Targeting cyclic di-AMP signaling through diadenylate cyclase inhibition reduces methicillin resistance in clinical MRSA isolates.

BACKGROUND

Methicillin-resistant Staphylococcus aureus (MRSA) remains a major clinical challenge due to persistent β-lactam resistance and biofilm-associated tolerance. While canonical resistance mechanisms are well defined, non-canonical pathways such as cyclic di-adenosine monophosphate (c-di-AMP) signaling remain poorly understood, and no effective inhibitors have yet been shown to reduce MRSA resistance.

METHODS

Clinical MRSA isolates were analyzed for c-di-AMP levels, gene expression, biofilm formation, and host immune responses under sub-inhibitory methicillin exposure. Structure-based virtual screening of FDA-approved compounds was performed to identify inhibitors of diadenylate cyclase (DacA), followed by biochemical validation, antimicrobial susceptibility testing, macrophage infection assays, and cytotoxicity evaluation.

RESULTS

Methicillin exposure and biofilm growth significantly increased intracellular c-di-AMP levels via upregulation of DacA and associated signaling genes. Elevated c-di-AMP promoted bacterial persistence and induced STING-dependent immune modulation, characterized by increased IFN-β and reduced IL-1β in infected macrophages. Tropinone and eucalyptol were identified as DacA inhibitors that reduced c-di-AMP production, disrupted biofilms, and restored methicillin susceptibility, achieving up to 4-8 fold reduction in MICs. Both compounds demonstrated low cytotoxicity and favorable predicted ADME profiles.

CONCLUSION

c-di-AMP signaling represents a key non-canonical regulator of methicillin resistance and pharmacological inhibition of DacA restores β-lactam susceptibility and represents a promising adjunctive strategy against MRSA.