2株广泛耐药结核分枝杆菌进行了全基因组序列对比,发现284个新突变基因,根据突变基因编码蛋白的生物学功能和氨基酸特性以及软件分析筛选出与耐多药/广泛耐药最可能相关的17个新突变基因,进一步分析最终获得3个关键的编码功能酶的新突变基因hemK、glyA1和MT3842。本项目拟在此基础上,通过PCR扩增和基因测序验证这3个基因在耐多药/广泛耐药结核分枝杆菌中的突变,选择突变率最高的1个基因进行克隆、蛋白质表达及纯化，利用Western Blot、ELISA及酶活性分析等技术检测基因突变对蛋白质功能的影响，并通过酶抑制剂和/或激活剂观察酶活性改变对耐药结核分枝杆菌药物敏感性的作用及其调控，以期了解结核分枝杆菌耐药的机制并为耐药结核病的治疗提供新的药物靶基因。

结核病是由结核分枝杆菌引起的目前除新冠病毒感染以外死亡人数最多的传染病，耐药结核病的出现和传播使全球公共卫生面临更为严峻的挑战。进一步阐明耐药结核病发生的机理以及寻求新的药物靶基因已迫在眉睫。本项目首先对前期筛选的新突变基因hemK（Rv1300）、glyA1（Rv1093）和MT3842进行验证，提示与耐药菌表型无明显关系。进一步分析前期相关候选基因，显示Rv2048c、Rv1527c和Rv3737突变与耐药菌表型相关。根据本项目同行专家建议，采用菌株的基因敲除及回补技术替代原方案中酶抑制剂及激活剂对靶基因的调控。随后通过构建Rv2048c、Rv1527c和Rv3737敲除株和/或过表达，观察敲除株对菌落形态、菌体生长、胞内存活能力、以及对现有12种抗结核药敏感性的影响，寻找新突变基因的药物靶标。结果（1）成功构建敲除株H37RvΔRv2048c、H37RvΔRv1527c和H37RvΔRv3737。（2）敲除株的胞内存活低于H37Rv-WT，提示Rv2048c、Rv1527c和Rv3737促进细菌在巨噬细胞内的存活。（3）H37RvΔRv2048c致乙硫异烟胺（ETH）和丙硫异烟胺（PTH）的MIC值降低，说明敲除株对ETH/PTH 的敏感性增加，提示ETH/PTH为Rv2048c的药物靶标。（4）H37Rv∆Rv1527c导致阿米卡星（AM）的敏感性增强，提示AM为Rv1527c 的药物靶标；通过临床分离菌全基因组测序、表型药敏及临床个案验证，显示结核分枝杆菌Rv1527c G1637A和 A1380P突变增加细菌对AM的敏感性。（5）H37RvΔRv3737可使AM MIC下降4倍且较为稳定；基因测序显示Rv3737基因突变与AM MIC水平无明显相关性，但临床分离菌Rv3737表达量随AM MIC提高而升高，提示Rv3737表达量升高可能与AM低水平耐药有关。（6）过表达株MS::Rv3737促进分枝杆菌在巨噬细胞内的存活，其机制可能与抑制结核分枝杆菌感染巨噬细胞自噬有关。结论：本项目筛选并鉴定了抗结核药物新靶基因，为进一步研究结核病发病机制及潜在药物靶标提供参考。