Chauhan, M.; Ivanova, I.; Sudnick, E. G.; Steere, R. W.; Tennant, J. R.; Hensley, J. A.; Arede, P.; Jensen, G. M.; Hatin, I.; Namy, O.; Bouloc, P.; Carroll, R. K.; Granneman, S.

Methicillin-resistant Staphylococcus aureus (MRSA) is a formidable human pathogen responsible for life-threatening infections worldwide. Central to its pathogenic success is the tightly coordinated regulation of virulence factors, including the phenol soluble modulins (PSM), short amphipathic toxins that drive cytolysis, immune evasion and biofilm maturation. We previously identified the conserved non-coding RNA RsaE as a putative regulator of the psm operon, but the biological significance of this interaction remained unclear. Here we show that RsaE and endoribonuclease Y jointly regulate psm transcript abundance at the post-transcriptional and transcriptional levels, the latter primarily through activation of the agr quorum sensing system. We show that the psm transcript is unusually stable and highly structured, with Shine-Dalgarno sequences of individual toxin-coding sequences differentially accessible, providing a mechanism for translational fine-tuning of individual PSM peptides. In vitro biofilm analyses revealed that RsaE deletion produces thinner biofilms with reduced extracellular DNA accumulation on the surface and transiently elevated cell viability. Strikingly, in a murine catheter infection model, {Delta}rsaE biofilms exhibited structural abnormalities and significantly reduced dissemination to kidneys. These findings identify the RsaE non-coding RNA as a key regulator linking central metabolism and quorum sensing to toxin expression, biofilm maturation and infection in MRSA.