Department of Microbiology, Vallabhbhai Patel Chest Institute, University of Delhi, Delhi-110007, India.
BMC Microbiol. 2011 Feb 23;11:41. doi: 10.1186/1471-2180-11-41.
The presence of four mammalian cell entry (mce) operons in Mycobacterium tuberculosis suggests the essentiality of the functions of the genes in these operons. The differential expression of the four mce operons in different phases of in vitro growth and in infected animals reported earlier from our laboratory further justifies the apparent redundancy for these genes in the genome.Here we investigate the extent of polymorphism in eight genes in the mce1 and mce4 operons of M. tuberculosis from four standard reference strains (H37Rv, H37Ra, LVS (Low Virulent Strain) and BCG) and 112 clinical isolates varying in their drug susceptibility profile, analysed by direct sequencing and Sequenom MassARRAY platform.
We discovered 20 single nucleotide polymorphisms (SNPs) in the two operons. The comparative analysis of the genes of mce1 and mce4 operons revealed that yrbE1A [Rv0167] was most polymorphic in mce1 operon while yrbE4A [Rv3501c] and lprN [Rv3495c] had the highest number of SNPs in the mce4 operon. Of 20 SNPs, 12 were found to be nonsynonymous and were further analysed for their pathological relevance to M. tuberculosis using web servers PolyPhen and PMut, which predicted five deleterious nonsynonymous SNPs. A mutation from proline to serine at position 359 of the native Mce1A protein was most deleterious as predicted by both PolyPhen and PMut servers. Energy minimization of the structure of native Mce1A protein and mutated protein was performed using InsightII. The mutated Mce1A protein showed structural changes that could account for the effects of this mutation.
Our results show that SNPs in the coding sequences of mce1 and mce4 operons in clinical isolates can be significantly high. Moreover, mce4 operon is significantly more polymorphic than mce1 operon (p < 0.001). However, the frequency of nonsynonymous substitutions is higher in mce1 operon and synonymous substitutions are more in mce4 operon. In silico modeling predict that nonsynonymous SNP at mce1A [Rv0169], a virulence gene could play a pivotal role in causing functional changes in M. tuberculosis that may reflect upon the biology of the bacteria.
结核分枝杆菌中有四个哺乳动物细胞进入（mce）操纵子，这表明这些操纵子中基因的功能至关重要。我们实验室之前报道的这些操纵子在体外生长的不同阶段和感染动物中的差异表达进一步证明了这些基因在基因组中的明显冗余。在这里，我们通过直接测序和 Sequenom MassARRAY 平台分析了来自四个标准参考菌株（H37Rv、H37Ra、LVS（低毒力株）和 BCG）和 112 株药物敏感性不同的临床分离株的 mce1 和 mce4 操纵子中八个基因的多态性程度。
我们在两个操纵子中发现了 20 个单核苷酸多态性（SNP）。mce1 和 mce4 操纵子基因的比较分析表明，mce1 操纵子中 yrbE1A [Rv0167] 最为多态，而 mce4 操纵子中 yrbE4A [Rv3501c] 和 lprN [Rv3495c] 具有最高数量的 SNP。在 20 个 SNP 中，有 12 个是非同义的，并使用 PolyPhen 和 PMut 网络服务器进一步分析了它们与结核分枝杆菌的病理相关性，这两种服务器预测了五个有害的非同义 SNP。在天然 Mce1A 蛋白的 359 位脯氨酸到丝氨酸的突变是最有害的，这是 PolyPhen 和 PMut 服务器都预测的。使用 InsightII 对天然 Mce1A 蛋白和突变蛋白的结构进行了能量最小化。突变的 Mce1A 蛋白显示出结构变化，可以解释这种突变的影响。
我们的研究结果表明，临床分离株中 mce1 和 mce4 操纵子的编码序列中的 SNP 可能显著较高。此外，mce4 操纵子比 mce1 操纵子的多态性更为显著（p < 0.001）。然而，mce1 操纵子中的非同义替换频率较高，而 mce4 操纵子中的同义替换频率较高。计算机建模预测，mce1A [Rv0169] 中的非同义 SNP 是一种毒力基因，可能在引起结核分枝杆菌功能变化中发挥关键作用，这可能反映了细菌的生物学特性。
