Shikonin potentiates amphotericin B activity against Aspergillus fumigatus through membrane homeostasis disruption

The escalating challenge of antifungal resistance in Aspergillus fumigatus underscores the critical need for novel therapeutic combinations. This study identifies the natural naphthoquinone shikonin (SHK) as a potent synergist of amphotericin B (AMB) against A. fumigatus. Checkerboard assays revealed a significant synergistic interaction (FICI ≤ 0.5), reducing the AMB MIC by up to 8-fold. Mechanistic investigations demonstrated that SHK exerts a multi-target effect: it effectively inhibites biofilm formation and disrupts key components of cell wall biosynthesis. Crucially, real-time imaging and propidium iodide uptake assays confirmed rapid and profound perturbation of membrane integrity. Molecular dynamics simulations provided a structural basis for this effect, indicating that SHK interacts directly with membrane ergosterol, promoting its disorganization and compromising membrane homeostasis. In vivo experiments showed that the combination of SHK and AMB could significantly improve the survival rate (65.2%) compared with any single drug. Our findings elucidate a unique multimodal mechanism by which SHK potentiates AMB, positioning it as a promising adjunctive therapy to combat AMB resistance and improve outcomes in invasive fungal infections.