Study On The Inhibitory Effect Of Dryocrassine On Staphylococcus Aures VWbp

Staphylococcus aureus(S.aureus,SA)is the major pathogen of hospital-acquired and community-acquired infections with high pathogenic.As a result of the strong selective pressure caused by the large number of antibiotics and other antimicrobial agents,S.aureus have evolved a series of drug resistance strategies.The evolution and the spread of drug-resistant pathogens,especially the emergence of super-resistant strains,pose a severe challenge to the treatment of infectious diseases and human health.Therefore,it is imperative to find and develop new strategies for the treatment of bacterial infections.The strong pathogenicity of S.aureus mainly depend on their virulence factors.S.aureus secreted Von Willebrand Factor-binding Protein(vWbp)is an important virulence factor affecting the pathogenicity of S.aureus.The vWbp have the ability to convert blood or plasma fibrinogen into fibrin deposited on the surface of the bacteria to accelerate the coagulation process.At the same time,the the formed fibrin barrier can resist the removal of the host immune system prompting infection limitations to form abscess lesions and thrombosis.In addition,vWbp can also promote the adhesion of S.aureus on the catheter surface and abscess formation.Therefore,it is of great significance to choose vWbp as an anti-virulence target to carry out the study of related inhibitors.Antiviral compounds are distinct from traditional antimicrobial agents that do not affect the growth activity of bacteria itself,but mainly target virulence factors of S.aureus to reduce pathogenicity.They can enable the invasive bacteria to be in a susceptible state,thereby enhancing the immune clearance or antibiotic-mediated bactericidal activity.Dryocrassine is a kind of phloroglucinol derivative with a variety of pharmacological effects such as anthelmintic,antiviral,antitumor and so on.It can also be used to treat bacterial infection.But can it affect the pathogenicity of S.aureus by affecting vWbp activity is still unknown.Objective:In this study,we screened the effective inhibitor(Dryocrassine)of vWbp,the target of drug action,from 200 kinds of traditional Chinese medicine monomers in our laboratory,and studied the effect of the inhibitor on the activity of vWbp protein and the effect on catheter-related bloodstream infections in vitro and the mouse renal abscess model in vivo caused by S.aureus.Taking vWbp as a therapeutic target,Dryocrassine can provide theoretical basis for helping body to resist S.aureus infection by acting on vWbp.Methods:1.In this study,we constructed the overexpression vector vWbp-p ET15 b containing vWbp gene,induced vWbp expression by IPTG and purified the protein by Ni column affinity chromatography.2.Screening vWbp inhibitors: The inhibitor of vWbp with no obvious antibacterial activity was screened from the Chinese medicine monomers in laboratory by S.aureus tube coagulation test;The traditional Chinese medicine monomer with the best inhibitory effect was selected as the research target in the later experiment.3.By drawing the growth curve of S.aureus Newman under the action of Dryocrassine,studied the antibacterial or bactericidal activity of the traditional Chinese medicine;The expression levels of vWbp protein were investigated by immunoblotting in the presence of different concentrations of drugs;The effect of the traditional Chinese medicine on the activity of vWbp protein was further studied by using S.aureus tube coagulation test and agarose plate experiment.4.To study whether there is a direct interaction between the traditional Chinese medicine monomer Dryocrassine and vWbp by the Drug Affinity Responsive Target Stability assay and Thermal Shift Assay.5.The actual therapeutic effect of the traditional Chinese medicine Dryocrassine on S.aureus infection was studied by constructing the model of catheter-related bloodstream infections in vitro and the mouse renal abscess model in vivo caused by S.aureus.Results:1.After the double digestion of recombinant vector vWbp-p ET15 b,there were two bands at 1527 bp and 5700 bp around;The result of SDS-PAGE showed a distinct band at 66 k Da after induction by IPTG,and a single band appeared at 66 k Da by Ni column affinity chromatography.2.The vWbp tube coagulation test results showed that the positive control group had completely coagulated within 10 mins,while the Dryocrassine group can significantly prolong the clotting time to 4 hours.3.The growth curve of S.aureus in the presence of Dryocrassine(512 ?g/m L)was not significantly different from that of wild strain.The results of Western blot showed that the expression of vWbp protein did not change significantly with the increased concentration of Dryocrassine.4.The result of DARTS showed that the amount of vWbp protein on SDS-PAGE increased with the concentration of Dryocrassine increasing,followed the digestive effect of Protase E on vWbp was decreased.On the contrary,no dosing group,vWbp protein was digested completely.5.The results of the thermal shift assay showed that the Tm of vWbp was 11? higher than that of the untreated group,which is far greater than 2?(There is a strong interaction between vWbp and Dryocrassine when the difference is greater than 2?).6.The results of scanning electron microscopy showed that the S.aureus adhered to the surface of the catheter was significantly reduced,and the biofilm formation of fibrin was also reduced.7.The pathology studies of S.aureus infection in vivo mouse renal abscess model had shown significant abscess formation in wild S.aureus group.The abscess significantly reduced with no obvious abscess formation in vWbp knockdown group(?vWbp).The effect of the Dryocrassine group was almost similar to that of the ?vWbp group.Conclusions:1.Dryocrassine(512 ?g/m L)couldn't inhibit the growth of S.aureus and couldn't affect vWbp protein expression.2.Dryocrassine had a significant inhibitory effect on the coagulation activity of S.aureus vWbp in a dose-dependent manner.3.Dryocrassine could directly interact with vWbp by affecting its protein activity and enhancing thermal stability.4.Dryocrassine could significantly reduce the adhesion and retention of S.aureus on the surface of the catheter and the formation of fibrillar structures.5.Dryocrassine had a therapeutic effect on the model of renal abscess in mices infected with S.aureus in vivo.6.Dryocrassine can reduce the activity of S.aureus by inhibiting vWbp activity.