Inhibitory Effect And Mechanism Of Sodium New Houttuyfonate Against Pseudomonas Aeruginosa

Pseudomonas aeruginosa is a gram-negative bacterium,which are often distributed in the soil,water,air,normal human skin,respiratory tract and intestinal tract and other environments.P.aeruginosa has a high incidence rate and mortality rate in the clinic,mainly due to their,high drug resistance,which brings certain difficulties to treat the infection caused by it.Sodium new houttuyfonate(SNH),whose chemical name is sodium lauryl acetaldehyde bisulfite,is a chemical composition of aldehyde components in the volatile oil of the whole plant of Houttuynia Cordata Thunb of Saururaceae.It has the functions of bacteriostasis and disinfection,improving immunity and so on.In addition,it has important significance to treat the infectious diseases.Previous studies have found that SNH has a certain inhibitory effect on P.aeruginosa,but the specific mechanism is still unclear.Therefore,the thesis mainly study the possible antibacterial mechanism by analyzed the effect of SNH on P.aeruginosa transcriptome.Objective: To explore the inhibitory mechanism of SNH against P.aeruginosa by analyzed the transcriptome of SNH against P.aeruginosa,the inhibitory effect of SNH against the biofilm of P.aeruginosa,the inhibitory effects of related genes and virulence factors,and the molecular docking between SNH and LasR protein.Methods: The minimum inhibitory concentration(MIC)of SNH was detected by microdilution assay,and the transcriptome of 1xMIC SNH against P.aeruginosa was sequenced by the Huada gene BGI-500 platform.The cell activity of SNH against P.aeroginosa was detectde by measuring its antibacterial activity,minimum bactericidal concentration(MBC),growth curve.The activity of virulence factors was identified by spectrophotometry and ELISA kit.The effect of SNH on P.aeruginosa biofilm was qualitative and quantitative detected by inverted microscope and crystal violet staining.The gene expression of P.aeruginosa biofilm,and virulence factors were detected RT-PCR and qRT-PCR,respectively.The binding ability between SNH and LasRprotein was examined by molecular docking technology.Results:(1)Under the action of 1xMIC SNH,the expression of 5940 genes in P.aeruginosa were detected by transcriptome sequencing,in which 1674 genes were DEGs,1454 genes were down-regulated and 220 genes were up-regulated;The most DEGs were associated with quorum sensing(QS)and biofilm formation were analyzed by the Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway.It suggested that the possible mechanism of SNH inhibiting the pathogenicity of P.aeruginosa is to inhibit biofilm formation and the synthesis of virulence factors via regulating QS system.(2)The experimental results show that SNH has a certain inhibitory effect on P.aeruginosa at 24 h.Enzyme-labeled assay showed that the antibacterial activity of P.aeruginosa gradually increased with the increase of SNH concentration.SNH was found to significantly inhibit the growth activity of P.aeruginosa in solid medium.Compared with the control group,total protease,LasA staphylococcus protease,LasB elastase,Lec A agglutinin,pyocyanin synthesis and exercise ability were inhibited by absorption spectrophotometry and ELISA kit.Compared with control group,SNH group with different concentrations could significantly destroy the formation of biofilm and showed concentration-dependent characteristics were observed by inverted microscope and crystal violet staining.RT-PCR and qRT-PCR results showed that SNH could not be only down-regulate lasR gene,but also down-regulate lasI,rhlI and pqsA genes.Several virulence genes lasA,lasB,lecA,phzM,and pilG were further detected by RT-PCR and qRT-PCR.The results showed that SNH had a down-regulation effect on them and showed concentration-dependent characteristics.(3)Molecular docking results showed that SNH has a certain binding capacity to LasR protein compared with natural ligand 3-oxo-C12-HSL,which indicates that SNH may play a role in inhibiting P.aeroginosa by binding to LasR protein.Conclusion: SNH can inhibit the growth of P.aeroginosa,the synthesis of virulence factors,the formation of biofilm and the expression of related genes.Its mechanism ofaction may be due to the combination of SNH and LasR protein,which inhibits the activity of LasR,thus inhibiting the P.aeroginosa QS system and further reducing the expression of related biofilm synthesis genes and virulence factors regulated by SNH.The results of this paper will provide experimental basis for clarifying the antibacterial mechanism of SNH and play an important role in expanding the new use of effective components of traditional Chinese medicine.