Abstract
Azoles inhibit ergosterol biosynthesis, resulting in ergosterol depletion and accumulation of toxic 14alpha-methylated sterols in membranes of susceptible yeast. We demonstrated previously that miconazole induces actin cytoskeleton stabilization in Saccharomyces cerevisiae prior to induction of reactive oxygen species, pointing to an ancillary mode of action. Using a genome-wide agar-based screening, we demonstrate in this study that S. cerevisiae mutants affected in sphingolipid and ergosterol biosynthesis, namely ipt1, sur1, skn1, and erg3 deletion mutants, are miconazole-resistant, suggesting an involvement of membrane rafts in its mode of action. This is supported by the antagonizing effect of membrane raft-disturbing compounds on miconazole antifungal activity as well as on miconazole-induced actin cytoskeleton stabilization and reactive oxygen species accumulation. These antagonizing effects point to a primary role for membrane rafts in miconazole antifungal activity. We further show that this primary role of membrane rafts in miconazole action consists of mediating intracellular accumulation of miconazole in yeast cells.
Highlights
The class of azole antimycotics constitutes the largest group of synthetic antifungal therapeutics currently in clinical use
We demonstrate that S. cerevisiae mutants affected in sphingolipid and ergosterol biosynthesis are resistant to miconazole, suggesting a possible involvement of membrane rafts in the mode of antifungal action of miconazole
We focused on the functional group of miconazole sensitivity genes implicated in sphingolipid and ergosterol biosynthesis, represented by IPT1, SKN1, SUR1, and ERG3
Summary
Yeast Strains, Plasmids, and Growth Media—Miconazole and methyl--cyclodextrin (MCD) were purchased from Sigma. Edelfosine was a kind gift from Prof. Acetonitrile was purchased from Fisher (Leicestershire, United Kingdom). The yeast strains used were S. cerevisiae strain BY4741 (wild-type (WT)) and the BY4741derived deletion mutant library (Invitrogen). These yeast strains were cultivated in yeast/peptone/dextrose (YPD; 1% yeast extract, 2% peptone, and 2% glucose). The plasmid encoding green fluorescent protein (GFP)-tagged Pma1p was a kind gift of Prof. Yeast strains transformed with this plasmid were cultured in 0.8 g/liter complete amino acid supplement mixture minus uracil (Bio 101, Inc.), 6.5 g/liter yeast nitrogen base, and 20 g/liter glucose. YPD, yeast/peptone/dextrose; GFP, green fluorescent protein; PBS, phosphate-buffered saline
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