Global Regulator Agr Regulates Vancomycin Resistance Of Staphylococcus Aureus Through Vra

Objectives The accessory gene regulator is a global regulator in Staphylococcus aureus,which can regulate cell metabolism and the expression of virulence factors.It has been reported that the function of accessory gene regulator(agr)is missing in clinical isolates of heterogeneous vancomycin-mediated S.aureus(hVISA)and vancomycin-mediated S.aureus(VISA),and agr knockout strains are more likely to develop into vancomycin-resistant strains than wild type,but the exact reason is still unclear.The vancomycin resistance-related regulatory system(vraSR)is a well-defined two component regulate system related to vancomycin resistance in Staphylococcus aureus.We speculate that agr may participate in the regulation of vancomycin resistance in Staphylococcus aureus through the vraSR two component regulate system.This study will test this hypothesis.Methods The heterogeneous vancomycin-mediated Staphylococcus aureus Mμ 3 strain was selected to construct the agr gene knockout strain(Δ agr)and gene complementation strain(CΔagr)by gene knockout and complementation techniques.The MIC value of vancomycin was detected by 96-well micro broth dilution method.The expression level of vraSR gene was tested by qRT-PCR method.β-galactosidase reporter gene experiment was performed to detect the regulatory effect of Agr on vraSR gene.Electrophoretic mobility shift assay indicated the AgrA protein can directly binds to the promoter region of vraSR.EMSA was performed with the truncated probes of vraSR to further identify the AgrA binding regions.Results Compared with the wild strain of Staphylococcus aureus M μ3,the vancomycin MIC of Mμ3-Δagr strain increased from 1.0 mg/L to 1.6 mg/L.The expression level of vraSR gene in Mμ 3-Δagr strain was 2.7 times higher than that in Mμ3 wild type strain(P<0.01),and Mμ3-CΔagr could restore the expression of vraSR gene.In the β-galactosidase reporter gene experiment,the β-galactosidase activity of Mμ 3Δagr+pOSl-vra strain was significantly higher than that of Mμ3+pOSl-vra strain(P<0.001),indicating that Agr negatively regulates vraSR gene expression.EMSA experiments show that AgrA protein can specifically bind to the promoter region of the vraSR gene,and the 63bp region at the 3’end of the promoter is the binding site.Conclusions In the heterogeneous vancomycin-mediated Staphylococcus aureus(hVISA)M μ3 strain,Agr down-regulates the expression of the vraSR gene by binding to the 3’end 63bp region of the promoter of the vraSR gene to inhibit the resistance of Staphylococcus aureus to vancomycin.