基于分子对接技术的结核分枝杆菌耐异烟肼和利福平的分子机制研究

Ponit mutation in the targeted gene is the main mechanism of drug resistance in Mycobacterium tuberculosis（MTB）. Five novel katG mutants (Trp191Stop, Thr271Pro, Trp328Phe, Leu546Pro and Asp695Gly), and six new allelesin the rpoB gene(Ile569Val, Ile572Met, Phe584Ser, Val615Met, Asp626Glu and Lys972Thr), were identified in our previous study(J Clin Microb.2012,7).However, it remains to be ascertained if these mutations are associated with INH or RFP resistance in Mycobacterium tuberculosis.To investigate the association between these mutations and INH or RFP resistance in Mycobacterium tuberculosis, the following studies will be performed: ①cloning wild katG gene and 5 novel katG mutants, and comparing their INH oxidase, peroxidase, and catalase activities; ②transforming the mutated rpoB and katG gene into wild-type Mycobacterium smegmatis and M.tuberculosis strain H37Ra,and determining the INH or RFP MIC（minimum inhibitory concentration）change of the recombinant Mycobacterium smegmatis and M.tuberculosis strain H37Ra;③ constructing the models of wild-type and mutated rpoB(katG) protein using the Protein Fold Recognition Server, docking wild-type and mutated rpoB (katG) protein with rifampin (isoniazid) using AutoDock 4.0, and comparing the molecular dynamics and conformational changes of the mutant rpoB-rifampin (KatG-isoniazid) complex with the wild-type rpoB-rifampin (KatG-isoniazid) complex. Moreover, to investigate the role of efflux pumps in MTB drug resistance, we will construct DNA microarrays containing probable drug efflux pump genes of MTB, and detect changes in the expression of these genes of the multidrug-resistant Mycobacterium tuberculosis clinical isolates on exposure of INH or RFP. The aim of this study is to explain the molecular mechanism of INH or RFP resistances conferred by the mutated katG or rpoB proteins, and provide useful information for discovering novel drug target points and developing the gene chips to detect drug resistance in Mycobacterium tuberculosis.

靶基因位点突变是结核分枝杆菌（MTB）产生耐药的主要原因。前期研究发现 MTB 5个KatG 和6个rpoB基因新突变位点(J Clin Microb.2012,7)。这些位点与MTB对异烟肼（INH）和利福平（RFP）耐药关系及其分子机制尚不清楚。本项目拟用分子克隆、分子对接、DNA微阵列等技术，进一步研究①野生型和突变型KatG蛋白的功能差异；②耻垢分枝杆菌和M.tuberculosis H37Ra导入重组突变型KatG和rpoB基因穿梭质粒的MIC变化；③野生型和诱变型KatG和rpoB蛋白与INH和RFP相互作用的差别；④耐多药株在药物诱导作用下的外排泵基因表达及活性的变化。本研究旨在明确上述突变位点与MTB对INH和RFP耐药的相关性，阐明相对应的KatG和rpoB变异蛋白引起MTB耐INH和RFP的分子机制。为结核病耐药检测基因芯片的开发和药物新靶点的发现提供理论和实验依据。

本课题构建了KatG基因单突变重组质粒，进行了突变蛋白诱导纯化及酶活性测定，研究结果提示：在5个KatG 突变位点TGG191TGA、ACT271CCT、TGG328TTT、CTC546CCC、GAC695GGC中，相比野生型，突变体Trp191stop 的KatG 蛋白酶活性完全丧失，而单突变Trp328Phe、Thr271Pro、Leu546Pro的KatG 蛋白的过氧化氢酶和过氧化物酶的活性分别下降30～50%和60～70%，而突变Asp695Gly的KatG蛋白的过氧化氢酶和过氧化物的活性无明显变化。分子对接分析表明：异烟肼与KatG蛋白质的作用是以疏水为主，辅以氢键作用。当KatG蛋白质Trp328Phe、Thr271Pro、Leu546Pro、Asp695Gly氨基酸残基突变后，活性区域结构发生改变，氢键作用引起异烟肼分子偏离活性区域，导致结合力下降。综上研究结果表明：单突变TGG191TGA、Trp328Phe、Thr271Pro、Leu546Pro的KatG蛋白酶活性缺失或明显下降，与异烟肼耐药相关。因此，这四个突变点可作为INH耐药菌株检测的标志，为临床筛查异烟肼耐药菌株、开发基因芯片诊断工具等提供理论依据。. 以PCR扩增野生型和突变型（Ile569Val, Ile572Met, Phe584Ser, Val615Met, Asp626Glu and Lys972Thr）rpoB全基因编码序列定向克隆入穿梭表达质粒pMV361获得重组穿梭表达质粒，将重组质粒电穿孔导入耻垢分枝杆菌，构建重组KatG基因的耻垢分枝杆菌，以琼脂平板稀释法检测各重组耻垢分枝杆菌对利福平药物敏感性变化。体内实验结果表明：rpoB-Ile569Val、Ile572Met、Phe584Ser、Val615Met、Asp626Glu和Lys972Thr突变菌株与野生型转化耻垢分枝杆菌对利福平药物敏感性比较并无明显改变，这提示六个rpoB基因突变体可能未涉及结核分枝杆菌对利福平的耐药性。