Requirement for Ergosterol in V-ATPase Function Underlies Antifungal Activity of Azole Drugs

Abstract

Ergosterol is an important constituent of fungal membranes. Azoles inhibit ergosterol biosynthesis, although the cellular basis for their antifungal activity is not understood. We used multiple approaches to demonstrate a critical requirement for ergosterol in vacuolar H+-ATPase function, which is known to be essential for fungal virulence. Ergosterol biosynthesis mutants of S. cerevisiae failed to acidify the vacuole and exhibited multiple vma − phenotypes. Extraction of ergosterol from vacuolar membranes also inactivated V-ATPase without disrupting membrane association of its subdomains. In both S. cerevisiae and the fungal pathogen C. albicans, fluconazole impaired vacuolar acidification, whereas concomitant ergosterol feeding restored V-ATPase function and cell growth. Furthermore, fluconazole exacerbated cytosolic Ca2+ and H+ surges triggered by the antimicrobial agent amiodarone, and impaired Ca2+ sequestration in purified vacuolar vesicles. These findings provide a mechanistic basis for the synergy between azoles and amiodarone observed in vitro. Moreover, we show the clinical potential of this synergy in treatment of systemic fungal infections using a murine model of Candidiasis. In summary, we demonstrate a new regulatory component in fungal V-ATPase function, a novel role for ergosterol in vacuolar ion homeostasis, a plausible cellular mechanism for azole toxicity in fungi, and preliminary in vivo evidence for synergism between two antifungal agents. New insights into the cellular basis of azole toxicity in fungi may broaden therapeutic regimens for patient populations afflicted with systemic fungal infections.

Alteration of complex sphingolipid composition and its physiological significance in yeast Saccharomyces cerevisiae lacking vacuolar ATPase

“…For example, in S. cerevisiae, the proton-pump activity of V-ATPase decreases on deletion of ELO3 or ORM1 and ORM2, all of which are involved in sphingolipid biosynthesis (Chung et al, 2003;Finnigan et al, 2011). In addition, impaired biosynthesis of ergosterol also causes a reduction in V-ATPase activity (Zhang et al, 2010). Since ergosterol together with complex sphingolipids is involved in the formation of lipid microdomains (Simons & Sampaio, 2011), it is likely that proper formation of lipid microdomains is required for the maintenance of V-ATPase activity.…”

Amphotericin primarily kills yeast by simply binding ergosterol

“…1A), was recently found to directly bind ergosterol but not to cause membrane permeabilization (21). Third, ergosterol is critical for many aspects of yeast cell physiology, including vacuole fusion (22), endocytosis (23), pheromone signaling (24), membrane compartmentalization (25), and the proper functioning of membrane proteins (26). Thus, binding alone of this multifunctional lipid should be sufficient for killing yeast cells.…”

Endosomal Trafficking Defects Can Induce Calcium-Dependent Azole Tolerance in Candida albicans

“…S everal important antifungal drugs inhibit the synthesis of ergosterol, a lipid that modulates the thickness, fluidity, and permeability of fungal cell membranes (1). These drugs include the azoles, which inhibit lanosterol demethylase (Erg11p).…”