Cucurbitacin B mitigates Staphylococcus aureus pathogenicity and reprograms macrophage responses to restore host defense

Methicillin-resistant Staphylococcus aureus (MRSA) remains a major health threat with limited therapeutic options. We identified cucurbitacin B (CuB), a plant-derived triterpenoid, as a concurrent, complementary adjunct that attenuates bacterial virulence while modulating host responses. Structure-guided docking together with electrophoretic mobility shift assay (EMSA) and lysate-based cellular thermal shift assay (CETSA) supported the engagement of the Staphylococcus exoprotein expression protein response regulator (SaeR) at the α4/β5 pocket, lowering SaeR-regulated toxins and immune responses factors at nonbactericidal exposures. In cell models, CuB improved intracellular bacterial control, alleviated MRSA-induced oxidative stress, restored the mitochondrial membrane potential, and shifted macrophages toward an anti-inflammatory, M2-like phenotype consistent with nuclear factor kappaB (NF-κB) modulation. Drug-pairing studies revealed class-selective, concentration-dependent synergy with glycopeptides (vancomycin (Van) and dalbavancin) and additivity with linezolid, indicating complementation rather than broad sensitization. Under repeated sub-minimum inhibitory concentration (MIC) exposure across forty passages, CuB alone maintained a stable MIC, whereas CuB combined with Van attenuated Van resistance trajectories; head-to-head competition between passage-zero and passage-forty lineages revealed no fitness advantage after passaging with CuB. In murine peritonitis, catheter-associated biofilm, and infected wound models, CuB lowered the bacterial burden, reduced inflammatory injury, and facilitated wound closure by controlling infection and inflammation, and tissue immunofluorescence via CD86 and CD206 provided spatial corroboration. On-target support came from the minimal incremental benefit of CuB in infections caused by a SaeR-deficient strain. Together, these findings position CuB as a SaeR-focused antivirulence-immunomodulatory adjunct that complements Van therapy and may improve outcomes while tempering resistance development, with claims limited to concurrent pathogen- and host-side activities rather than a mechanistically integrated dual mechanism. • First identification of Cucurbitacin B as a direct inhibitor of SaeR within the SaeRS two-component system. • CuB suppresses MRSA virulence gene expression and biofilm formation without affecting bacterial growth. • CuB reprograms macrophages toward an anti-inflammatory, reparative M2 phenotype by restoring mitochondrial function and inhibiting NF-κB activation. • CuB demonstrates strong therapeutic efficacy in multiple murine infection models, reducing bacterial burden, tissue damage, and promoting healing. • CuB synergizes with vancomycin, enhancing bacterial clearance and reducing resistance development.