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

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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.

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