Staphylococcus aureus is a major cause of skin, soft tissue, respiratory and endovascular infections. The emergence of antibiotic-resistant strains, including methicillin-resistant S. aureus (MRSA), has limited the treatment options to last-line antibiotics like vancomycin, and increased the burden on the health-care system. Populations of MRSA with intermediate levels of vancomycin resistance (VISA) have emerged and are commonly associated with vancomycin treatment failure. These strains are associated with distinct physiological changes including cell wall thickening and reduced autolysis. Transcriptional profiling of MRSA isolates following exposure to antibiotics indicate that regulatory small RNA (sRNA) expression is correlated with antibiotic stress, although the precise function of these gene regulators is unknown.
Here we have performed UV crosslinking, ligation and sequencing of hybrids (CLASH) to profile the in vivo sRNA-mRNA interaction network and identify functions for regulatory sRNAs in the MRSA isolate JKD6009. Multiple sRNA-mRNA interactions were verified experimentally, confirming the robustness of our approach. Using CRISPR interference (dCas9) to knock down expression of sRNAs interacting with cell wall synthesis or antibiotic tolerance-associated mRNAs, we have screened for vancomycin sensitivity phenotypes. One sRNA, here termed the vancomycin-intermediate small RNA (visA), exhibited severe vancomycin and teicoplanin sensitivity, suggesting a glycopeptide-specific sRNA response. Vancomycin sensitivity was recapitulated in a ∆visA mutant and vancomycin tolerance was restored in a ∆visA::visA chromosomal repair strain confirming that visA is required for vancomycin tolerance in VISA isolates. Additionally, the identified visA regulon includes both novel mRNAs and sRNA sponging interactions. One such mRNA interaction identified by CLASH was the lytic transglycosylase isaA involved in cell wall expansion and turnover. Knock down of isaA also rendered cells vancomycin-sensitive resembling the ∆visA strain. Finally, we show ∆visA has significantly decreased cell wall thickness when compared to wild-type, which most likely contributes to the increased vancomycin sensitivity seen. These results demonstrate a novel mechanism of antibiotic tolerance through sRNA visA regulation of isaA and cell wall turnover during antibiotic stress.