Non-canonical stringent response signaling mediates antimicrobial fatty acid sensitivity in Staphylococcus aureus

Staphylococcus aureus is a leading cause of skin and soft tissue infections, yet colonization of healthy skin is limited by multiple defenses, including antimicrobial fatty acids (AFAs) produced by host cells and the resident microbiota. The mechanisms by which S. aureus overcomes these lipid-based defenses remain incompletely understood. Here, we show that mutations truncating the essential stringent response regulator Rel confer broad tolerance to both host-and microbially-derived AFAs in diverse S. aureus strains. Unlike classical stringent response activation, C-terminal Rel truncations do not induce typical (p)ppGpp-dependent transcriptional changes but instead enhance activity of the alternative sigma factor SigB and the staphylococcal accessory regulator SarA. This SigB-SarA regulatory cascade promotes transcriptional remodeling, including upregulation of pyrimidine biosynthesis genes, and coincides with alterations to cell envelope structure. Moreover, in the absence of SigB or SarA, tolerance can be restored through mutations in the serine/threonine phosphatase Stp1, highlighting additional pathways that modulate cell envelope-mediated resistance to AFAs. These findings identify a previously unrecognized consequence of Rel mutation in S. aureus; whereby small truncations may promote survival in AFA-rich host environments facilitating skin colonization and infection. ImportanceStaphylococcus aureus is a major cause of skin and soft tissue infections, and persistent skin colonization is a significant risk factor for infection. Antimicrobial fatty acids (AFAs), produced by microbes and host cells on human skin, normally limit S. aureus colonization. The mechanisms that allow this pathogen to overcome these lipid defenses are incompletely understood. We show that mutations truncating the essential stringent response regulator Rel enable S. aureus to tolerate both host-and microbially derived AFAs. These Rel variants, which have been identified in clinical isolates, alter cell envelope properties through the transcriptional regulator SarA rather than activating a classical stringent response. Our findings reveal a previously unrecognized adaptation that may facilitate S. aureus survival on the skin and promote infection.