Purine nucleosides interfere with c-di-AMP levels and act as adjuvants to re-sensitize MRSA to β-lactam antibiotics

Elucidating the complex mechanisms controlling mecA/PBP2a-mediated {beta}-lactam resistance in methicillin resistant Staphylococcus aureus (MRSA) has the potential to identify new drug targets with therapeutic potential. Here, we report that mutations that interfere with de novo purine synthesis (pur operon), purine transport (NupG, PbuG and PbuX) and the nucleotide salvage pathway (DeoD2, Hpt) increased {beta}-lactam resistance in MRSA strain JE2. Extrapolating from these findings, exogenous guanosine and xanthosine, which are fluxed through the GTP branch of purine biosynthesis were shown to significantly reduce MRSA {beta}-lactam resistance. In contrast adenosine, which is fluxed to ATP, significantly increased oxacillin resistance, whereas inosine, which can be fluxed to ATP and GTP via hypoxanthine, only marginally reduced the oxacillin MIC. Increased oxacillin resistance of the nupG mutant was not significantly reversed by guanosine, indicating that NupG is required for guanosine transport, which in turn is required to reduce {beta}-lactam resistance. Suppressor mutants resistant to oxacillin/guanosine combinations contained several purine salvage pathway mutations, including nupG and hpt. Microscopic analysis revealed that guanosine significantly increased cell size, a phenotype also associated with reduced levels of c-di-AMP. Consistent with this, guanosine significantly reduced levels of c-di-AMP, and inactivation of GdpP, the c-di-AMP phosphodiesterase negated the impact of guanosine on {beta}-lactam susceptibility. PBP2a expression was unaffected in nupG or deoD2 mutants suggesting that guanosine-induced {beta}-lactam susceptibility may result from dysfunctional c-di-AMP-dependent osmoregulation. These data reveal the therapeutic potential of purine nucleosides as {beta}-lactam adjuvants that interfere with the normal activation of c-di-AMP required for high-level {beta}-lactam resistance in MRSA. ImportanceThe clinical burden of infections caused by antimicrobial resistant (AMR) pathogens is a leading threat to public health. Maintaining the effectiveness of existing antimicrobial drugs or finding ways to reintroduce drugs to which resistance is widespread is an important part of efforts to address the AMR crisis. Predominantly the safest and most effective class of antibiotics are the {beta}-lactams, which are no longer effective against methicillin resistant Staphylococcus aureus (MRSA). Here we report that the purine nucleosides guanosine and xanthosine have potent activity as adjuvants that can resensitise MRSA to oxacillin and other {beta}-lactam antibiotics. Mechanistically, exposure of MRSA to these nucleosides significantly reduced the levels of the cyclic dinucleotide c-di-AMP, which is required for {beta}-lactam resistance. Drugs derived from nucleotides are widely used in the treatment of cancer and viral infections highlighting the clinical potential of using purine nucleosides to restore or enhance the therapeutic effectiveness of {beta}-lactams against MRSA and potentially other AMR pathogens.