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. 2010 Jun 3;6(6):e1000939.
doi: 10.1371/journal.ppat.1000939.

Requirement for ergosterol in V-ATPase function underlies antifungal activity of azole drugs

Yong-Qiang Zhang  1 , Soledad Gamarra, Guillermo Garcia-Effron, Steven Park, David S Perlin, Rajini Rao
Affiliations

Requirement for ergosterol in V-ATPase function underlies antifungal activity of azole drugs

Yong-Qiang Zhang et al. PLoS Pathog. .

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 Ca(2+) and H(+) surges triggered by the antimicrobial agent amiodarone, and impaired Ca(2+) 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.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. erg24Δ exhibits multiple vma− phenotypes.
Growth of WT, vma2Δ, erg24Δ strains under different conditions. Values were normalized to growth of each strain under control condition (A and C), to growth of each strain at pH 4.3 (B), to growth of each strain at pH 6 without FK506 (D), or to growth of WT under the two conditions with non-fermentable carbon source (F). (E) Growth of the strains in YPD or YPD supplemented with Calcofluor white. All measurements were in triplicate, and means and standard deviations are plotted. p values of two-tailed t-test are shown.
Figure 2
Figure 2. ERG24 deletion impairs the function of V-ATPase but not Pma1p.
(A) Vacuolar pH of WT, erg24Δ and vma2Δ strains measured with pH sensitive fluorophore BCECF-AM, which accumulates in the yeast vacuole. (B) Initial H+ pumping rate was calculated from ΔA490–540 during the first 60 s after initiating the reaction. ATPase activity was calculated from drop of A340 between 3 and 6 minutes after initiating the reaction. Means and standard deviations are plotted from data for at least three independent vacuolar vesicle preparations. p values of two-tailed t-test are shown. (C) Medium acidification by Pma1 upon glucose activation was measured as described in Methods. Extracellular pH (pHout) was recorded after glucose was added to 2% at time 0.
Figure 3
Figure 3. Expression and localization of V-ATPase in erg24Δ.
Vph1p (A) and Vma5p (B) were tagged with C-terminal GFP at their chromosomal loci in WT and erg24Δ strains. Vacuolar membranes were stained with FM4-64 for 30 min in YPD and chased for another 30 min with fresh YPD. (C) Immunoblotting of Vph1p and Vma2p in vacuolar vesicles isolated from WT and erg24Δ. (D) Vma2p to Vph1p ratio in WT was designated as V1/Vo ratio of 1. Calculation was based on data from three independent vacuolar vesicle preparations for each strain. Means of the ratios and standard deviation are plotted.
Figure 4
Figure 4. Ergosterol is critical for V-ATPase function.
(A) WT vacuolar vesicles were incubated with MβCD or MβCD preloaded with cholesterol (cholesterol to MβCD ratio 1∶20 by weight) at 4°C for 30 min. Vacuolar vesicles were spun down and analyzed for ATPase function. (B) WT vacuolar vesicles treated with MβCD for 30 min at 4°C were spun down and used for immunoblotting to assess the abundance of Vph1p and Vma2p.
Figure 5
Figure 5. Fluconazole treatment disrupts V-ATPase function.
(A) Vacuolar pH of WT (BY4742) cultures treated with fluconazole at specified concentrations for 6 hours in YPD. (B) Proton pumping rate and ATPase activity of WT (BY4742) cultures treated with or without fluconazole for 6 hours in YPD. Three independent batches of vacuolar vesicles were isolated and used to assay V-ATPase function. Cultures of upc2-1 mutant, WYP361, were treated with fluconazole (100 µg/ml), ergosterol (50 µM) or their combination at OD 0.1. Growth (C, representative of three independent experiments) was assessed at 8 hour post-treatment, and vacuolar pH (D) was assessed at 6 hour post-treatment. (E) WT C. albicans cells (SC5314) were grown in YPD with or without fluconazole for 5 hours. FM4-64 was added to the cultures to stain vacuoles for 30 min. Cells were chased with fresh YPD with or without fluconazole for 20 min followed by quinacrine addition for another 5 min. Fluorescence microscopic images of the cells were taken immediately after washing. Means and standard deviations are plotted. p values of two-tailed t-test are shown.
Figure 6
Figure 6. Synergy between fluconazole and amiodarone.
(A) Early log phase cells expressing pHluorin were grown for 6 hours to mid log phase (OD ∼1) in SC minus leucine medium with or without fluconazole. Amiodarone (10 µM) was injected at the arrow. Measurement was done in triplicate, and the means and standard deviations are plotted . (B) Early log phase WT cells expressing aequorin were grown with or without fluconazole for 4 hours. Amiodarone (10 µM) was injected at the arrow. Ca2+-dependent Aequorin luminescence was measured in triplicate as previously described . For 45Ca uptake, early log phase WT cells of S. cerevisiae (C) and C. albicans (D) were grown with or without fluconazole (20 µg/ml for S. cerevisiae, 1 µg/ml for C. albicans) for 6 hours. Vacuolar vesicles isolated from these cultures were assayed for MgATP-dependent 45CaCl2 uptake as described in Materials and Methods . Concanamycin A was added to assess V-ATPase dependence of CaCl2 uptake. p values of two-tailed t-test were shown. (E) Mice infected with C. albicans (ATCC 36082) were treated with vehicle, fluconazole, amiodarone, and their combination intraperitoneally. On day 4 post-infection, mouse kidneys (5 per group) were removed, weighed, homogenized, serially diluted and then plated onto YPD agar plates containing chloramphenicol and ampicillin. CFU were counted after 48 hours. Means of CFU per gram of kidney under each treatment and standard errors are plotted.
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