The emergence of multidrug-resistant Staphylococcus aureus (MRSA) is a major public health concern. Current treatment is dependent on last line antibiotics like vancomycin. However, MRSA isolates that exhibit intermediate resistance to vancomycin are increasingly detected worldwide and are associated with treatment failure. These vancomycin-intermediate S. aureus (VISA) isolates arise from the acquisition of a disparate series of point mutations that lead to physiological changes including cell wall thickening and reduced autolysis.
Transcriptional profiling has revealed that antibiotic treatment drives conserved changes in small RNA (sRNA) expression in S. aureus and may contribute to the VISA phenotype. However, the function of hundreds of sRNAs in S. aureus are still poorly understood. Here, we used the endoribonuclease RNase III, which processes sRNA-RNA duplexes, as a scaffold to capture sRNA-RNA interactions in VISA using a proximity-dependant ligation and sequencing technique termed CLASH. RNase III-CLASH recovered 215 unique sRNA-RNA interactions in vivo and these are enriched for functions associated with cell division, citrate transport, and cellular responses to oxidative stress.
To identify sRNA-mRNA interactions that regulate mRNA translation, we correlated transcript abundance, ribosome occupancy, and protein levels. We used Self-Organising Maps to cluster genes with similar transcription and translation patterns and identified a cluster of mRNAs that appeared to be post-transcriptionally repressed. By overlaying our sRNA interactome on these clusters we identified sRNAs that may be mediating this post-transcriptional repression. Two of these sRNA-mRNA interactions are mediated by RsaOI, a sRNA that is highly upregulated in response to vancomycin. CRISPRi knockdown of RsaOI resulted in increased vancomycin sensitivity in two different VISA strains. Further, we confirmed that RsaOI post-transcriptionally represses the sugar phospotransferase component PtsH, Arginase, and the vancomycin-associated autolysin Atl. Together, this multi-omics analyses has provided insights into how sRNA-responsive networks induce changes in S. aureus to adapt to antibiotic stress.