Study On The Effect Of Aspirin On Biofilm Formation Of Staphylococcus Xylosus By ITRAQ

Dairy Mastitis is not only harmful to dairy industry,but also one of the diseases that lead to cow elimination.The infection of coagulase negative Staphylococcus like Staphylococcus xylosus is on the rise,which hinders the future development of dairy industry.However,CNS has traditionally been considered a minor mastitis-causing pathogen.In recent years,the prevalence of CNS-associated IMI has increased in several regions of the globe.And Staphylococcus xylosus can form a mature biofilm(biofilm,BF),and BF can adhere to each other or adhere to the surface of bioactive or inactive objects.Furthermore,biofilms offer protection against hostile environments,such as the immune response and drug resistance.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 work.Aspirin(acetylsalicylic acid)is a very popular antipyretic,anti-inflammatory,and analgesic that is the most common active component of non-steroidal anti-inflammatory drugs.Additionally,it also affects biofilm formation by various microorganisms,including Candida albicans,Staphylococcus epidermidis,Escherichia coli,and Pseudomonas aeruginosa.However,the study of aspirin inhibiting biofilm formation of S.xylosus has not been found.For seek its mechanism that aspirin inhibits the biofilm formation of Staphylococcus xylosus,so in this study,we used iTRAQ technology to screen for differential proteins formed by the biofilm of Staphylococcus xylosus with 1/2 MIC(0.625 mg/mL)aspirin and search for possible protein targets of aspirin.On the basis of this,we further studied whether the imidazole glycerol phosphate dehydratase is the target of aspirin.The main results of this study are as follows:(1)Using crystal violet staining and scanning electron microscopy,subinhibitory concentrations of aspirin on biofilm formation of staphylococcus xylosus is studied.The results show that 1/2 MIC(0.625 mg/mL),1/4 MIC(0.3125 mg/mL),1/8 MIC(0.15625 mg/mL)and 1/16 MIC(0.078125 mg/mL)significantly reduced the biofilm formation of Staphylococcus xylosus and caused the disappearance of the three-dimensional structure of the biofilm.It indicated that aspirin can inhibit the biofilm formation of Staphylococcus xylosus.(2)By iTRAQ,we screen the differentially expressed protein according to the ratio >1.2 or <0.8(p <0.05).178 differentially expressed proteins were screened and identified,67(37.6%)of which up-regulated and 111(62.4%)down-regulated.In the above proteins,data analysis can be used to produce important proteins that associated with biofilm formation and the metabolic process of bacteria,including: imidazoleglycerol-phosphate-dehydratase(HisB,A0A068E9J3),histidine ammonia-lyase(huth,A0A060MSD4),imidazolonepropionase(hutI,A0A068E2P9),urocanate hydratase(hutu,A0A068E633),glutamine synthetase(GlnA,A0A068E8L1),glyceraldehyde-3-phosphate dehydrogenase(GAPDH,A0A060MH08),phosphoglucomutase(PGM,C6ZDJ7),Ketol-acid-reductoisomerase(KARI,A0A068E468),3-isopropylmalate-dehydrogenase(LeuB,A0A068E3H6)and isocitrate dehydrogenase(IDH,A0A090K7P3).Gene ontology analysis indicated the majority of the differentially expressed proteins enrichment in metabolic processes.We then used the Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway database to analyze a large number of differentially expressed proteins and identified genes involved in biosynthesis of amino acids pathway,carbon metabolism(pentose phosphate and glycolytic pathways,tricarboxylic acid cycle)and nitrogen metabolism(histidine metabolism).These novel proteins represent candidate targets in aspirin-mediated inhibition of S.xylosus biofilm formation at sub-MIC levels.In the above differential proteins,the imidazoleglycerol phosphate dehydratase is involved in the histidine biosynthesis in nitrogen metabolism,and histidine has a significant effect on the biofilm formation.Therefore,this study suggests that the imidazoleglycerol phosphate dehydratase may be the target of aspirin.At the same time,it was used to verify that there is no interaction between aspirin and imidazoleglycerol phosphate dehydratase in vitro by biacore.In a word,it indicated that aspirin can clearly inhibit biofilm formation of Staphylococcus xylosus,and aspirin also affects nitrogen metabolism and carbon metabolism.Although IGPD plays an important role in histidine biosynthesis,and it can significantly affect biofilm formation.However,the interaction between aspirin and imidazoleglycerol phosphate dehydratase did not bind in vitro by Biacore,indicating that imidazolidase dehydratase is not the target of aspirin.