Evolving MRSA: High-level β-lactam resistance in Staphylococcus aureus is associated with RNA Polymerase alterations and fine tuning of gene expression

Most clinical MRSA (methicillin-resistant S. aureus) isolates exhibit low-level beta -lactam resistance (oxacillin MIC 2-4 mu g/ml) due to the acquisition of a novel penicillin binding protein (PBP2A), encoded by mecA. However, strains can evolve high-level resistance (oxacillin MIC >= 256 mu g/ml) by an unknown mechanism. Here we have developed a robust system to explore the basis of the evolution of high-level resistance by inserting mecA into the chromosome of the methicillin-sensitive S. aureus SH1000. Low-level mecA-dependent oxacillin resistance was associated with increased expression of anaerobic respiratory and fermentative genes. High-level resistant derivatives had acquired mutations in either rpoB (RNA polymerase subunit beta) or rpoC (RNA polymerase subunit beta') and these mutations were shown to be responsible for the observed resistance phenotype. Analysis of rpoB and rpoC mutants revealed decreased growth rates in the absence of antibiotic, and alterations to, transcription elongation. The rpoB and rpoC mutations resulted in decreased expression to parental levels, of anaerobic respiratory and fermentative genes and specific upregulation of 11 genes including mecA. There was however no direct correlation between resistance and the amount of PBP2A. A mutational analysis of the differentially expressed genes revealed that a member of the S. aureus Type VII secretion system is required for high level resistance. Interestingly, the genomes of two of the high level resistant evolved strains also contained missense mutations in this same locus. Finally, the set of genetically matched strains revealed that high level antibiotic resistance does not incur a significant fitness cost during pathogenesis. Our analysis demonstrates the complex interplay between antibiotic resistance mechanisms and core cell physiology, providing new insight into how such important resistance properties evolve. Author summary Methicillin resistant Staphylococcus aureus (MRSA) places a great burden on human healthcare systems. Resistance is mediated by the acquisition of a non-native penicillin-binding protein 2A (PBP2A), encoded by mecA. MRSA strains are resistant to virtually all beta -lactam antibiotics, and can shift from being low- to high-level resistant. Prior studies have revealed the involvement of components of the core genome in increased resistance, but the underlying mechanism is still unknown. In this study, we have found that increased resistance is associated with mutations in either rpoB (RNA polymerase subunit beta) or rpoC (RNA polymerase subunit beta') resulting in slower growth and elevated levels of PBP2A. Furthermore, transcript profiling revealed that insertion of mecA triggered metabolic imbalance by altering anaerobic and fermentative gene expression, accompanied by low-level resistance whereas, acquisition of rpoB and rpoC mutations reversed gene expression to wild-type level and enabled cells to become highly-resistant. The mutations also affected RNA polymerase activity. A set of matched strains revealed that changes in antibiotic resistance levels do not have a significant cost in terms of pathogenic potential. Our study reveals a novel effect of mecA acquisition on central metabolism and sheds light on potential pathways essential for high-level resistance.