Protein-mediated stabilization of amphotericin B increases its efficacy against diverse fungal pathogens

Amphotericin B (AMB), a potent and broad-spectrum antifungal agent, faces solubility and toxicity challenges in clinical use. In this study, we explored the ability of DewY and EAS_Δ15, class I fungal hydrophobin proteins with unique amphipathic properties and self-assembly capabilities, to stabilize AMB in solution. UV-visible spectroscopy confirmed the ability of hydrophobin proteins to stabilize the monomeric state of AMB in aqueous solution for up to 48 h. Further assays revealed that this effect was not exclusive to hydrophobins, however, as non-hydrophobin proteins provided similar stabilizing effects. AMB-protein combinations exhibited enhanced efficacy against diverse clinically relevant fungal pathogens, with 4- to 32-fold reductions in the effective <i>in vitro</i> dosage compared to AMB alone. Microscopic analyses found fungal cells treated with AMB alone and in combination with proteins had identical morphological changes, suggesting that protein interactions do not alter the mode of action of AMB. Instead, our results indicate that the monomeric state of AMB is stabilized in aqueous solution by non-specific interactions with hydrophobic areas on proteins. We suggest that this protein-mediated enhancement of solubility could reduce the required dose of AMB, providing a basis for optimizing AMB-based antifungal therapies.IMPORTANCEFungal infections are a growing global health concern, yet effective antifungal treatments remain limited by toxicity and poor solubility. AMB, a potent broad-spectrum antifungal, is highly effective but suffers from severe side effects and formulation challenges. Our study demonstrates that proteins, including fungal hydrophobins, can stabilize AMB in its monomeric form, significantly enhancing its solubility and efficacy against a range of fungal pathogens. These findings suggest that protein-mediated stabilization could enhance the effectiveness of AMB by reducing the required dosage and potentially lowering its toxic side effects. This approach offers a promising strategy for optimizing AMB therapies and improving treatment options, especially in resource-limited settings where fungal infections impose a significant health burden.