Functional Study Of Staphylococcus Aureus Transcription Factors SarS,SarV And VraR In Bovine Mastitis

The healthy breeding of bovine provides a new solution for the sustainable development of animal husbandry in China.In the traditional breeding industry,the unstandardized feeding and management practices aggravate the breeding environment,which leads to the increasing incidence of bovine mastitis.Bovine mastitis is the most costly disease for the dairy industry.In addition,bovine mastitis can affect human health due to the antibiotic residue in milk and bacterial antibiotic resistance.At present,antibiotics are still the main way to treat bovine mastitis.However,due to the abuse of antibiotics,multiple resistant strains are constantly emerging and lead to the low cure rate of bovine mastitis.Therefore,it is necessary to study the pathogenic bacteria in bovine mastitis.Staphylococcus aureus is one of the main pathogens causing bovine mastitis owning to its highly pathogenic nature,strong environmental adaptability and antibiotic tolerance.In our previous study in the laboratory,transcriptome data of S.aureus strain BA01611 in the presence or absence of oxacillin treatment have showed the differential expression of 3 genes sarS,sarV and vraR.In this study,we first validated the transcriptome results by qRT-PCR.Then,we knocked out the sarS,sarV and vraR genes in order to investigate their functions.Finally,we also attempted to find some downstream genes which were regulated by vraR.The results were shown as follows:1.Using the qRT-PCR method,our results showed that the expression levels of sarS,sarV and vraR have increased by 0.56,0.79,and 15.45 times after treatment with oxacillin,respectively.Thus,our qRT-PCR results confirmed the transcriptome results.2.In this study,the sarS,sarV,and vraR knockout bacteria BA01611-?sarS,BA01611-?sarV,BA01611-?vraR were successfully constructed using the method of homologous recombination using a E.coli and S.aureus shuttle vector pKZ2.3.This study found that SarS was not related to the resistance of S.aureus to oxacillin,vancomycin and teicoplanin,while SarV and VraR increased the resistance of S.aureus to oxacillin.In addition,SarS,SarV and VraR were not related to the resistance of S.aureus to vancomycin.However,SarV and VraR increased the resistance of S.aureus to the teicoplanin.Moveover,SarS weakened the biofilm formation ability of S.aureus,while SarV and VraR did not affect the biofilm formation of S.aureus.SarS,SarV and VraR weakened the hemolysis of bacterias;while their effects on the ability of S.aureus to coagulate plasma was not significantly changed.4.Real-time PCR was used to explore the downstream genes which were regulated by vraR.There were no differences of the expression of mecA among BA01611?WT?,BA01611-?vraR and BA01611-?vraR-complement strains.On the other hand,8 other genes were regulated by vraR,including vraX,vraC,vraS,msrR,murA2,mgt,SAOUHSC₀1761,and SAOUHSC₀2101.In summary,this study used gene knockout method to explore the function of SarS,SarV and VraR of S.aureus.In addition,this study also found that 8 downstream genes were regulated by vraR.