Sharma Mukul, Vedithi Sundeep Chaitanya, Das Madhusmita, Roy Anindya, Ebenezer Mannam
Department of Biotechnology, Indian Institute of Technology, Hyderabad, Telangana, India.
Schieffelin Institute of Health Research and Leprosy Center, Vellore, Tamil Nadu, India; Department of Biochemistry, University of Cambridge, Cambridge CB2 1GA, UK.
Int J Mycobacteriol. 2017 Oct-Dec;6(4):365-378. doi: 10.4103/ijmy.ijmy_111_17.
Survival of Mycobacterium leprae, the causative bacteria for leprosy, in the human host is dependent to an extent on the ways in which its genome integrity is retained. DNA repair mechanisms protect bacterial DNA from damage induced by various stress factors. The current study is aimed at understanding the sequence and functional annotation of DNA repair genes in M. leprae.
T he genome of M. leprae was annotated using sequence alignment tools to identify DNA repair genes that have homologs in Mycobacterium tuberculosis and Escherichia coli. A set of 96 genes known to be involved in DNA repair mechanisms in E. coli and Mycobacteriaceae were chosen as a reference. Among these, 61 were identified in M. leprae based on sequence similarity and domain architecture. The 61 were classified into 36 characterized gene products (59%), 11 hypothetical proteins (18%), and 14 pseudogenes (23%). All these genes have homologs in M. tuberculosis and 49 (80.32%) in E. coli. A set of 12 genes which are absent in E. coli were present in M. leprae and in Mycobacteriaceae. These 61 genes were further investigated for their expression profiles in the whole transcriptome microarray data of M. leprae which was obtained from the signal intensities of 60bp probes, tiling the entire genome with 10bp overlaps.
It was noted that transcripts corresponding to all the 61 genes were identified in the transcriptome data with varying expression levels ranging from 0.18 to 2.47 fold (normalized with 16SrRNA). The mRNA expression levels of a representative set of seven genes ( four annotated and three hypothetical protein coding genes) were analyzed using quantitative Polymerase Chain Reaction (qPCR) assays with RNA extracted from skin biopsies of 10 newly diagnosed, untreated leprosy cases. It was noted that RNA expression levels were higher for genes involved in homologous recombination whereas the genes with a low level of expression are involved in the direct repair pathway.
This study provided preliminary information on the potential DNA repair pathways that are extant in M. leprae and the associated genes.
麻风分枝杆菌是麻风病的致病菌，其在人类宿主中的存活在一定程度上取决于其基因组完整性的维持方式。DNA修复机制可保护细菌DNA免受各种应激因素诱导的损伤。本研究旨在了解麻风分枝杆菌中DNA修复基因的序列和功能注释。
利用序列比对工具对麻风分枝杆菌的基因组进行注释，以鉴定在结核分枝杆菌和大肠杆菌中有同源物的DNA修复基因。选择一组已知参与大肠杆菌和分枝杆菌科DNA修复机制的96个基因作为参考。其中，基于序列相似性和结构域结构，在麻风分枝杆菌中鉴定出61个。这61个基因被分为36个已鉴定的基因产物（59%）、11个假设蛋白（18%）和14个假基因（23%）。所有这些基因在结核分枝杆菌中都有同源物，在大肠杆菌中有49个（80.32%）。麻风分枝杆菌和分枝杆菌科中存在一组12个在大肠杆菌中不存在的基因。利用从10例新诊断的未经治疗的麻风病患者皮肤活检组织中提取的RNA，通过定量聚合酶链反应（qPCR）分析，进一步研究了这61个基因在麻风分枝杆菌全转录组微阵列数据中的表达谱。该微阵列数据由60bp探针的信号强度获得，探针以10bp重叠覆盖整个基因组。
注意到在转录组数据中鉴定出了与所有61个基因相对应的转录本，其表达水平各不相同，范围为0.18至2.47倍（以16SrRNA标准化）。使用qPCR分析从10例新诊断的未经治疗的麻风病患者皮肤活检组织中提取的RNA，分析了一组7个代表性基因（4个已注释基因和3个假设蛋白编码基因）的mRNA表达水平。注意到参与同源重组的基因的RNA表达水平较高，而表达水平较低的基因参与直接修复途径。
本研究提供了关于麻风分枝杆菌中现存的潜在DNA修复途径及其相关基因的初步信息。
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