Study On The Inhibitory Effect And Mechanism Of Isovanillic Acid On Staphylococcus Aures VWbp

Objective: To investigate the inhibitory effect of isovanillic acid on the activity of the von Willebrand factor-binding protein(vWbp)secreted by Staphylococcus aureus(S.aureus)and to investigate the mechanism of action behind it.Methods: Through MIC and growth curve experiments,evaluate the effect of isovanillic acid on the growth state of S.aureus;In vitro test tube coagulation and blood plate experiments to verify the inhibitory effect of isovanillic acid on vWbp activity;Western blot to evaluate the effect of isovanillic acid on vWbp protein expression;Thermal shift assay verify whether isovanillic acid has a direct interaction with vWbp;Molecular docking and point mutation to find the key amino acid residues of isovanillic acid and vWbp binding.In addition,the protective and therapeutic effects of isovanillic acid on mice were evaluated by in vivo animal experiments.Results: 1.The growth curve results showed that isovanillic acid did not affect bacterial growth at a concentration of 128 μg/m L.2.The test tube coagulation and blood plate experiments show that isovanillic acid can inhibit the activity of vWbp.3.Western blot and thermal shift assay proved that isovanillic acid does not affect the expression of vWbp protein,and it has a direct interaction with vWbp.4.Molecular docking and point mutation experiments show that isovanillic acid may inhibit coagulation through the combination of residues GLN-383 and TYR-386 with vWbp.5.The results of animal experiments show that isovanillic acid can improve the survival rate of pneumonia mice,reduce the pathological changes of lung tissue,and reduce the bacterial load in the lungs.In addition,the combination of isovanillic acid and vancomycin was more effective than vancomycin alone.Conclusion: 1.Isovanillic acid is a direct inhibitor of coagulase vWbp,which inhibits the activity of vWbp in a concentration-dependent manner.2.Isovanillic acid may directly bind to vWbp through residues GLN-383 and TYR-386,and play an inhibitory role in the way of reducing protein stability.3.Isovanillic acid reduces the virulence of S.aureus and protects mice from lethal pneumonia.In addition,the combination of isovanillic acid and vancomycin can enhance the anti-infective effect of the latter.