Study On The Inhibitory Effect And Mechanism Of Galangin On Staphylococcus Aureus VWbp

Staphylococcus aureus(S.aureus)is a common gram-positive pathogen that is highly pathogenic and causes various infections such as soft tissue infections,endocarditis,pneumonia,and osteomyelitis.In recent years,the emergence and spread of multidrug resistance in S.aureus and the lag in antibacterial drugs have made clinical treatment of bacterial infections difficult,and necessitate the development of new antiinfective strategies to reduce increasingly serious infections with resistant bacteria.S.aureus causes diseases via a large group of virulence factors,and these virulence factors are not necessary for bacterial growth.Therefore,the pathogenicity of S.aureus can be effectively reduced without affecting the growth of the bacteria and reducing the occurrence of drug resistance by inhibiting the virulence factors of the bacteria.vWbp is a coagulase secreted by S.aureus,as an important virulence factor that affects the pathogenicity of S.aureus,vWbp bind to prothrombin in blood,leading to the formation of active staphylothrombin,which induces the generation of fibrin that may induce coagulation and reduce granulocyte activation and phagocytosis,promote the survival of bacteria.Therefore,the development of antivirulent drugs that targeting vWbp may be a promising approach to cope with S.aureus-related infections.Galangin is a kind of flavonoids which has anti-tumor,anti-inflammatory,antiaging and other pharmacological activities.It also has anti-viral and anti-bacterial activities.But whether it can treat the infections caused by S.aureus by inhibiting the vWbp is still unknown.Objective:In this experiment,the virulence factor vWbp secreted by S.aureus was used as the research target.We screened galangin from different Chinese herbal extracts,which could inhibit the coagulation activity of vWbp.This study investigated the inhibitory effect and mechanism of galangin on vWbp,and studied the actual therapeutic effect of galangin in vivo by constructing a mouse model of S.aureus pneumonia.It provides a new theoretical basis for further development of therapeutic drugs to treat the infections of S.aureus.Methods:1.The vWbp protein was induced and expressed by the existing vector vWbppET15 b in the laboratory,and the protein was separated and purified by Ni-NTA affinity chromatography.2.The purified protein was used as the research object,and we screened inhibitors of vWbp from different Chinese herbal extracts by the tube coagulase activity assay.3.Growth curve experiment and Western Blot experiment were used to study the effect of growth of S.aureus Newman and the expression of vWbp protein incubated with galangin.4.The tube and plate coagulase activity assays were used to study the inhibitory effect of different concentrations of galangin on the coagulation activity of vWbp protein.5.The thermal shift assay and bio-layer interferometry assay were used to investigate whether galangin binds specifically to vWbp protein.6.The binding mode of galangin and vWbp protein was simulated by molecular docking and molecular dynamics experiments,and the key sites of vWbp protein binding to galangin were speculated by point mutation experiment of amino acids combined with fluorescence quenching and tube coagulation assays.7.Constructing a mouse model of S.aureus pneumonia,the therapeutic effect of galangin on pneumonia in mice was studied through the survival rate curves,the numbers of lung colonies and the pathological changes of lung tissues.Results:1.After identification by SDS-PAGE,we successfully induced the expression of vWbp protein with a molecular weight of 66 kDa.The high purity vWbp protein was obtained after Ni-NTA affinity chromatography.2.The tube coagulase activity assay showed that galangin can significantly inhibit coagulation activity of vWbp and prolong coagulation time.3.Growth curve analysis and Western Blot experiment showed that galangin had no effect on the growth of S.aureus Newman and the expression of vWbp protein.4.The tube and plate coagulase activity assays found that galangin has a significant inhibitory effect on coagulation activity of vWbp protein,and the inhibitory effect increases with increasing concentrations.5.The thermal shift assay showed that galangin can increase the thermal stability of vWbp protein,indicating that they are specifically bound;The bio-layer interferometry assay showed that the changes in the layer thickness of the biosensor combined with the vWbp protein after the binding of galangin,which indicated that galangin binds strongly with vWbp.6.Molecular dynamic simulations and verification experiments of mutant proteins revealed that the Trp-64 and Leu-69 residues may be necessary for the binding of galangin to vWbp.7.In vivo experiments showed that the survival rate of mice were improved,the numbers of lung colonies were significantly reduced and the pathological changes in lung tissues were reduced after treatment of galangin,which showed that galangin has a good therapeutic effect on S.aureus pneumonia in mice.Conclusions:1.We successfully screened out the Chinese herbal extracts,galangin,without inhibiting the growth of S.aureus Newman,which significantly inhibited the clotting activity of vWbp in a dose-dependent manner.2.Galangin,as an effective inhibitor of S.aureus vWbp,can be firmly combined with vWbp.3.Trp-64 and Leu-69 residues of vWbp protein may be the key sites for galangin binding to vWbp.4.Galangin can reduce the virulence of S.aureus by inhibiting the activity of vWbp,and has a good therapeutic effect on S.aureus pneumonia in mice.