Loss of the Ap ₄ A hydrolase YqeK impairs stress adaptation and virulence gene expression in Staphylococcus aureus

The nucleotide diadenosine tetraphosphate (Ap ₄ A) accumulates during stress across organisms and cell types and is widely hypothesized to be an alarmone or second messenger. While Gram-negative bacteria use ApaH-family hydrolases to degrade Ap ₄ A and other dinucleoside tetraphosphates (Ap ₄ Ns), Gram-positive bacteria, including Staphylococcus aureus, use YqeK. Inactivation of Ap ₄ A hydrolases and corresponding Ap ₄ A accumulation cause diverse phenotypic effects in both Gram-negative and Gram-positive bacteria, ranging from increased sensitivity to antimicrobials to reduced virulence. However, the physiological role of YqeK in S. aureus remains uncharacterized. Here, we constructed an isogenic yqeK mutant in S. aureus and showed that Δ yqeK was sensitive to nitrosative and organic acid stress. We used a luminescence-based assay to show that Δ yqeK had ∼1000-fold higher relative Ap ₄ N levels than wild-type even during unstressed growth, and all phenotypes were restored by complementation. Transcriptomics revealed that Δ yqeK exhibited stress-specific dysregulation of translation, nucleotide metabolism, central metabolism, iron acquisition, and stress response genes. In contrast, Δ yqeK had few transcriptional differences relative to wild-type during unstressed growth despite the large Ap ₄ N accumulation, suggesting that the effects of Ap ₄ Ns are contingent on the cellular stress state. Unexpectedly, we also found that the entire agr quorum sensing operon and numerous additional virulence genes, including hemolytic toxins, had reduced expression in Δ yqeK , correlating with reduced hemolytic activity in the mutant even in the absence of stress. Our data reveal YqeK to be a critical metabolic determinant of S. aureus stress resistance and virulence and position this hydrolase as a promising candidate for anti-virulence drug development.