Purpose: The purpose of this study was to define the transcriptional response of MRSA JE2 to osmotic stress and elucidate the role of the stress response regulator σB using RNA-Seq.
Methods: A nonpolar transposon was used to disrupt transcription of σB encoded by rpoF. The minimum inhibitory concentration (MIC) of NaCl was defined for wildtype and rpoF::TnT. RNA was extracted from early log phase (OD600 = 0.4) wildtype cells with and without exposure to 10% NaCl and rpoF::TnT cells exposed to 10% NaCl. RNA-Seq was performed on the Illumina HiSeq 2500 platform in triplicate. Reads were normalized and mapped to the MRSA FPR3757 genome using Tophat (v 2.0.11). The EdgeR package in R (v. 3.1.1) was used to identify differential expression (adj. P-value < 0.05) between wildtype cells with and without exposure to NaCL and wildtype and rpoF::TnT cells exposed to NaCl.
Results: The MIC for wildtype and rpoF::TnT exposed to NaCl was 13.8% and 9.4%, respectively. A total of 68 genes were differentially expressed when wildtype cells were exposed to NaCl, including up-regulation of virulence genes asp23 and rpoF. Comparison of wildtype and rpoF::TnT exposed to NaCl identified 371 differentially expressed genes and 16 ncRNAs. Virulence regulators agr and sar were upregulated in rpoF::TnT, while antibiotic resistance genes femA, epiF, and epiP were downregulated.
Significance: This study defines the global transcriptome of MRSA during osmotic stress and increases understanding of the σB regulatory network in S. aureus. Understanding stress response networks in MRSA may lead to the development of better control strategies and novel drug targets.