基于RNA-seq技术分析黄芩苷抑制铜绿假单胞菌生物膜的分子机制

Analysis of molecular mechanisms of baicalin in inhibiting Pseudomonas aeruginosa biofilm based on RNA-seq technique

目的探讨黄芩苷抑制铜绿假单胞菌生物膜的分子机制,为黄芩苷用于铜绿假单胞菌感染的辅助治疗提供理论依据和数据支撑。方法使用结晶紫染色、光学显微镜和荧光显微镜观察亚抑菌浓度黄芩苷对铜绿假单胞菌PAO1生物膜的影响,同时利用转录组测序(RNA-seq)技术分析PAO1基因表达情况,对差异表达基因进行功能富集和信号通路分析,并通过实时荧光聚合酶链反应(qPCR)和表型试验对涉及多个生物过程和代谢途径的差异基因进行验证。结果黄芩苷作用下,RNA-seq共筛选出差异表达基因1821个,其中下调基因1054个,上调基因767个,主要在生物膜形成、吩嗪类生物合成、群体感应系统及双组分系统等信号通路富集。黄芩苷对PAO172 h内形成的生物膜均具有抑制作用;qPCR和表型验证结果显示,黄芩苷可有效下调生物膜关键组分(包括胞外多糖和蛋白质)及其调控基因(包括pelA、pslA、algR、algD、pilA和filC)的表达水平;同时下调蛋白酶LasA、弹性蛋白酶LasB、绿脓菌素的产生和转录以及群体感应系统相关基因(包括lasR、rhlR、pqsR)和Ⅵ型分泌系统相关基因(hcp1、hcp2、hcp3)的转录水平。结论黄芩苷可通过下调群体感应系统和Ⅵ型分泌系统调控铜绿假单胞菌生物膜相关组分的表达和转录,进而抑制铜绿假单胞菌生物膜的形成,具有多靶点和多途径的特点。

OBJECTIVE To explore the molecular mechanisms of baicalin in inhibiting the biofilm of Pseudomonas aeruginosa so as to provide theoretical basis and data support for adjuvant treatment of P.aeruginosa infection with baicalin.METHODS The effect of sub-inhibitory concentration of baicalin on P.aeruginosa PAO1 biofilm was observed by using crystal violet staining,light microscope and fluorescence microscope.The expression of PAO1 gene analyzed by transcriptome sequencing(RNA-seq)technique,the differentially expressed genes were subjected to functional enrichment and signaling pathway analysis,and the key genes involved in multiple biological processes and metabolic pathways were validated through qPCR and phenotype trial.RESULTS The result of RNA-seq indicated that under the effect of baicalin,totally 1821 differentially expressed genes were identified,including 1054 down-regulated genes and 767 up-regulated genes.These genes were predominantly enriched in signaling pathways associated with biofilm formation,production of phenazine compounds,regulation of quorum sensing system and two-component system.Baicalin had inhibiting effect on the PAO1 biofilm that was formed within 72 hours;the findings from the qPCR and phenotypic validation experiments suggested that baicalin presented a significant potential to decrease the expression of biofilm-related components such as extracellular polysaccharides and proteins,as well as their regulating genes(pelA,pslA,algR,algD,pilA and filC).Furthermore,LasA,LasB and phzM which were responsible for producing LasA,LasB,and pyocyanin respectively,as well as the transcription levels of quorum sensing systems(lasR,rhlR and pqsR)and typeⅥsecretion systems(hcp1,hcp2 and hcp3),were all downregulated when exposed to baicalin.CONCLUSION Baicalin inhibits the formation of P.aeruginosa biofilm by downregulating the expression and transcription of quorum sensing systems and typeⅥsecretion systems to regulate the biofilm-related components,which acts in a muti-target and multi-pathway manner.