Staphylococcus aureus is an opportunistic pathogen that may cause severe disease including sepsis, endocarditis, and pneumonia. Methicillin-resistant S. aureus (MRSA) is emerging as a major cause of hospital-acquired infections, and these isolates often acquire resistance to multiple antibiotics limiting treatment options. Current last-line options for treatment of MRSA include vancomycin, linezolid, and tigecycline however, S. aureus strains with resistance to last-line antibiotics have emerged. S. aureus with vancomycin intermediate susceptibility (VISA), defined as a vancomycin minimal inhibitory concentration between 4 and 8 µg/mL, is a major cause of vancomycin treatment failure. The molecular basis of vancomycin-intermediate susceptibility is not fully understood although phenotypic features such as increased cell wall thickness have been linked to VISA strains,.
Bacteria respond to many acute external stresses through regulatory non-coding RNAs. Cis-regulating ncRNAs control transcription and translation of genes within the same locus and include attenuators that regulate transcription by promoting early termination in response to specific ligands or ribosomal pausing. Previously, the Term-seq method was developed to detect RNA termination sites and has been used to identify antibiotic responsive attentuators in Bacillus subtilis (Dar et al.,2016).
In the present study, we have used dRNA-seq to identify RNA 5’ ends and Term-seq to identify RNA 3’ ends, providing a map of transcriptome architecture into S. aureus. We have additionally mapped RNA 3’ ends in cultures treated sub-lethal dosages of vancomycin, linezolid, or tigecycline for 10 minutes. A computational pipeline was written to predict antibiotic-responsive attenuators using a combination of the Term-seq and dRNA-seq data. This pipeline will be used to find transcripts with condition-dependent termination, and regulated termination sites in 5’-untranslated (UTR) regions of genes. Collectively these data provide a high-resolution map of the transcriptome in MRSA and will identify novel antibiotic-responsive RNA structures in this important human pathogen.