Combining Colistin and Fluconazole Synergistically Increases Fungal Membrane Permeability and Antifungal Cidality

[Image: see text] The increasing emergence of drug-resistant fungal pathogens, together with the limited number of available antifungal drugs, presents serious clinical challenges to treating systemic, life-threatening infections. Repurposing existing drugs to augment the antifungal activity of well-tolerated antifungals is a promising antifungal strategy with the potential to be implemented rapidly. Here, we explored the mechanism by which colistin, a positively charged lipopeptide antibiotic, enhances the antifungal activity of fluconazole, the most widely used orally available antifungal. In a range of susceptible and drug-resistant isolates and species, colistin was primarily effective at reducing fluconazole tolerance, a property of subpopulations of cells that grow slowly in the presence of a drug and may promote the emergence of persistent infections and resistance. Clinically relevant concentrations of colistin synergized with fluconazole, reducing fluconazole minimum inhibitory concentration 4-fold. Combining fluconazole and colistin also increased survival in a C. albicans Galleria mellonella infection, especially for a highly fluconazole-tolerant isolate. Mechanistically, colistin increased permeability to fluorescent antifungal azole probes and to intracellular dyes, accompanied by an increase in cell death that was dependent upon pharmacological or genetic inhibition of the ergosterol biosynthesis pathway. The positive charge of colistin is critical to its antifungal, and antibacterial, activity: colistin directly binds to several eukaryotic membrane lipids (i.e., l-α-phosphatidylinositol, l-α-phosphatidyl-l-serine, and l-α-phosphatidylethanolamine) that are enriched in the membranes of ergosterol-depleted cells. These results support the idea that colistin binds to fungal membrane lipids and permeabilizes fungal cells in a manner that depends upon the degree of ergosterol depletion.