| Pseudomonas aeruginosa(PA)is a common gram-negative pathogenic bacteria,which can cause chronic infection in patients with low immunity,and the infection is easy to relapse and difficult to remove.Because PA is resistant to many common antibiotics,the infection is difficult to control and cure.PA can not only form biofilm,but also secrete a variety of virulence factors.At present,the studies on PA biofilm and virulence factors are mainly about antibiotics,while the inhibitory effect of other drugs on PA is rarely reported.According to other reports,the formation and maintenance of biofilms and the synthesis and release of virulence factors are mainly regulated by density sensing system,namely quorum sensing(QS).Umbelliferone is a kind of coumarin compound,which has good antibacterial,antihypertensive and anticancer effects.According to our experimental study,umbelliferone has obvious inhibitory effect on PA,but its mechanism is not clear.This paper mainly studied and analyzed the virulence factors and biofilm effects of umbelliferol on PA,and expected to clarify the mechanism of inhibiting the biofilm and pathogenicity of PA by umbelliferol.Aims: To explore the effect of umbelliferone on PAO1 biofilm and virulence factors,and to explore its antibacterial mechanism.Methods: According to the established screening system,CV026 plate color reaction,the preliminary screening for antibacterial drugs.The changes of PAO1 swimming ability and virulence factor production were observed after treatment with different concentrations of umbelliferone.qPCR was used to detect the changes of gene expression related to quorum sensing system.The microstructure of PAO1 biofilm and the content of PAO1 biofilm were determined by semi-quantitative method.Molecular simulation docking test simulated the binding state of receptor protein and umbellactone.Resistance of PAO1 to umbelliferol was observed by simulating the induced resistance in vitro.Results: Umbelliferone significantly inhibited the production of LasA protease with 30.3,41.8,69.8,% and LasB protease with 31.3,34.4,39.3% at sub-MICs respectively.The production of pyocyanin was reduced by 14.9,16.19,18.4% and rhamnolipids by 28.5,54.2,62.8% at sub-MICs.And biofilm formation reduced by 25.3,34.6,43.8 % at sub-MICs.It was also showed 4.0,10.4,23.5% surpression in cell surface hydrophobicity(CSH)and 24.7,40.8,48.3% surpression in extracellular DNA(eDNA)production at sub-MICs.In addition,real-time qPCR revealled that umbelliferone down-regulated the expression of lasI(34.8-86.8%),lasR(39.3-85.4%),rhlI(45.3-85.6%)and rhlR(48.2-86.8%).From the molecular docking studies,umbelliferone could bind tightly to QS receptors(LasI,LasR and RhlR)Conclusion: Umbelliferone can inhibit the synthesis of virulence factors and affect the formation and dispersion of biofilm.It can bind closely to receptor proteins related to the QS system,which can delay or inhibit the resistance of PAO1. |
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