表皮葡萄球菌SE1457与vraSR突变株转录组测序比较分析

Comparison analysis on the transcriptome sequencing of Staphylococcus epidermidis SE1457 and its isogenic vraSR mutant strain

目的筛选和验证VraSR调控的下游靶基因,为表皮葡萄球菌vraSR生物学功能研究提供参考。方法在加与未加万古霉素(Van+/Van-)两种条件下,抽提表皮葡萄球菌SE1457及其同源性vraSR突变株RNA,构建cDNA文库(每个菌株3个独立样品),采用Illumina HiSeq进行转录组测序,差异表达基因筛选标准为Q value<0.05,且表达值变化倍数FC≥2(FDR校正P<0.05,t检验),对差异表达基因进行GO及KEGG分析,并进行qRT-PCR验证。结果与SE1457野生株相比,Van+条件下vraSR突变株有39个差异表达基因(16个下调,23个上调),Van-条件下vraSR突变株有61个差异表达基因(35个下调,26个上调),差异表达基因主要涉及糖代谢、磷酸戊糖途径、三羧酸循环等。Van+/Van-2种条件下qRT-PCR检测生物膜形成相关基因icaA相对表达量分别为0.44±0.06和0.17±0.05(P<0.01),icaR为4.35±0.79(P<0.01)和9.27±4.4(P<0.05),未发现耐药相关基因(pbp2,sgtB,murZ)转录水平改变(P>0.05)。结论表皮葡萄球菌VraSR可能通过ica途径调控生物膜形成,并通过调节肽聚糖合成相关基因的转录间接影响耐药性。

Objective To explore the target genes regulated by VraSR and provide the reference for the biological function study of VraSR in Staphylococcus epidermidis.Methods The RNA samples of S.epidermidis SE1457 and its allelic vraSR mutant cultured with or without vancomycin(Van+/Van-)were extracted,and the cDNA library was constructed.Transcriptomic sequencing was performed by Illumina Hi Seq system.The screening criteria for differentially expressed genes(DEGs)were Q value<0.05,and transcriptional level more than 2 folds(FDR correction P<0.05,T test).DEGs were analyzed using GO and KEGG enrichment analysis,and verified using qRT-PCR.Results Compared with those in SE1457 parent strain,39 DEGs(16 down-regulated genes,23 up-regulated genes)were found in the vraSR deletion mutant under Van+condition,whereas 61 DEGs(35 down-regulated genes,26 up-regulated genes)under Van-condition,these DEGs were mainly involved in glucose metabolism,pentose phosphate pathway,tricarboxylic acid cycle,etc.The results of qRT-PCR under both Van+/Van-conditions further indicated that the relative expression of icaA was 0.44±0.06 and 0.17±0.05(P<0.01),respectively,and icaR was 4.35±0.79(P<0.01)and 9.27±4.4(P<0.05).However,the transcriptional levels of the drug-resistance related genes such as pbp2,sgtB,and murZ showed no significant changes(P>0.05).Conclusion S.epidermidis VraSR may regulate biofilm formation through ica pathway,and it also indirectly affect drug resistance by regulating the transcription of genes related to peptidoglycan synthesis.