VraCP Regulates Cell Wall Metabolism And Antibiotic Resistance In Vancomycin-intermediate Staphylococcus Aureus Strain Mu50

The infection caused by methicillin-resistant Staphylococcus aureus(MRSA)is a global threat to public health.Vancomycin is a glycopeptide antibiotic used for the treatment of clinical MRSA infections,whereas vancomycin-intermediate Staphylococcus aureus(VISA)is increasingly being reported as associated with treatment failure.However,the molecular mechanism underlying the generation of VISA is not yet understood.Previous studies have shown that vraC and vraP may be involved in vancomycin resistance,although the molecular mechanism remains elusive.In this study,we defined the role of the vraC,vraP and vraCP in cell wall metabolism and antibiotic resistance in clinical VISA strains Mu50.Results indicated that vraC and vraP are co-transcribed,induced by vancomycin in vraSR-dependent manner.And VraP can form stable complex with VraC to keeping their own steadily.The vraC,vraP and vraCP mutants exhibited thinner cell walls and increased susceptibility to the cell wall-associated antibiotics compared with the WT strain.Especially,mutants showed significant decreases in the vancomycin MIC(from 8 to 2 mg/L),and became known as vancomycin sensitive S.aureus(VSSA).Consistent with these phenotypes,RT-qPCR revealed downregulated transcription of glyS,sgtB,ddl and alr2,which are involved in cell wall biosynthesis.Moreover,the transcription of cell wall hydrolysis genes,including sceD,lytM and isaA,were significantly downregulated,supporting the finding that mutants exhibited reduced autolysis rates.EMSA(electrophoretic mobility shify assay)confirmed that both VraC and VraCP can directly bind to the sceD,lytM and isaA promoter regions containing the consensus sequence(5’-TTGTAAN2AN3TGTAA-3’),which is crucial for the binding of VraCP with target genes.GFP-reporter assays further revealed VraC and VraCP can enhance promoter activity of sceD to positively regulate its expression.In conclusion,our data revealed the molecular mechanisms in which vraC,vraP and vraCP contribute to cell wall metabolism and cell wall-associated antibiotics in S.aureus Mu50 strain.Deletion of vraC,vraP and vraCP leads to a series of phenotypic alterations,including increased susceptibility to the cell wall-associated antibiotics,reduced cell wall thickness and decreased autolysis.Furthermore,differential expression genes involved in cell wall metabolism were also detected,supporting phenotypic differences as hereinbefore described.These results deepen our knowledge of the promotion of vancomycin resistance in Mu50 and provide new insights into the generation and development of VISA strains.