Mechanism Of Pterostilbene Inhibits Aerolysin And Biofilm Of Aeromonas Hydrophila

Aeromonas hydrophila(A.hydrophila)is a widely hosted pathogenic which can several infections both in human and animals,particular aquatic animals.A.hydrophila is an opportunistic pathogen in freshwater aquaculture,which is responsible for diseases such as bacterial septicemia and enteritis.Antibiotics are the main approach for treating infections caused by A.hydrophila.However,irrational use of antibiotics leads to the emergence of drug resistance.In addition,antibiotic residues in aquatic products might affect the health of consumers.Therefore,there is an urgent need to develop drugs with novel mechanism against A.hydrophila infection.With increasing knowledge of the pathogenesis of A.hydrophila,anti-virulence strategies have attracted extensive attention.Development of anti-infective drugs targeting virulence factors has good application prospects.Aerolysin is the chief virulence factor secreted by A.hydrophila,with hemolytic,enterotoxic and cytotoxic activities.The toxin is related to the pathogenicity of A.hydrophila,and can be used as an ideal target for developing drugs against A.hydrophila infections.Here,we investigated the inhibitory effects of pterostilbene on the pathogenicity of A.hydrophila in vitro by inhibiting aerolysin and biofilm.The results are as follows:1.According to the results of MIC,growth curves,hemolytic activity,Westernblot and biofilm formation,pterostilbene had no role on the growth of A.hydrophila at concentrations between 0 to 16 μg/m L.Pterostilbene could significantly decrease the hemolysis of bacterial supernatants at concentrations higher than 2 μg/m L,biofilm formation were remarkably inhibited at concentrations higher than 4 μg/m L.Moreover,the hemolysis of purified aerolysin could be significantly reduced with 4g/m L pterostilbene2.Molecular docking and dynamics simulations were carried out to analyze the binding sites and binding modes of pterostilbene and aerolysin.We found that pterostilbene could bind to domain 3 of aerolysin by π-alkyl.The potential binding sites of were Val189 and Ile302 by calculation of binding free energy.3.According to the results of quantitative PCR,pterostilbene at 16 μg/m L could significantly inhibit the transcription of quorum sensing encoding genes ahy I and ahy R,and aerolysin encoding gene aer A,thereby inhibiting the expression of aerolysin and the formation of biofilm.Then the main binding sites of pterostilbene and aerolysin were confirmed by site-directed residue mutagenesis and fluorescence quenching assays.According to the aerolysin oligomerization assay,heptamers of aerolysin was inhibited by addition of pterostilbene.4.To determine the inhibitory effect of pterostilbene on aerolysin-mediated cytotoxicity,LDH release and live/dead cell staining assays were performed.The results showed that pterostilbene could significantly reduce LDH release and cell death in A549 cells caused by aerolysin.In summary,pterostilbene inhibits the expression of aerolysin and biofilm formation by inhibiting the quorum sensing system.The activity of aerolysin was inhibited by inhibiting the assemble of aerolysin heptamer.It can protect A549 cells from aerolysin-mediated cell injury.The study demonstrated the mechanism of pterostilbene inhibits A.hydrophila aerolysin and biofilm,and provided a promising candidate for drug development anti-virulence strategies.