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

Viralkumar V. Panchal, Caitlin Griffiths, Hamed Mosaei, Bohdan Bilyk, Joshua A.F. Sutton, Oliver T. Carnell, David P. Hornby, Jeffrey Green, Jamie K. Hobbs, William L. Kelley, Nikolay Zenkin, Simon J. Foster*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

9 Citations (Scopus)

Abstract

Most clinical MRSA (methicillin-resistant S. aureus) isolates exhibit low-level â-lactam resistance (oxacillin MIC 2-4 μg/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 μg/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 β) or rpoC (RNA polymerase subunit β') 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.

Original languageEnglish
Article numbere1008672
JournalPLoS Pathogens
Volume16
Issue number7
DOIs
Publication statusPublished - Jul 2020
Externally publishedYes

Bibliographical note

Funding Information:
This work was supported by the 2022 Futures Initiative, University of Sheffield; Wellcome Trust (212197/2/18/2, 102851/Z/13/Z and 217189/ Z/19/Z; https://wellcome.ac.uk/, SJF, JKH, NZ), Medical Research Council (MR/T000740/1; https:// mrc.ukri.org/, NZ), Engineering and Physical Sciences Research Council (EP/T002778/1; https:// epsrc.ukri.org/; SJF, JKH, NZ) and the Swiss National Science Foundation (WLK 310030- 146540 and 10030-166611). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Publisher Copyright:
© 2020 Panchal et al.

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