Esculetin mitigates MRSA pathogenicity via innovative multitarget inhibition of Staphylococcus aureus sortase a, coagulase, and von Willebrand factor-binding protein.

The global proliferation of antibiotic-resistant Staphylococcus aureus, particularly methicillin-resistant Staphylococcus aureus (MRSA), highlights the urgent need for innovative antivirulence strategies. The redundancy and multiplicity of virulence factors produced by S. aureus necessitate interventions capable of concurrently targeting multiple virulence mechanisms. In this study, we identified esculetin, a natural compound, through comprehensive screening of a compound library. Esculetin uniquely targets sortase A (SrtA), coagulase (Coa), and von Willebrand factor-binding protein (vWbp) without inhibiting bacterial growth in vitro. Esculetin significantly attenuated SrtA-mediated MRSA USA300 strain invasion and biofilm formation while also inhibiting the coagulase activities of both vWbp and Coa. Using fluorescence resonance energy transfer (FRET), fluorescence quenching, and thermal shift assays, we confirmed direct binding interactions between esculetin and SrtA, Coa, and vWbp. In vivo studies demonstrated that esculetin significantly reduced MRSA virulence in Galleria mellonella larvae. Notably, the combination of esculetin and vancomycin markedly enhanced the therapeutic efficacy against MRSA USA300-induced pneumonia and skin infections in murine models, providing superior protection compared with vancomycin monotherapy. This study presents the first comprehensive demonstration of a natural compound capable of simultaneously inhibiting multiple virulence factors of S. aureus. The multitarget inhibition strategy of esculetin represents a promising advancement in combating antibiotic-resistant S. aureus infections by enhancing the therapeutic potential of existing antibiotics and reducing the likelihood of resistance development.