Saikosaponin D from Bupleurum targets SaeR to disrupt biofilm formation and pathogenic programs for the treatment of MRSA pneumonia.

Staphylococcus aureus pneumonia, particularly methicillin-resistant Staphylococcus aureus (MRSA), remains a major clinical challenge due to biofilm formation, complex regulatory programs, and rapid acquisition of antimicrobial resistance (AMR). The SaeRS two-component system is a central regulatory node controlling multiple infection-related pathways, making its response regulator SaeR an attractive therapeutic target. Targeting such upstream regulators provides an alternative strategy to conventional bactericidal approaches. Through a network pharmacology-guided screen, we identified saikosaponin D (SSD), an oleanane-type pentacyclic triterpenoid saponin from Bupleurum, as a candidate compound. Biophysical and biochemical assays, including cellular thermal shift assay (CETSA), surface plasmon resonance (SPR), and electrophoretic mobility shift assay (EMSA), demonstrated that SSD directly binds SaeR and impairs its DNA-binding capacity. This interference repressed SaeR-dependent transcription, resulting in reduced α-hemolysin and Panton-Valentine leucocidin (PVL) production, diminished hemolytic activity, decreased bacterial adhesion and invasion, and disruption of biofilm integrity through suppression of extracellular matrix components. In vivo, SSD conferred significant protection in both invertebrate and mammalian infection models. In Galleria mellonella, SSD improved survival, and in a murine model of MRSA pneumonia, SSD reduced pulmonary bacterial burden, alleviated inflammation and edema, and enhanced overall survival. Collectively, these findings establish SaeR as a druggable upstream regulator and highlight SSD as a natural product scaffold with translational potential for therapeutic development against MRSA infections.