The diadenosine tetraphosphate hydrolase YqeK controls fitness, biofilm formation, staphyloxanthin production and virulence in Staphylococcus aureus

Diadenosine tetraphosphate (Ap4A) is a nucleotide metabolite, which is degraded by the YqeK hydrolase in Staphylococcus aureus in vitro. In this study, we analyzed the phenotypes of the yqeK mutant under stress, antibiotics, biofilm and macrophage infection conditions to investigate the functions of Ap4A in S. aureus COL. Using nucleotide metabolomics, we confirmed that Ap4A levels are 105-fold higher in the yqeK mutant, accompanied by lower adenylate and guanylate nucleotide pools. The yqeK mutant showed a delayed growth in LB, TSB and RPMI medium, and decreased survival under lethal oxidative and quinone stress. Transcriptome analysis revealed the upregulation of the sspABC operon and the CodY, T-box Met and G-box regulons indicating increased amino acids and GTP biosynthesis, whereas the AgrA, Fur, PurR, and T-box Cys regulons were downregulated in the yqeK mutant. These gene expression changes could be restored to WT level in the yqeK complemented strain, resulting also in a DNA damage response as revealed by the induction of LexA regulon members and mobile genetic elements (pathogenicity island SAPI3 and prophage L54a). Moreover, the yqeK mutant showed enhanced biofilm formation, higher intracellular iron levels and lower staphyloxanthin levels. Using infection assays, we demonstrated a decreased survival of the yqeK mutant inside J774A.1 murine macrophages, supporting a link between Ap4A and pathogenicity regulation via Agr-controlled virulence factors in S. aureus. Future research should be directed to understand how Ap4A regulates nucleotide, iron and amino acid metabolism as well as biofilm and virulence phenotypes in S. aureus. IMPORTANCE: S. aureus is an important human pathogen, which can cause life-threatening infections especially in immunocompromised patients. Due to the prevalence of multidrug resistant strains, the search for new drug targets is an urgent goal. Diadenosine tetraphosphate (Ap4A) has been shown to contribute to stress responses, antibiotic resistance, biofilm development and virulence in bacteria. In this work, we showed that Ap4A is upregulated upon deletion of yqeK encoding the Ap4A hydrolase in S. aureus COL. Moreover, the yqeK mutant was impaired in growth and survival during oxidative stress and after infection of murine macrophages, indicating that Ap4A contributes to the host-pathogen interactions in S. aureus. Transcriptome analyses revealed alterations of the nucleotide, amino acid and iron metabolism as well as the downregulation of Agr-controlled cytotoxins, contributing to the lower virulence of the yqeK mutant. Altogether, our results provide leads for the design of inhibitors against YqeK to combat S. aureus infections.