RseA基因影响耻垢分枝杆菌药物敏感性的生物信息学分析与初步验证

Bioinformatics analysis and preliminary verification of the antibiotic sensitivity of Mycobacterium smegmatis,as affected by the RseA gene

目的筛选RseA在耻垢分枝杆菌中可能调控药物敏感性的基因或通路,并进行初步验证。方法利用分子克隆技术构建RseA基因过表达耻垢分枝杆菌组和空质粒对照组;利用转录组测序技术和生物信息学方法筛选2组细菌间的差异表达基因(DEGs),分析其基因本体论(GO)及京都基因和基因组百科全书(KEGG)富集情况;并构建蛋白质-蛋白质相互作用(PPI)网络。利用结核分枝杆菌复苏促进因子E(RpfE)促使非复制持留菌复苏,测定复苏指数(RI)对筛选出的关键靶向基因或通路进行初步验证。结果RseA基因过表达后,筛选出2403个DEGs,其中2335个表达上调,68个表达下调。GO分析结果表明,DEGs主要参与氮化合物代谢过程的调控、RNA代谢过程的调控、转录因子活性和序列特异性DNA结合过程调控等。KEGG分析结果显示,DEGs主要参与萜类骨架的生物合成、果糖和甘露糖代谢和同源重组等通路。从PPI网络MCODE模块中筛选出前8个hub基因rpsG、rplM、rpsO、rpsT、rpmH、rplS、rpsJ、rplT,并从这些基因的分析结果得知,其主要参与了核糖体通路。使用RpfE促进复苏后,RseA组复苏指数明显低于对照组。结论RseA调控了多个靶向基因,参与核糖体的结构组成和核糖体通路,增强耻垢分枝杆菌对抗结核药物的敏感性,为进一步的机制研究奠定了基础。

This study aimed to screen the potential genes or pathways regulated by RseA in Mycobacterium smegmatis and to provide preliminary verification of the results.M.smegmatis strains overexpressing RseA or a plasmid control were constructed through molecular cloning technology.Transcriptome sequencing and bioinformatics methods were used to compare the differentially expressed genes(DEGs)between the groups;to assess the DEG functional enrichment through gene ontology(GO)analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis;and to construct a protein-protein interaction(PPI)network.M.tuberculosis resuscitation factor E(RpfE)was used to promote the recovery of non-replicating retained bacteria,and the resuscitation index(RI)was measured to perform preliminary verification of the screened key targeted genes or pathways.A total of 2403 DEGs in the RseA overexpression group relative to the control were identified,comprising 2335 up-regulated genes and 68 down-regulated genes.GO functional enrichment analysis showed that the DEGs mainly participated in the following categories:regulation of nitrogen compound metabolic process,RNA metabolic process,transcription factor activity and sequence-specific DNA binding.KEGG pathway analysis revealed enrichment in the pathways of terpenoid backbone biosynthesis,fructose and mannose metabolism and homologous recombination.A screen of the top eight hub genes(rpsG,rplM,rpsO,rpsT,rpmH,rplS,rpsJ and rplT)from the MCODE module of the PPI network revealed that these genes are involved in ribosome pathways.This study revealed that the RseA can regulate multiple targeted genes,affect the ribosome structure and ribosome pathways,and enhance the sensitivity of M.smegmatis to anti-tuberculosis drugs.These findings lay a foundation for further mechanistic research.