Abstract

Diadenosine tetraphosphate (Ap₄A) is an evolutionarily conserved alarmone, or small intracellular signaling molecule that rapidly accumulates in response to stress, reprograming cellular physiology to promote survival over growth. Ap4A is implicated in the stress response of bacteria, yet its role remains largely uncharacterized in Staphylococcus aureus. In S. aureus, YqeK encodes a putative Ap₄A hydrolase, implicated in regulating alarmone metabolism and stress responses by controlling intracellular Ap₄A levels. In this study, a luminescence-based assay to quantify Ap₄A accumulation in wild-type, ΔyqeK, and a genetically complemented strain of S. aureus was developed. Cultures were normalized, lysed, and processed to remove interfering nucleotides, followed by selective enzymatic treatment to distinguish Ap₄A from other adenylated species. Resulting nucleotide pools were analyzed using an ATP-dependent luciferase readout, allowing indirect but sensitive detection of Ap₄A levels. Using this approach, ΔyqeK demonstrated an increased level of more than 1000-fold Ap₄A concentration relative to wild-type and complemented strains, supporting a central role for the yqeK gene in Ap₄A regulation in S. aureus. Notably, accumulation of Ap₄A was elevated in the ΔyqeK strain, indicating that Ap₄A levels are stress-responsive and exacerbated in the absence of functional YqeK. This assay provides a reliable method for probing dinucleotide alarmone metabolism and the evaluation of genetic and chemical perturbations affecting Ap₄A metabolism.

We define a reliable approach to measure Ap₄A in Staphylococcus aureus and identify yqeK as a key regulator of its levels. Our findings link Ap₄A signaling to adaptation under infection-relevant stress, highlighting YqeK and this pathway as potential vulnerabilities in drug-resistant strains such as MRSA. As an extension of this research, development of an endocardial vegetation model is underway to examine Ap₄A regulation in a disease-relevant context.

Keywords

Staphylococcus aureus, Ap4A, yqeK

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