Targeting Cyclic di-AMP Signaling through Diadenylate Cyclase Inhibition Reduces Methicillin Resistance in Clinical MRSA Isolates
- David Ojcius
- 4 hours ago
- 1 min read
HIGHLIGHT
Cyclic-di-AMP is a non-canonical driver of methicillin resistance in MRSA
Sub-MIC methicillin and biofilms elevate c-di-AMP via DacA upregulation
Elevated c-di-AMP activates STING and skews macrophage responses
FDA approved drugs Tropinone and eucalyptol identified as DacA inhibitors
DacA inhibition restores β-lactam susceptibility in MRSA
Abstract
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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