Abstract
The antifungal compound miconazole inhibits ergosterol biosynthesis and induces reactive oxygen species (ROS) in susceptible yeast species. To further uncover the mechanism of miconazole antifungal action and tolerance mechanisms, we screened the complete set of haploid Saccharomyces cerevisiae gene deletion mutants for mutants with an altered miconazole sensitivity phenotype. We identified 29 S. cerevisiae genes, which when deleted conferred at least 4-fold hypersensitivity to miconazole. Major functional groups encode proteins involved in tryptophan biosynthesis, membrane trafficking including endocytosis, regulation of actin cytoskeleton, and gene expression. With respect to the antifungal activity of miconazole, we demonstrate an antagonism with tryptophan and a synergy with a yeast endocytosis inhibitor. Because actin dynamics and induction of ROS are linked in yeast, we further focused on miconazole-mediated changes in actin cytoskeleton organization. In this respect, we demonstrate that miconazole induces changes in the actin cytoskeleton, indicative of increased filament stability, prior to ROS induction. These data provide novel mechanistic insights in the mode of action of a ROS-inducing azole.
Highlights
The aim of the present study was to further unravel the mode of antifungal action of miconazole and tolerance mechanisms against miconazole in yeast
Identification of Miconazole-hypersensitive S. cerevisiae Deletion Mutants—To identify genes that may contribute to miconazole tolerance or its antifungal action, we screened the haploid set of S. cerevisiae mutants individually deleted for 4853 nonessential yeast genes for hypersensitivity or resistance to miconazole
Most genes fall into a limited set of functional classes and define specific areas of cellular biology, including (i) tryptophan biosynthesis, (ii) membrane trafficking including endocytosis, (iii) regulation of actin cytoskeleton, and (iv) regulation of gene expression
Summary
The aim of the present study was to further unravel the mode of antifungal action of miconazole and tolerance mechanisms against miconazole in yeast. We screened the haploid set of Saccharomyces cerevisiae deletion mutants in non-essential genes for both hypersensitivity and resistance to miconazole by determining the minimal inhibitory miconazole concentration (MIC) for all individual yeast knock-out mutants using 2-fold dilution series of miconazole in liquid YPD medium. In this way, no miconazole-resistant yeast mutants could be identified. The following major functional families of miconazole tolerance genes could be distinguished: (i) tryptophan biosynthesis, (ii) membrane trafficking, including endocytosis, (iii) regulation of actin cytoskeleton, and (iv) regulation of gene expression These genetic data were confirmed by biochemical tests using excess Trp or the endocytosis inhibitor compound 5235236 (C5235236) [5]. Because the actin cytoskeleton in yeast has been demonstrated to regulate ROS induction and apoptosis or programmed cell death in yeast via interactions with the mitochondria [6], we further focused on the effect of the ROS-inducing azole miconazole on actin cytoskeleton in yeast
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