François Isabelle E J A, Bink Anna, Vandercappellen Jo, Ayscough Kathryn R, Toulmay Alexandre, Schneiter Roger, van Gyseghem Elke, Van den Mooter Guy, Borgers Marcel, Vandenbosch Davy, Coenye Tom, Cammue Bruno P A, Thevissen Karin
Centre of Microbial and Plant Genetics, Katholieke Universiteit Leuven, 3001 Heverlee, Belgium.
J Biol Chem. 2009 Nov 20;284(47):32680-5. doi: 10.1074/jbc.M109.014571. Epub 2009 Sep 25.
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.
唑类药物抑制麦角固醇的生物合成，导致麦角固醇耗竭，并在敏感酵母的细胞膜中积累有毒的14α-甲基化固醇。我们之前证明，咪康唑在诱导活性氧之前会诱导酿酒酵母中的肌动蛋白细胞骨架稳定，这表明存在一种辅助作用模式。在本研究中，我们通过全基因组琼脂筛选证明，在鞘脂和麦角固醇生物合成中受影响的酿酒酵母突变体，即ipt1、sur1、skn1和erg3缺失突变体，对咪康唑具有抗性，这表明膜筏参与了其作用模式。膜筏干扰化合物对咪康唑抗真菌活性以及对咪康唑诱导的肌动蛋白细胞骨架稳定和活性氧积累的拮抗作用支持了这一点。这些拮抗作用表明膜筏在咪康唑抗真菌活性中起主要作用。我们进一步表明，膜筏在咪康唑作用中的这一主要作用包括介导咪康唑在酵母细胞中的细胞内积累。
