Study On The Rutin Inhibit Biofilm Formation Of Staphylococcus Xylosus Targeting IGPD

Staphylococcus xylosus(S.xylosus)is the main strains of coagulase-negative Staphylococcus in dairy cow mastitis.Its isolation rate is not only rising,but also has a strong ability of biofilm formation.The presence of biofilm can enhance the survival pressure of bacteria to cope with environmental changes,enhance bacterial resistance and cause persistent infection of the host.Therefore,how to interfere or eliminate the formation of bacterial biofilm has become a hot topic for researchers.Imidazole glycerophosphate dehydratase(IGPD)plays an important role in the formation of biofilm of S.xylosus.IGPD is one of the most important metalloenzymes in L-histidine synthesis pathway.It exists widely in the cells of lower plants and microorganisms,so it is considered as a potential drug target.Rutin is a flavonoid drug with many pharmacological activities,but whether it can interfere with the formation of biofilm of S.xylosus and the mechanism of intervention have not been reported.In this study,we investigated the effect of rutin on the biofilm formation of S.xylosus.Based on molecular docking,surface plasmon resonance and amino acid site-directed mutagenesis,the binding of rutin to some sites of IGPD was elucidated and its target was determined..We investigated the effects of rutin on the expression of his B gene,the expression of IGPD protein,the content of L-histidine and the activity of enzymes.The results are as follows:(1)The results of crystal violet staining and scanning electron microscopy showed that rutin at 0.8 mg/m L for 6,12 and 24 h could significantly interfere with the formation of biofilm of S.xylosus.(2)The optimal binding conformation of rutin to IGPD protein was determined by molecular docking technology.Ile27 and His36 were predicted to be the key amino acids for rutin to bind to IGPD.Root mean square deviation and interaction energy of protein-ligand complex were analyzed by molecular dynamics.The results showed that the binding of rutin to IGPD was stable and the interaction energy decreased continuously.(3)Surface plasmon resonance,enzyme activity analysis and amino acid point mutation technique were used to verify the binding sites of rutin to IGPD.The results showed that rutin could bind to IGPD and inhibit the activity of IGPD.Ile27 and His36 were binding sites of rutin to IGPD.(4)Western blot and q PCR were used to reveal the regulation of rutin on IGPD.The results showed that rutin could significantly reduce the transcription level of his B gene and the expressionof IGPD.At the same time,although the ability of his B gene deleted strains to biofilm was significantly reduced,rutin still had an intervention effect on the formation of biofilm of his B gene deleted strains of S.xylosus.The results showed that rutin could significantly reduce the expression of his B gene.Intervention of biofilm formation by affecting IGPD may exist,but other mechanisms need to be studied.(5)In addition,the determination of L-histidine content showed that rutin could significantly reduce the L-histidine content of S.xylosus,which showed that rutin could inhibit the formation of biofilm by reducing the L-histidine content.In summary,our study took the point of view of IGPD target as the starting point,reveals the mechanism of rutin intervention in the formation of biofilm of dairy cow mastitis S.xylosus from two levels of regulation and direct combination,which not only lays the foundation for solving the problem of dairy cow mastitis that can not be cured for a long time,but also provides a new idea for the research and development of IGPD inhibitors to control bacterial biofilm infection.