Staphylococcus aureus is a major opportunistic human pathogen and a leading cause of bacteraemia, endocarditis, and medical device-related infections. The emergence of multidrug-resistant S. aureus (MRSA) that have developed resistance to several antibiotics is a public health concern. Current treatment is dependent on the efficacy of 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 appear to arise from the acquisition of a disparate series of point mutations that lead to physiological changes including cell wall thickening and increased autolysis.
Transcriptional profiling has revealed that changes in small RNA (sRNA) expression in S. aureus are correlated with antibiotic treatment 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 using a proximity-dependant ligation and sequencing technique termed CLASH. RNase III-CLASH recovered 204 sRNA-RNA interactions in vivo and ontological analyses revealed that these sRNA-mRNA interactions are enriched for functions associated with reproduction, citrate transport, and stress response.
Small RNA interaction networks have been found to contain a proportion of bona fide but ‘non-functional’ RNA-RNA interactions. Discriminating between functional and non-functional RNA-RNA interactions remains a challenge. To identify sRNA-mRNA interactions that post-transcriptionally regulate mRNA expression, we correlated transcript abundance, ribosome occupancy, and protein levels for mRNAs targeted by sRNAs in our CLASH network. We used Self-Organising Maps to cluster genes with similar expression patterns and identified a cluster of genes enriched with mRNAs that are known to be post-transcriptionally repressed by sRNAs. Together, this analysis will provide insight into how sRNA-responsive networks induce changes in S. aureus to adapt to antibiotic stress.