异烟肼干预耻垢分枝杆菌的转录组差异分析

Comparative transcriptome analysis of Mycobacterium smegmatis following an intervention with isoniazid

目的探究异烟肼(INH)对耻垢分枝杆菌(MS)MC2155的基因表达调控水平的干预作用,以期发现INH诱导的耐药基因突变形成的新分子机制。方法实验组使用1/2最小抑菌浓度(MIC)的INH处理,对照组用相同稀释倍数的二甲基亚砜(DMSO),培养72 h后,提取各组MS MC2155的总RNA,用illumina Hiseq平台进行全转录组测序。用bowtie2软件将测定结果同MS MC2155标准株的参考基因组序列进行比对,获得INH干预前后的差异基因(different expression gene, DEG),用GO及KEGG数据库注释上述获得的unigene。结果基因表达差异分析显示,在INH干预后发现1537个DEG(P≤0.05,|log2FC|≥1),其中805个上调(52.37%),732个下调(47.63%)。GO富集分析显示差异基因主要涉及结构分子活动、囊泡等分子功能以及细胞大分子合成、细胞酰胺代谢等生物学过程,包括whiB4、rplM、gmK和nuoC基因;KEGG富集分析显示差异表达基因主要集中在万古霉素耐药通路,包括whiB4、nrdB、rpmB-3和pth;以及核糖体合成、氧化磷酸化和肽聚糖合成等生物合成过程的通路。STRING分析显示,共有391个基因编码的蛋白质存在相互作用,其中处于中心节点的蛋白包括rpsD、rpsB和rplM。结论全转录组水平分析表明INH能促进MS MC2155对万古霉素耐药相关基因的表达,并可抑制细胞壁合成相关基因的表达,可为进一步阐明INH在结核病治疗过程中诱导形成耐药突变的分子机制提供参考。

Objective To explore the effectiveness of isoniazid(INH) as an intervention in the regulation of gene expression in Mycobacterium smegmatis(MS) MC2155, in order to discover novel mechanisms of drug resistance induced by INH. Methods The experimental group was treated with INH at a half of the minimum inhibitory concentration(MIC) against MS MC2155, while the control group was cultured with dimethyl sulfoxide(DMSO) at the same dilution for 72 h. Total RNA was extracted from each group and sequenced on the illumine Hiseq platform. The results were compared to the reference genome of MS MC2155 using the software bowtie2, and differentially expressed genes(DEGs) between the experimental and control groups were ascertained. The obtained unigene was annotated using the GO and KEGG databases. Results DEG analysis revealed 1,537 DEGs(P<0.05, |log2FC| > 1) in MS MC2155 treated with INH. Of those, 805 DEGs were up-regulated(52.37%) and 732 were down-regulated(47.63%). GO enrichment analysis indicated that the DEGs were mainly involved in structural molecular activities, vesicles and other molecular functions, as well as biological processes such as cellular macromolecular synthesis and cellular amide metabolism. The DEGs included whiB4, rplM, gmK, and nuoC;KEGG enrichment analysis also indicated significant enrichment in the vancomycin resistance pathway, including whiB4, nrdB, rpmB-3, and pth. Enrichment also included the pathways of biosynthesis processes such as ribosome synthesis, oxidative phosphorylation, and peptidoglycan synthesis. STRING analysis revealed that 391 proteins interact with each other, and genes including rpsD, rpsB, and rplM were located at the central node. Conclusion Transcriptome analysis indicated that INH could upregulate the level of expression of genes related to vancomycin resistance in MS MC2155 and downregulate the level of expression of genes related to cell wall synthesis. This research could provide a reference to further elucidate the molecular mechanisms of drug resistance mutations induced by INH during tuberculosis treatment.