Staphylococcus aureus is a major opportunistic human pathogen and a leading cause of bacteraemia, infective endocarditis, and medical device-related infections. The advent and use of antibiotics made it possible to treat S. aureus infections effectively. However, the acquisition of antibiotic resistance has led to the emergence of drug resistant clones of S. aureus, termed multidrug-resistant S. aureus (MRSA). MRSA isolates that exhibit intermediate resistance to vancomycin, a last line antibiotic used to treat multi-drug resistant Gram-positive pathogens, are increasingly detected worldwide. Vancomycin-intermediate S. aureus (VISA) appear to arise from the acquisition of a disparate series of point mutations that lead to physiological changes including cell wall thickening and decreased autolysis.
Transcriptional profiling has revealed that changes in small RNA expression in S. aureus are correlated with antibiotic treatment and may contribute to the VISA phenotype. However, the functions of the hundreds of small RNAs in S. aureus are still poorly understood. The endoribonuclease RNase III processes sRNA-mRNA duplexes and we have used this protein as a scaffold to capture the sRNA-mRNA interaction network after antibiotic treatment using RNase-CLASH (cross-linking, ligation and sequencing of hybrids). To understand how these vancomycin-responsive sRNA interactions drive changes in the proteome, we will use Ribo-seq to map the precise position of translating ribosomes (ribosome protected fragments, RPFs) after vancomycin stress. Analysis of RPFs will elucidate how mRNA translation rates, protein abundance and ribosome occupancy are affected by vancomycin-induced sRNAs. Together, the above analyses will provide insight into the post-transcriptional and translational changes induced by sRNA-responsive networks in S. aureus to generate the VISA phenotype and adapt to antibiotic stress.