Daphnoretin as a potent sortase A inhibitor effectively disarms drug-resistant Staphylococcus aureus infections.

The rapid spread of drug-resistant Staphylococcus aureus (S. aureus) has severely undermined the efficacy of existing antibiotics, highlighting the urgent need to explore novel antivirulence strategies to address this global public health challenge. Sortase A (SrtA) is a critical virulence factor of S. aureus that covalently anchors multiple surface proteins to the bacterial cell wall, thereby mediating adhesion, invasion, immune evasion, and biofilm formation, playing a pivotal role in pathogenesis. In this study, potential inhibitors were screened using a fluorescence resonance energy transfer (FRET) assay, and daphnoretin was identified as a potent SrtA inhibitor with a half-maximal inhibitory concentration (IC₅₀) of 8.501 µg/mL. In vitro experiments demonstrated that daphnoretin effectively suppressed SrtA-mediated pathogenic phenotypes, including bacterial adhesion, invasion, and biofilm formation, thereby attenuating virulence. Further fluorescence quenching and molecular docking analyses revealed the binding mechanism between daphnoretin and SrtA. In vivo, daphnoretin significantly improved host survival in Galleria. mellonella infection model induced by the methicillin-resistant S. aureus (MRSA) and alleviated joint damage and infection symptoms in a rat model of septic arthritis while markedly reducing the bacterial burden in infected tissues. Collectively, this study is the first to elucidate the mechanism of daphnoretin as a SrtA-targeting inhibitor and its antivirulence potential, providing important experimental evidence for the development of novel therapeutic strategies against drug-resistant S. aureus.