Miconazole Induces Changes in Actin Cytoskeleton prior to Reactive Oxygen Species Induction in Yeast

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.Miconazole belongs to the azole antifungals, which are currently the most widely used antimycotics. Azoles inhibit ergosterol biosynthesis, resulting in accumulation of toxic methylated sterol intermediates and, subsequently, fungal cell growth arrest (1). Recently, an additional mode of antifungal action was uncovered for miconazole. Apart from inhibition of ergosterol biosynthesis, miconazole induces reactive oxygen species (ROS) 6 in susceptible fungi, leading to fungal cell death (2, 3).The aim of the present study was to further unravel the mode of antifungal action of miconazole and tolerance mechanisms against miconazole in yeast. Information regarding drug tolerance mechanisms is invaluable for antifungal therapy: theoretically, miconazole action can be increased by using inhibitors of identified yeast tolerance mechanisms. To our knowledge, this study is the first report on yeast tolerance mechanisms against imidazoles, such as miconazole, and their mode of action using a genome-wide screening approach in yeast. In this study, 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. However, we could identify 29 S. cerevisiae genes or open reading frames that upon deletion result in at least 4-fold hypersensitivity to miconazole and hence are involved in miconazole tolerance. The following major functional families of miconazole tolerance genes could be distinguished: (i) tryptophan biosynthesis, (ii) membrane trafficking, including endocytosis, (iii) regulation of ac...

Membrane Rafts Are Involved in Intracellular Miconazole Accumulation in Yeast Cells

“…Apart from inhibition of ergosterol biosynthesis, miconazole induces accumulation of reactive oxygen species (ROS) 3 in susceptible fungi, leading to fungal cell death (2,3). Moreover, we have demonstrated that miconazole induces actin stabilization prior to this ROS accumulation (4). These data point to an ancillary mode of action for this azole, as was already suggested in the 1970s (5).…”

Trafficking through the Late Endosome Significantly Impacts Candida albicans Tolerance of the Azole Antifungals

“…Using a high-throughput screening assay, we recently identified a variety of imidazole antifungals, including miconazole and ketoconazole as disrupting the integrity of the C. albicans vacuole (unpublished results). Some imidazoles have been proposed to possess a secondary antifungal mechanism that is independent of their inhibition of ergosterol biosynthesis and that accounts for their fungicidal activity at high concentrations (34)(35)(36)(37). We therefore tested whether the fungistatic triazole fluconazole caused similar perturbation of the C. albicans vacuole.…”

Phytosphingosine-1-Phosphate Is a Signaling Molecule Involved in Miconazole Resistance in Sessile Candida albicans Cells

“…Besides this fungistatic mechanism of action, recent data indicate a fungicidal effect of miconazole (an imidazole) against Candida species cells in suspension and in young and mature biofilms (17,28). The accumulation of reactive oxygen species appears to be involved in this process, although it is likely that other mechanisms are also involved in the fungicidal activity (10,16,27). Despite the observed fungicidal activity of miconazole also against biofilms, 1% to 10% of sessile C. albicans cells survive exposure to high levels of this antifungal agent (28).…”