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 gram-negative and gram-positive species, ranging from increased antimicrobial sensitivity 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 ∆ yqeK was sensitive to nitrosative and organic acid stress, with all phenotypes restored by complementation. We used a luminescence-based assay to quantify relative Ap ₄ Ns and found that ∆ yqeK had ~1,000 fold higher signal than wild-type even during unstressed growth. Transcriptomics revealed that ∆ yqeK exhibited stress-specific dysregulation of translation, nucleotide metabolism, central metabolism, iron acquisition, and stress response genes. In contrast, ∆ yqeK had few transcriptional differences relative to the 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, the agr quorum sensing operon and numerous virulence genes showed reduced expression in ∆ yqeK , correlating with reduced hemolytic activity. This represents the first reported link between Ap ₄ N accumulation and quorum sensing suppression in a gram-positive pathogen. 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.

IMPORTANCE

Staphylococcus 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 Ap ₄ A, rendered S. aureus more susceptible to infection-relevant stress conditions but had little impact on normal growth. Ap ₄ Ns 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 .