| Increasing cases of infections caused by methicillin-resistant Staphylococcus aureus (MRS A) strains in healthy individuals have raised concerns worldwide. MRS A strains are resistant to almost the entire family of P-lactam antibiotics due to the acquisition of an extra penicillin-binding protein PBP2a, which exhibits a remarkably lower affinity for β-lactams than PBP2. Besides the expression of PBP2a, which is responsible for higher level of P-lactam resistance, several additional native genes have been identified as being essential to the full expression of oxacillin resistance. Most of these genes, called fem (factor essential for methicillin resistance) or aux (auxiliary) genes, have a direct or indirect role in peptidoglycan metabolism/structure.Studies have shown that spoVG is involved in oxacillin resistance, while the regulatory mechanism remains elusive. Here, we have found that SpoVG plays a positive role in oxacillin resistance through promoting cell wall synthesis and inhibiting cell wall degradation in MRS A strain N315. Deletion of spoVG in strain N315 led to a significant decrease in oxacillin resistance and ceftizoxime resistance. Autolysis is commonly associated with the killing mechanism of penicillin and P-lactams. We also found that the spoVG mutant had a dramatic increase in Triton X-100-induced autolytic activity.Many genes involved in cell wall synthesis or cell wall degradation can influence methicillin resistance or autolysis. To determine whether the expression of these genes was altered in the spoVG mutant, we performed real-time quantitative reverse transcription-PCR (qRT-PCR) to examine the mRNA levels of 17 potential target genes, among which 8 genes were associated with cell wall synthesis or oxacillin resistance (femA, mecA, blaZ, pbpl, pbp2, pbp3, pbp4, and vraS) and 9 genes were implicated in cell wall degradation (e.g., atl, lytN, lrgA, arlS, slel, lytS, cidA, lytM, and sarA). qRT-PCR revealed that the expression of 8 genes related to cell wall metabolism or oxacillin resistance was altered in the spoVG mutant. The expression of femA, mecA, and blaZ involved in cell wall synthesis or methicillin resistance were decreased. The mRNA level of lytN (coding for murein hydrolase) was significantly increased in the spoVG mutant, while the transcript levels of IrgA (coding for antiholin), slel (coding for murein hydrolase), and regulatory loci (lytS and arlS) were significantly reduced in the spoVG mutant. Electrophoretic mobility shift assay indicated that SpoVG can directly bind to the putative promoter regions of lytN,femA, and lytSR (the two-component system). In addition, we have also found at least two 5’-TAATTT/A-3’ sequences existing in the putative promoter regions of lytN,femA, and lytSR.In summary, our findings suggest a molecular mechanism in which SpoVG modulates oxacillin resistance by regulating cell wall metabolism in the presence of an intact mecA. While the molecular mechanism of mecA expression regulated by mecR1-mecl has been widely recognized, the exact mechanism of antibiotic resistance caused by auxiliary factors remains elusive. Our study reveals a new auxiliary factor and provides a better understanding of the regulatory pathway of fem/aux genes. |
