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

Cow mastitis is a serious hazard to the dairy industry,especially in recent clinical cases.The economic losses caused by recessive cow mastitis are increasing year by year,plaguing the further development of the dairy industry.Staphylococcus xylosus?S.xylosus?is an important opportunistic pathogen of recessive cow mastitis.When it forms a biofilm?BF?,its drug resistance is obviously enhanced and it also avoids host immune clearance.A derivative result is persistent infection among cows and it will be hardly healed.Therefore,the research on the regulation mechanism of bacterial biofilms and the search for new drug targets have become one of foucs in current research.Cefquinome is the fourth-generation cephalosporin antibiotic for only animal using with wide antibacterial spectrum and has strong antibacterial activity.It has been reported that cefquinome can inhibit the biofilm formation of Staphylococcus aureus,but S.xylosus has not been reported.For seeking its mechanism that cefquinome inhibits the biofilm formation of S.xylosus,we used iTRAQ technology to screen out differential proteins formed by the biofilm of S.xylosus with 1/2MIC?0.125?g/mL?cefquinome and search for possible protein targets of cefquinome.Through the molecular interaction technology,we found the imidazole glycerophosphate dehydratase?IGPD?as a potential target.On the basis of this,the hisB gene mutant and complement strains were obtained by gene knockout and complementation techniques,it demonstrates that IGPD plays an important role in biofilm formation.Finally,we used molecular docking,point mutation technology,and molecular interaction technology to further confirm it.The main results of this study are as follows:?1?Using crystal violet staining and scanning electron microscopy to detect the ability of biofilm formation,the results showed:1/2 MIC?0.125?g/mL?,1/4 MIC?0.0625?g/mL?,and 1/8MIC?0.03125?g/mL?cefquinome significantly reduced the biofilm formation of S.xylosus and caused the disappearance of the three-dimensional structure of the biofilm.It indicated that cefquinome can inhibit the biofilm formation of S.xylosus.?2?Based on iTRAQ technology,1/2 MIC?0.125?g/mL?cefquinome was used to quantify the differential proteomes of S.xylosus biofilms,revealing the protein target of cefquinome.Based on the differential protein selection criteria,ie ratio>1.2 or<0.8?p-value<0.05?,164 differentially expressed proteins were screened and identified.The GO enrichment and KEGG pathway database analysis showed that differentially expressed proteins was connected with amino acid metabolism?histidine metabolism?,carbon metabolism and nitrogen metabolism pathways.These proteins may be candidates for cefquinome to inhibit the biofilm formation of S.xylosus.Among the above differential proteins,five proteins were involved in the histidine metabolic pathway,?A0A068E2P9?Imidazolonepropionase?hutI?,?A0A068E547?Formimidoylglutamate,?A0A068E633?Urocanatehydratase?hutU?,?A0A068E4P8?1-?5-phosphoribosyl?-5-[?5-phosphoribosylamino?methylideneamino]imidazole-4-carboxamideisomerase?HisA?,?A0A068E9J3?Imidazoleglycerol-phosphate dehydratase?IGPD,down-regulated 3.97 fold?were significantly down-regulated.The IGPD protein and its coding gene hisB were quantitatively analyzed by Western-blot and Real-time PCR experiments.It was found that the IGPD protein expression and hisB gene level were consistent with the iTRAQ data,which indicated that cefquinome may be down-regulated IGPD protein level.So in this study,we speculated that IGPD may be the target of cefquinome.?3?Based on gene knockout and complement techniques to detect the founction of IGPD.It was found that the ability of the hisB gene-deleted strain to form a biofilm(OD_595nm=0.896)was significantly lower than that of the wild strain(OD_595nm=2.231)?p<0.05?;The complement strain biofilm formation ability(OD_595nm=1.347)was restored compared with the gene-deleted strain?p<0.05?,which indicated that IGPD plays an important role in the formation of the biofilm of S.xylosus.?4?The effect of histidine biosynthesis on biofilm formation was investigated by histidine content assays.The results showed that 1/2 MIC and 1/4 MIC cefquinome reduced the histidine content of the wild-type strain OD_476nm=0.188,0.389,p<0.05(Control group histidine OD_476nm=0.588),and were able to reduce it's biofilm formation;however,1/2 MIC and 1/4 MIC cefquinome had no significant effect on the histidine content of the hisB gene-deleted strain OD_476nm=0.122,0.125,p<0.05(Control histidine OD_476nm=0.131),and no significant effect on it's biofilm formation;when adding histidine?0.5 mM,1 mM,5 mM?at different concentrations to hisB gene-deleted and 1/2 MIC?0.125?g/mL?cefquinome treated wild-type strains,the ability of the biofilm formation was significantly increased?p<0.05?.The results indicated that histidine biosynthesis plays an important role in biofilm formation,and cefquinome inhibited biofilm formation by reducing the biosynthesis of histidine.?5?By crystal violet staining and IGPD enzyme activity assay to explore whether IGPD is the target of cefquinome.The results showed that 1/2 MIC?0.125?g/mL?cefquinome reduced the biofilm formation of wild-type strains(OD_595nm=0.623,p<0.05),but had no significant effect on biofilm formation of hisB gene-deleted strains(OD_595nm=0.601,p>0.05);1/2 MIC?0.125?g/mL?and 1/4 MIC?0.0625?g/mL?of cefquinome reduced IGPD enzyme activity(OD_{280 nm}=0.183,0.376,p<0.05),while the deletion strain was not detected IGPD activity.The results suggested that cefquinome reduces biofilm formation by decreasing IGPD activity,so we considered IGPD as the target of cefquinome.?6?Based on molecular docking technology,BIAcore technology and IGPD enzyme activity assays to predict and prove the possible combination of cefquinome and IGPD.Molecular docking results showed that cefquinome and IGPD enzyme can directly bind,histidine 36 and histidine 63were major binding sites;1mM cefquinome was detected by BIAcore test;it had a strong binding force,and RU value reached 73;the kinetic curve showed a good gradient change,which showed that IGPD can be combined with cefquinome closely;Through IGPD histidine 36 and histidine 63group detection of the enzyme activity of mutated proteins,it revealed that 1/2 MIC?0.125?g/mL?of cefquinome had no significant change in the activity of the mutant protein compared to the control group?p>0.05?,which indicated that these two sites were the major binding sites.In summary,IGPD plays an important role in the biofilm formation of S.xylosus;cefquinome not only inhibits IGPD enzyme activity and reduces histidine synthesis,but on the other hand it also associates with the histidine 36 and histidine 63 directly binds to IGPD,it plays a role in the biofilm formation of S.xylosus.In conclusion,IGPD is one of the targets cefquinome that inhibiting biofilm formation of S.xylosus.