Base On Glutamine Synthetase Target To Investigate Mechanism Of Erythromycin Against Staphylococcus Xylosus Biofilm Formation

Cow mastitis on dairy industry not only is very serious harm,but also it is one of cow eliminated disease.Cow mastitis is caused by cow mastitis pathogenic bacteria such as Staphylococcus aureus,Streptococcus agalactiae,Escherichia coli.Besides these cow mastitis pathogenic bacteria,coagulase negative staphylococci such as Staphylococcus xylosus infection was a rising trend,which has become a new characteristic of cow mastitis.Staphylococcus xylosus produce multimeric complexes matrix surrounded itself,and adhere to the non active or living surface.Staphylococcus xylosus form a certain structural biofilm.Biofilm can resist host immune and antimicrobial.It is difficult to cure the infection caused by mastitis repeated attacks,and it result in persistent infection of cow mastitis.Staphylococcus xylosus infection has caused some scholar’s attention,and it has become a new research hotspot in the field of cow mastitis prevention and treatment.Therefore,all scholars both here and abroad generally focus the research on Staphylococcus xylosus biofilm control mechanism and new drug targets to improve cow mastitis prevention and treatment.Erythromycin is a macrolide drug,and it not only has inhibitory action to gram positive bacteria,such as Staphylococcus aureus,Streptococcus pyogenes,etc.,but also erythromycin can decrease biofilm formation of Streptococcus suis,Pseudomonas aeruginosa and Staphylococcus epidermidis,erythromycin can increase biofilm formation of Corynebacterium diphtheria and Staphylococcus epidermidis,erythromycin can not affect biofilm formation of Porphyromonas gingivalis and Corynebacterium urealyticum.In conclusion,due to the different bacterial species,the effects of erythromycin on biofilm are different.At present,there is no study about the effect of erythromycin on biofilm formation of staphylococcus xylosus,and there is also no study about its mechanism.Therefore,in this study,we investigated the effect of erythromycin on biofilm formation of staphylococcus xylosus,we carried out comparative proteomic analyses of cells at two different conditions(1/2 MIC of erythromycin treated cells and nontreated cells)to elucidate the protein target of erythromycin by i TRAQ technology.The differential expression proteins were analyzed by gene knockout technique to find new drug targets for control of staphylococcus xylosus biofilm infection.Base on differential protein expression analysis,we further research on glutamine synthetase whether is the targets of erythromycin against staphylococcus xylosus biofilm.Therefore,the glnA gene knockout strain prove that glutamine synthetase plays a role in biofilm formation and is erythromycin target.The main results are as follows:(1)By crystal violet staining,scanning electron microscopy and confocal laser scanning microscopy,subinhibitory concentrations of erythromycin on biofilm formation of staphylococcus xylosus is studied.The results show that 1/2MIC(0.8μg/mL),1/4MIC(0.4μg/m L),1/8MIC(0.2μg/mL)and 1/16MIC(0.1μg/m L)of erythromycin clearly decrease biofilm formation of Staphylococcus xylosus,viable bacteria,and biofilm structure.(2)By i TRAQ proteome quantitative analysis technology,we carried out comparative proteomic analyses of cells at two different conditions(1/2 MIC of erythromycin treated cells and nontreated cells)to screen the differentially expressed protein according to the ratio >2.0 or <0.5(p-value <0.05).On the basis of the two criteria,24 differentially expressed proteins were identified,4(16.7%)of which displayed increased and 20(83.3%)displayed decreased abundance.In this study,the expression of Preprotein translocase subunit Sec G(A0A060MLT7),Fmt B protein(A0A068E3T6),Gluconate permease(A0A068E1W8),Sodium/proline symporter(A0A068E9F7),Poly-gamma-glutamate synthase subunit Pgs B/Cap B(A0A068E9C1),Oligopeptide transport system permease protein Opp B(A0A060MF11)and Sodium/alanine symporter family protein(A0A068E6R7)changed and these proteins belong to membrane proteins and cell-surface proteins.These proteins might affect bacterial adhesion.Thus,these proteins might play a significant role in biofilm formation.In this study,the expression of Poly-gamma-glutamate synthase subunit Pgs B/Cap B(A0A068E9C1)changed.The protein is involved in capsule polysaccharide biosynthetic process.Biosynthesis of capsular polysaccharide affects biofilm formation.Erythromycin might affect the biosynthesis of capsule polysaccharide.In this study,the expression of Glutamine synthetase(A0A068E8L1)changed.The protein is involved in biosynthesis of glutamine.Subinhibitory concentrations of erythromycin can reduce the activity of glutamine synthetase,which leads to decrease glutamine biosynthesis.In conclusion,glutamine synthetase plays an important role in biofilm formation,and it can be directly combined with erythromycin.(2)Based on gene knockout and molecular docking methods,it is shown that glutamine synthetase not only plays a role in biofilm formation,but also acts as a target for erythromycin.The biofilm formation ability of staphylococcus xylosus glnA gene deletion strain was significantly reduced.However,when the culture medium was supplemented with glutamine,the biofilm formation ability of staphylococcus xylosus glnA gene deletion strain was significantly increased.Erythromycin reduced biofilm formation of wild strain,reduced glutamine synthetase activity and reduced glutamine biosynthesis,however erythromycin did not affect biofilm formation of glnA gene deletion strain,and it also failed to change glutamine synthetase biological activity and glutamine contents.Molecular docking results showed that erythromycin and glutamine synthetase were directly combined with the active groups,which resulted in glutamine synthetase activity reduction.The major binding site are 260 bit histidine(His),261 bit leucine(Leu),346 bit arginine(ARG),and 347 phenylalanine(PHE).In conclusion,glutamine synthetase plays an important role in biofilm formation,and it can be directly combined with erythromycin.In summary,the study results show that erythromycin clearly decreases biofilm formation of Staphylococcus xylosus,and erythromycin also affects biosynthesis of membrane proteins,cell surface proteins and capsular polysaccharides.Glutamine synthetase is involved in glutamine biosynthesis and plays an important role in biofilm formation.By reducing glutamine synthetase activity,erythromycin can reduce the glutamine biosynthesis and interfere with biofilm formation.Glutamine synthetase is target of erythromycin.