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

Abstract

Elucidating the complex mechanisms controlling mecA/PBP2a-mediated β-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 β-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 β-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 β-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 β-lactam susceptibility. PBP2a expression was unaffected in nupG or deoD2 mutants suggesting that guanosine-induced β-lactam susceptibility may result from dysfunctional c-di-AMP-dependent osmoregulation. These data reveal the therapeutic potential of purine nucleosides as β-lactam adjuvants that interfere with the normal activation of c-di-AMP required for high-level β-lactam resistance in MRSA.

Metabolic reprogramming and flux to cell envelope precursors in a pentose phosphate pathway mutant increases MRSA resistance to β-lactam antibiotics

“…The OX-induced increase in MRSA cell size, which we and others have previously reported (31,53,(59)(60)(61), was more significant in wild-type JE2 and pglcomp than the pgl mutant (Fig. 3C).…”

“…Reduction in cell size may correlate with increased b-lactam resistance of pgl, as previously reported for a c-di-AMP phosphodiesterase gdpP mutant (41). In addition to the previously reported OX-induced increase in cell size (31,53,59,61), a dramatic cell lysis phenotype was also observed in wild-type JE2 grown in CDMG with subinhibitory OX (0.05 µg/ml), and not in the pgl mutant.…”

“…We and others have previously reported that purine nucleotide homeostasis plays a key role in the regulation of b-lactam resistance in MRSA (49)(50)(51)(52)(53). Mutations in the pur operon and purine salvage pathway were associated with increased resistance, whereas exposure of MRSA to the purine nucleosides guanosine or xanthosine (53).…”

“…Three independent biological replicates were performed for each strain and the resulting data plotted using GraphPad Prism software V9. guidelines (87) and as previously described (53) with the slight modifications. Briefly, overnight cultures were diluted into 5 ml fresh TSB and grown for 3 h at 37°C with shaking at 200 rpm.…”

“…We and others have previously reported that purine nucleotide homeostasis plays a key role in the regulation of b-lactam resistance in MRSA (49)(50)(51)(52)(53). Mutations in the pur operon and purine salvage pathway were associated with increased resistance, whereas exposure of MRSA to the purine nucleosides guanosine or xanthosine reduced b-lactam resistance (53). The purine-derived second messenger signalling molecules (p)ppGpp and c-di-AMP regulate b-lactam resistance, and exposure to exogenous guanosine downregulated c-di-AMP levels in S. aureus (53).…”