Studies On The Mechanism Of Sodium Houttuyfonate Against Pseudomonas Aeruginosa Biofilm Targeting To Quorum Sensing Molecule

Pseudomonas aeruginosa, a Gram-negative opportunistic pathogen,, is a typical biofilm-forming microbe wihich allows it to thrive in a diverse range of natural and nosocomial niches. P. aeruginosa is one of the most common causes of severe infections such as wound, eye and lung, and is often difficult to cure due to the high prevalence of multi-drug resistance.Sodium houttuyfonate(SH) is a product of the addition reaction of sodium bisulfite and houttuynin that is one of the main and most effective extracts from Houttuynia cordata Thunb. SH is used in traditional Chinese medicine for the treatment of a wide range of infectious diseases and regulation effects on the immune system. Previously founds from our research indcate that SH can effectively inhibit growth, especially the prevent biofilm formation research indcate of P. aeruginosa. However, the antimicrobial mechanisms of SH are still remain unknown. The quorum sensing(QS) system is a population density-dependent regulatory system and that enables cell-to-cell communication and coordinated control of gene expression in microbes. Co-ordination of gene expression through QS is an important regulatory mechanism of virulence and drug-resistance in P. aeruginosa.Thus, interfering with QS will inhibit microbial pathogenicity. Therefore, we investigated the anti-QS activity to explore the antimicrobial mechanisms of SH against P. aeruginosa in this study.Objective: Investigates the mechanism of Sodium houttuyfonate against Pseudomonas aerμginosa biofilm targeting quorum sensing system.Methods: We identified the best synthesis time and extraction method of AHLs in P.aeruginosa by plate assay of Chromobacterium violaceum CV026 strains, and investigated the influences of AHLs production of on P. aeruginosa by SH treatment;plate assay was used to observe the changes of motility activities of P. aeruginosa during the different SH concentrations; semi-quantitative and quantitative RT-PCR were used to investigated expression of QS and related genes of P. aeruginosa; Western blotblot was applied to analysze the bdl A protein expression changes of P. aeruginosa in biofilm dispersion state; morphogical changes of P. aeruginosa biofilm were detected by SEM Results:(1) The CV026 plate assay results showed that the largest production of signals molecule AHLs was secreted at 72 h compared with any other time in P.aeruginosa. We establishes the approach to extract and detect AHLs.(2) We firstly found that SH effectively inhibits the three types virulence related motility activities of P. aeruginosa, i.e., swimming, twitching and swarming. The presented results showed that the inhibition of SH on motility of P. aeruginosa is dose-dependent.Furthermore, we found that the expression of structural genes flg B and pil G was down-regulated by SH, implying that mechanism of inhibitory effects of SH against motility of P. aeruginosa may be due to the inhibition of flagella and pili of P.aeruginosa by SH.(3) The RT-PCR and q RT-PCR results showed that the expression of AHL biosynthesis gene, las I, and key regulatory gene, las R, were down-regulated by SH. Furthermore, we found that las A and psl A, which are regulated by Las R, were also down-regulated in response to SH in a concentration-dependent manner. Unexpectedly, we found that the expression rhl I was up-regulated by SH. In addition, we also detected the expression of las B, gac A, rsm As, and mex A related in virulence factor, virulence regulation and drug resistance under the SH treatment. However, expression of these four genes is not significantly affected by SH.(4) SEM observation results indicate that biofilm dispersion of SH group was significantly suppressed at 100 min of biofilm formation, and biofilm structure was destroyed by SH treatment after 24 h compared with the control group.(5) Western bloting test result indicates that the expression of Bdl A involving in P.aeruginosa biofilm dispersal was down-regulated after SH treatment. q RT-PCR results showed that the genes expression of biofilm dispersalbdl A, rhl A, tpb A and tpb B were down-regulated, and pel A and pel D were up-regulated after SH treatment. These resultssuggestedthat SH could inhibit the biofilm dispersion of P. aeruginosa by affecting OS regulatory system.Conclusion: The present results demonstrate that SH possess anti-QS property to inhibit the biofilm formation and virulence factor production of P. aeruginosa. In addition, due to the close chemical structure of SH and AHL, the possible inhibitory mechanism of SH against QS in P. aeruginosa may be the competitive binding activity to Las R with AHLs(3-oxo-C12-HSL) and thus specifically inhibit the sensing of this molecule to reduce the enzyme activity of Las R, and related genes and virulence factors express were inhibited by against Las system on P. aeruginosa, then play a role of antibacterial by this means.