Prim-O-glucosylcimifugin targets Staphylococcus aureus caseinolytic protease P to inhibit α-hemolysin expression and promote healing of MRSA-induced diabetic skin infections.

Targeting bacterial virulence factors is considered a promising strategy because it poses a lower risk of promoting antibiotic resistance. This study investigated the therapeutic potential and underlying mechanism of prim-O-glucosylcimifugin (POG) against Staphylococcus aureus (S. aureus). In a diabetic mouse model, POG significantly reduced bacterial burden and accelerated wound healing, which was accompanied by enhanced tissue regeneration and collagen deposition, but no detectable toxicity. It also improved survival in a Galleria mellonella model infected with methicillin-resistant S. aureus. In vitro, POG did not inhibit bacterial growth, indicating an antivirulence rather than bactericidal mode of action. Transcriptomic analysis revealed that POG downregulated multiple virulence-associated genes, including α-hemolysin (hla), and disrupted quorum sensing, stress response, and metabolic pathways. Consistently, POG treatment suppressed hemolytic activity and the production of Hla. Molecular docking, molecular dynamics simulations, and cellular thermal shift assays confirmed the direct binding of POG to S. aureus caseinolytic protease P (SaClpP). Furthermore, fluorescence resonance energy transfer assays demonstrated that POG inhibited SaClpP activity in a dose-dependent manner, with an IC50 of 14.27 μg/mL. In a clpP knockout strain, the suppressive effects of POG on Hla production and hemolytic activity were abolished, confirming SaClpP as the key target responsible for its antivirulence effects. Collectively, these results demonstrate that POG is a promising antivirulence agent that attenuates S. aureus pathogenicity by targeting SaClpP.