Vedithi Sundeep Chaitanya, Lavania Mallika, Kumar Manoj, Kaur Punit, Turankar Ravindra P, Singh Itu, Nigam Astha, Sengupta Utpal
Stanley Browne Laboratory, The Leprosy Mission Community Hospital, Nand Nagri, New Delhi, 110093, India,
Med Microbiol Immunol. 2015 Apr;204(2):193-203. doi: 10.1007/s00430-014-0354-1. Epub 2014 Sep 9.
Presence of point mutations within the drug resistance determining regions of Mycobacterium leprae (M. leprae) genome confers molecular basis of drug resistance to dapsone, rifampin and ofloxacin in leprosy. This study is focused on the identification of mutations within the rpoB gene region of M. leprae that are specific for rifampin interaction, and further in silico analysis was carried out to determine the variations in the interactions. DNA and RNA were isolated from slit skin scrapings of 60 relapsed leprosy patients. PCR targeting rpoB gene region and amplicon sequencing was performed to determine point mutations. mRNA expression levels of rpoB and high-resolution melt analysis of mutants were performed using Rotor Gene Q Realtime PCR. Molecular docking was performed using LigandFit Software. Ten cases having point mutations within the rpoB gene region were identified and were clinically confirmed to be resistant to rifampin. A new mutation at codon position Gln442His has been identified. There is a 9.44-fold upregulation in the mRNA expression of rpoB gene in mutant/resistant samples when compared with the wild/sensitive samples. In silico docking analysis of rifampin with wild-type and Gln442His mutant RpoB proteins revealed a variation in the hydrogen-bonding pattern leading to a difference in the total interaction energy and conformational change at position Asp441. These preliminary downstream functional observations revealed that the presence of point mutations within the rifampin resistance determining regions of rpoB gene plays a vital role in conferring genetic and molecular basis of resistance to rifampin in leprosy.
麻风分枝杆菌（M. leprae）基因组耐药性决定区域内的点突变赋予了麻风病对氨苯砜、利福平和氧氟沙星耐药的分子基础。本研究聚焦于鉴定麻风分枝杆菌rpoB基因区域内与利福平相互作用特异性相关的突变，并进一步进行了计算机模拟分析以确定相互作用的差异。从60例复发型麻风病患者的皮肤刮片中分离出DNA和RNA。进行靶向rpoB基因区域的PCR及扩增子测序以确定点突变。使用Rotor Gene Q实时PCR进行rpoB的mRNA表达水平测定及突变体的高分辨率熔解分析。使用LigandFit软件进行分子对接。鉴定出10例rpoB基因区域存在点突变的病例，临床确诊为对利福平耐药。已鉴定出密码子位置Gln442His处的一个新突变。与野生型/敏感型样本相比，突变体/耐药型样本中rpoB基因的mRNA表达上调了9.44倍。利福平与野生型和Gln442His突变型RpoB蛋白的计算机模拟对接分析显示氢键模式存在差异，导致总相互作用能和Asp441位置的构象变化有所不同。这些初步的下游功能观察结果表明，rpoB基因利福平耐药性决定区域内的点突变在赋予麻风病对利福平耐药的遗传和分子基础方面起着至关重要的作用。
