Pyrimidine sufficiency is required for Sae two-component system signaling in Staphylococcus aureus.

Nucleotide metabolism in pathogens is essential for their virulence, supporting their growth, survival, and immune evasion during infection. Virulence in Staphylococcus aureus is driven by the production of virulence factors that facilitate nutrient acquisition and promote immune evasion and subversion. One key virulence regulatory system is the Sae two-component system, which upregulates the production of various virulence factors. The sensor histidine kinase SaeS, a member of the intramembrane family of histidine kinases (HKs), lacks a signal-binding domain, leaving the mechanisms by which these HKs sense signals and regulate gene expression unclear. We report that pyrimidine sufficiency is essential for maintaining Sae activity. Disruption of genes involved in pyrimidine biosynthesis reduces Sae-dependent promoter activity under pyrimidine-limited conditions. Phos-tag electrophoresis confirmed that pyrimidine limitation impacts SaeS kinase activity directly. The effect of pyrimidine limitation on SaeS was abrogated in a strain producing only the catalytic domain, suggesting that pyrimidines regulate SaeS activity at the membrane. Additionally, pyrimidine limitation results in cell envelope defects, specifically increased lipoteichoic acids, and incorporation of free fatty acids into the membrane. While both cell envelope aberrations are detrimental to Sae activity, we found that removal of the accumulated free fatty acids restored Sae activity. Our study highlights the interplay between nucleotide metabolism and membrane integrity in regulating virulence factor expression through signal transduction systems in pathogens.IMPORTANCEVirulence is often correlated with nutrient depletion, but our understanding of this coordination is incomplete. In Staphylococcus aureus, the Sae two-component system is a major regulator of virulence factor production and secretion, but as the sensor histidine kinase SaeS lacks an obvious domain to perceive its inducing signal, basic questions surrounding how the kinase is triggered persist. Here, we aimed to investigate the mechanism by which pyrimidines act to promote the activity of the SaeS kinase in S. aureus and further expand on the importance of the roles of pyrimidines in regulating envelope biogenesis. Understanding this intersection between nucleotide metabolism and virulence regulation opens up the possibility for the development of targeted anti-virulence strategies against S. aureus infections.