McGavin, M. J. | Search bioRxiv

bioRxiv2026

The RND family efflux pump FemT contributes to lipid homeostasis in Staphylococcus aureus

The RND efflux pump FemT encoded by SAUSA300_2213 of Staphylococcus aureus USA300 is co-transcribed with femX which has an essential role in synthesizing the Lipid II precursor of peptidoglycan. Anticipating that this arrangement reflects a critical accessory role for femT, we constructed USA300{Delta}femT to assess its function. Although growth of USA300{Delta}femT in tryptic soy broth (TSB) was not impaired, transcriptomic data revealed a mild cellular stress response, accompanied by reduced expression of ohyA and crt genes involved in fatty acid metabolism and carotenoid lipid synthesis respectively. Accordingly, USA300{Delta}femT exhibited impaired growth on exposure to saturated and unsaturated fatty acids, and exposure to subinhibitory 50 {micro}M palmitic acid promoted accumulation of reactive oxygen species, reduced respiratory activity, and altered membrane function and morphology. The transcriptome of cells grown under this condition revealed strongly attenuated expression of ohyA and crt, and several genes required for oxidative and anaerobic respiration, concomitant with strongly enhanced expression of several stress response pathways. Cellular metabolites were also profoundly altered. Finally, lipidomic analysis of USA300{Delta}femT exposed to oleic acid revealed increased incorporation of oleic acid into phosphatidylglycerol, accompanied by a significant reduction in undecaprenol C55 lipid carrier, and respiratory quinones MK-7 and MK-8. Our data are consistent with a role for FemT in maintaining cellular lipid homeostasis by promoting efflux of isoprenoid and carotenoid lipids that are prone to oxidative damage, including C55 and menaquinones that undergo cyclic reactions in peptidoglycan synthesis and electron transport.

microbiology↗

bioRxiv2026

Differential Pathogenic and Commensal Responses of Staphylococcus aureus and Staphylococcus epidermidis Towards Chemical Signals of Human Skin

Pathogenic Staphylococcus aureus and commensal Staphylococcus epidermidis encounter acidic pH and C16 fatty acids on human skin, but S. aureus uniquely has a complete fad pathway for metabolism of saturated C16:0 palmitic acid. We now report on significant differences in their response to C16 fatty acids during growth at pH 5.5. Unsaturated palmitoleic acid C16:1 was more toxic to S. aureus, but toxicity was mitigated by saturated C16:0. Consistent with a functional fad pathway, C16:0 conferred enhanced growth to S. aureus, but not S. epidermidis. Acidic pH and C16 fatty acids stimulated SspA serine protease production in S. aureus but repressed the orthologous Esp protease in S. epidermidis. Although S. aureus biofilm formation was stimulated by acidic pH and C16:0, this effect was abrogated by 25 {micro}M C16:1 which promotes protease production, whereas S. epidermidis maintained enhanced biofilm in presence of C16:1. Exogenous C16:0 was directly incorporated into phospholipid by S. epidermidis but was extended to C18:0 and C20:0 in S. aureus prior to incorporation. This may account for differential signaling through the GraSR two component sensor, which is required for SspA production in S. aureus at acidic pH. Notably, singular graSR dependent phenotypes in S. aureus graS/R deletions were restored by S. epidermidis graS/R at acidic pH alone, whereas growth at pH 5.5 with 25 {micro}M C16:1 could only be restored with S. aureus graS/R. These findings provide important new insight into how members of the Staphylococcal genus are differentially influenced by common environmental signals on human skin. IMPORTANCEHuman skin is a chemically hostile environment, with acidic pH and antimicrobial fatty acids that challenge microbial survival. Understanding how closely related Staphylococcus epidermidis and Staphylococcus aureus navigate these conditions is critical for distinguishing commensal behavior from pathogenic potential. Our research reveals that S. aureus and S. epidermidis, though genetically similar, employ markedly distinct adaptive mechanisms in response to identical skin-derived cues. Specifically, each species remodels its membrane phospholipids in unique ways under acidic pH and C16 fatty acid exposure. These environmental factors also differentially modulate their biofilm formation and protease activity. Together, our findings highlight how the same host-derived chemical signals of skin can activate virulence-associated traits in S. aureus while supporting commensal persistence in S. epidermidis.

microbiology↗