Department of Microbiology, Jahrom Branch, Islamic Azad University, Jahrom, Iran.
Department of Microbiology, Jahrom Branch, Islamic Azad University, Jahrom, Iran.
Microb Pathog. 2022 Jan;162:105366. doi: 10.1016/j.micpath.2021.105366. Epub 2021 Dec 27.
Mycobacterium tuberculosis Beijing genotype was associated with tuberculosis outbreaks and increased transmissibility. To understand the variation in virulence between Beijing and non-Beijing clinical isolates of M.tuberculosis genotypes, the esat-6 gene sequencing, and its expression was compared in the macrophage environment.
MATERIALS & METHODS: Among 64 nonrepetitive, culture-positive M.tuberculosis, DNA extraction of 24 and 40 pure confirmed Beijing and non-Beijing isolates was accompanied by the boiling method. esat-6 gene PCR amplification and their sequencing were carried out by specific primers and its expression was performed on human macrophage cell line U937 after 6, 12, and 18 h of exposure to bacilli. The esat-6 mRNA transcription and expression in M. tuberculosis treated macrophage by Real-Time PCR and Western blot method.
Data analysis based on sequencing of the east-6 gene PCR product showed that this gene exists in all isolates and there are no changes or single nucleotide variation between the Beijing and non-Beijing isolates. In Beijing strains, the esat-6 expression was increased during the study times, but it was constant in non-Beijing isolates. esat-6 gene expression in Beijing isolates reached to about 44.9 times more than non-Beijing isolates after 18 h incubation on the macrophages cell line.
esat-6 is a conserved gene both in Beijing and non-Beijing isolates of M.tuberculosis. More expression of the east-6 gene in the macrophage model may indicate that this gene is likely to play a more important role in increasing the pathogenicity of Beijing strains.
结核分枝杆菌北京基因型与结核病暴发和传染性增加有关。为了了解北京型和非北京型结核分枝杆菌基因型临床分离株之间毒力的差异，我们比较了巨噬细胞环境中 esat-6 基因的测序及其表达情况。
在 64 例非重复、培养阳性的结核分枝杆菌中，24 例和 40 例纯确认的北京和非北京分离株采用煮沸法提取 DNA。采用特异性引物进行 esat-6 基因 PCR 扩增和测序，并将其在暴露于细菌 6、12 和 18 小时后在人巨噬细胞系 U937 上进行表达。采用实时 PCR 和 Western blot 法检测结核分枝杆菌处理巨噬细胞中的 esat-6 mRNA 转录和表达。
基于 esat-6 基因 PCR 产物测序的数据分析表明，该基因存在于所有分离株中，北京和非北京分离株之间没有变化或单个核苷酸变异。在 Beijing 株中，esat-6 表达在研究期间增加，但在非 Beijing 分离株中保持不变。在巨噬细胞系上孵育 18 小时后，北京分离株中 esat-6 基因的表达量比非 Beijing 分离株增加了约 44.9 倍。
esat-6 是北京和非北京结核分枝杆菌分离株中的保守基因。巨噬细胞模型中 esat-6 基因的高表达可能表明该基因可能在增加 Beijing 株的致病性方面发挥更重要的作用。
