Loss of the Ap4A hydrolase YqeK impairs stress adaptation and virulence gene expression in Staphylococcus aureus

The nucleotide diadenosine tetraphosphate (Ap4A) 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 Ap4A and other dinucleoside tetraphosphates (Ap4Ns), Gram-positive bacteria, including Staphylococcus aureus, use YqeK. Inactivation of Ap4A hydrolases and corresponding Ap4A 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 {Delta}yqeK was sensitive to nitrosative and organic acid stress. We used a luminescence-based assay to show that {Delta}yqeK had [~]1000-fold higher relative Ap4N levels than wild-type even during unstressed growth, and all phenotypes were restored by complementation. Transcriptomics revealed that {Delta}yqeK exhibited stress-specific dysregulation of translation, nucleotide metabolism, central metabolism, iron acquisition, and stress response genes. In contrast, {Delta}yqeK had few transcriptional differences relative to wild-type during unstressed growth despite the large Ap4N accumulation, suggesting that the effects of Ap4Ns 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 {Delta}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. ImportanceS. aureus is a leading cause of antibiotic-resistant bacterial infections worldwide and is resistant to many components of the host immune response. Here, we discovered that deletion of YqeK, an enzyme that degrades a stress-associated nucleotide signaling molecule called Ap4A, rendered S. aureus more susceptible to infection-relevant stress conditions but had little impact on normal growth. Ap4Ns accumulated in the yqeK mutant and caused major stress-specific changes in gene expression, including reduced expression of key virulence genes. This correlated with a reduction in the destruction of red blood cells, a measure of bacterial toxicity toward host cells. Our data suggest that YqeK represents a promising target for new drugs aimed at reducing the virulence of S. aureus.