RNase III CLASH in MRSA uncovers sRNA regulatory networks coupling metabolism to toxin expression

Methicillin-resistant Staphylococcus aureus (MRSA) is a bacterial pathogen responsible for high levels of human morbidity and mortality. MRSA co-ordinates expression of an array of bacterial factors involved in antimicrobial resistance, nutrient acquisition and immune evasion in the host. Post-transcriptional regulation by small RNAs (sRNAs) has emerged as an important mechanism for the control of MRSA virulence. However, the function of the majority of sRNAs during infection is unknown. To address this gap in understanding, we performed UV cross-linking, ligation and sequencing of hybrids (CLASH) in MRSA to unravel sRNA-RNA interactions under conditions that mimic the host environment. Using double stranded ribonuclease III (RNase III) as a bait we not only uncovered known interactions but also hundreds of novel sRNA-RNA pairs. Strikingly, our results suggest that the production of small membrane-permeabilizing toxins is under extensive sRNA-mediated regulation and that their expression is intimately connected to metabolism. Importantly, we discovered that two sRNAs, RNAIII and RsaE, control the expression of at least four cytolytic toxins that are important for MRSA virulence. Taken together, we present a comprehensive analysis of sRNA-target interactions in S. aureus and provide detail on how these contribute to the control of virulence in response to changes in metabolism. Overall design: CLASH was performed on RNase III in S. aureus USA300 and S. aureus JKD6009 backgrounds. As a control, CLASH was performed on the untagged parental strains. S. aureus was grown in rich medium, TSB, until the post-exponential phase and was then shifted to either RPMI medium (to mimic the human bloodstream) or LPM medium at either pH 5 or pH 7 (to mimic the intracellular host environment). S. aureus was incubated in these medium for 15 minutes and then harvested and used as inputs for CLASH. Additionally, RNA sequencing was performed on USA300 after the shifts to RPMI and LPM to understand transcriptome-level changes. For RNA sequencing, samples were taken after 5, 10, 15 and 30 minutes of exposure to the media.