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

Nucleotide metabolism in pathogens is essential for their virulence, supporting 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 (TCS), which upregulates the production of various virulence factors. The sensor histidine kinase SaeS, a member of the intramembrane family of histidine kinases (IM-HKs), lacks a signal-binding domain, leaving the mechanisms by which these HKs sense signals and regulate gene expression unclear. We report that de novo pyrimidine biosynthesis 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. 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, defective pyrimidine biosynthesis caused membrane defects and increased incorporation of free fatty acids into the membrane. Further, providing an extracellular sink for free fatty acids restored Sae activity in these mutants. Our study highlights the interplay between nucleotide metabolism and membrane integrity in regulating virulence factor expression through signal transduction systems in pathogens.