基于RNA-Seq技术分析亚抑菌浓度的四季青对耐药铜绿假单胞菌运动能力的抑制作用

Inhibitory Effect of Sijiqing(四季青)at Sub-Minimum Inhibitory Concentration on the Motility of Drug-resistant Pseudomonas aeruginosa Based on RNA-seq

目的:通过转录组测序(RNA-Seq)技术探究亚抑菌浓度的四季青对耐药铜绿假单胞菌的影响并进行验证。方法:使用Illumina测序平台Illumina HiSeq 3000对四季青处理的耐药铜绿假单胞菌和正常对照组进行转录组测序,采用DEGseq软件分析两组细菌间差异表达基因;通过GOseq R包软件和KOBAS数据库中对差异表达基因进行GO生物富集和KEGG通路分析;体外测定亚抑菌浓度的四季青对耐药铜绿假单胞菌浮泳和蹭性运动抑制作用。结果:与正常对照组比较,亚抑菌浓度的四季青处理后的耐药铜绿假单胞菌有44个基因上调,304个基因下调;这些基因主要富集于鞭毛和纤毛依赖性细胞运动、细胞运动定位、趋化性、细胞呼吸等生物过程以及鞭毛装配、细菌趋化性、双组分系统、氧化磷酸化、TCA循环等通路;体外验证试验中,与正常对照组比较,亚抑菌浓度的四季青对耐药铜绿假单胞菌典型的鞭毛和纤毛介导的运动如浮泳和蹭行运动有显著抑制(P<0.05或P<0.01)。结论:四季青通过多靶点、多通路影响耐药铜绿假单胞菌,其中抑制鞭毛、纤毛及其介导的运动、趋化性等是关键作用机制,阐明了四季青在影响临床耐药铜绿假单胞菌致病性和耐药性等方面的应用潜力,为四季青作为抗耐药菌药物的开发和临床应用提供参考。

Objective:To explore and verify the effect of Sijiqing(四季青)at sub-minimum inhibitory concentration on drug-resistant Pseudomonas aeruginosa by RNA-Seq.Methods:The transcriptome of P.aeruginosa treated with Sijiqing and the control group was sequenced by Illumina HiSeq 3000,and the differentially expressed genes between the two groups were analyzed by DEGseq.GOseq R package and KOBAS database were employed for Gene Ontology(GO)term enrichment and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment of the differentially expressed genes.The inhibitory effect of Sijiqing at sub-minimum inhibitory concentration on swimming and swarming motility of resistant P.aeruginosa were determined in vitro.Results:A total of 44 genes were up-regulated and 304 genes were down-regulated in P.aeruginosa treated with Sijiqing at sub-minimum inhibitory concentration compared with the control group.These genes were mainly involved in flagellum and cilium-dependent cell movement,cell movement localization,chemotaxis,cell respiration and other biological processes,as well as flagellum assembly,bacterial chemotaxis,two-component system,oxidative phosphorylation,tricarboxylic acid(TCA)cycle,and other pathways.In verification test in vitro,Sijiqing at sub-minimum inhibitory concentration significantly inhibited the typical flagellum-medicated and cilium-mediated movement of resistant P.aeruginosa,such as swimming and swarming motility.Conclusion:Sijiqing can interfere with resistant P.aeruginosa through multiple targets and multiple pathways,and the key mechanism is the likelihood that it inhibits flagella,cilia,and their mediated movement and chemotaxis.The potential of Sijiqing for influencing the pathogenicity and drug resistance of clinical resistant P.aeruginosa is clarified,which can serve as a reference for the development and clinical application of antibacterial agent against resistant P.aeruginosa with Sijiqing.