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

Open AccessPublished:June 06, 2007DOI:https://doi.org/10.1074/jbc.M608505200
      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 (
      • Vanden Bossche H.
      • Marichal P.
      • Willemsens G.
      • Bellens D.
      • Gorrens J.
      • Roels I.
      • Coene M.C.
      • Le Jeune L.
      • Janssen P.A.
      ). Recently, an additional mode of antifungal action was uncovered for miconazole. Apart from inhibition of ergosterol biosynthesis, miconazole induces reactive oxygen species (ROS)
      The abbreviations used are: ROS, reactive oxygen species; MIC, minimal inhibitory concentration of miconazole; MM, minimal medium.
      6The abbreviations used are: ROS, reactive oxygen species; MIC, minimal inhibitory concentration of miconazole; MM, minimal medium.
      in susceptible fungi, leading to fungal cell death (
      • Kobayashi D.
      • Kondo K.
      • Uehara N.
      • Otokozawa S.
      • Tsuji N.
      • Yagihashi A.
      • Watanabe N.
      ,
      • François I.E.J.A.
      • Cammue B.P.A.
      • Borgers M.
      • Ausma J.
      • Dispersyn G.D.
      • Thevissen K.
      ).
      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. A similar approach was previously used by Parsons et al. (
      • Parsons A.B.
      • Brost R.L.
      • Ding H.
      • Li Z.
      • Shang C.
      • Sheikh B.
      • Brown G.W.
      • Kane P.M.
      • Hughes T.R.
      • Boone C.
      ) to uncover yeast tolerance mechanisms against various chemical compounds, including the triazole fluconazole. However, miconazole and fluconazole are characterized by different activity features: fluconazole does not seem to induce ROS to the same extent as miconazole in susceptible fungi (
      • Kobayashi D.
      • Kondo K.
      • Uehara N.
      • Otokozawa S.
      • Tsuji N.
      • Yagihashi A.
      • Watanabe N.
      ,
      • François I.E.J.A.
      • Cammue B.P.A.
      • Borgers M.
      • Ausma J.
      • Dispersyn G.D.
      • Thevissen K.
      ).
      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 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) (
      • Toenjes K.A.
      • Munsee S.M.
      • Ibrahim A.S.
      • Jeffrey R.
      • Edwards J.E.
      • Johnson D.I.
      ). Regarding miconazole antifungal action, we could demonstrate an antagonism with Trp on one hand and a synergy with C5235236 on the other hand. Moreover, 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 (
      • Gourlay C.W.
      • Ayscough K.R.
      ), we further focused on the effect of the ROS-inducing azole miconazole on actin cytoskeleton in yeast.

      EXPERIMENTAL PROCEDURES

      Yeast Strains and Media—Yeast strains used are S. cerevisiae strain BY4741, the BY4741-derived deletion mutant library (4853 mutants; Invitrogen), S. cerevisiae strain W303-1A (MATa leu2–3/112 ura3-1 trp1-1 his3–11/15 ade2-1 can1– 100), and Candida albicans strain SC5314 CAI (
      • Fonzi W.A.
      • Irwin M.Y.
      ). The media used were YPD (1% yeast extract, 2% peptone, 2% glucose), MM-Trp (0.8 g/liter complete supplement mixture-Trp, complete amino acid supplement mixture minus tryptophan, Bio 101 Systems; 6.5 g/liter yeast nitrogen base; 20 g/liter glucose), minimal medium (MM)-Tyr or MM-Phe (6.5 g/liter yeast nitrogen base; 20 g/liter glucose; 20 mg/liter adenine sulfate; 20 mg/liter uracil; 20 mg/liter histidine; 20 mg/liter arginine; 20 mg/liter methionine; 20 mg/liter tryptophan; 30 mg/liter leucine; 30 mg/liter isoleucine; 30 mg/liter lysine; 100 mg/liter glutamic acid; 100 mg/liter aspartic acid; 150 mg/liter valine; 200 mg/liter threonine; 400 mg/liter serine, supplemented with either 50 mg/liter phenylalanine or 30 mg/liter tyrosine). The endocytosis inhibitor compound 5235236 (C5235236) was purchased from Chem Bridge (San Diego, CA).
      Screening of a Yeast Deletion Mutant Library for Altered Miconazole Sensitivity—The minimal inhibitory concentration of miconazole for the individual deletion mutants (MIC) was determined in YPD medium in sealed 96-well microplates and compared with MIC of S. cerevisiae BY4741 wild type. To this end, 80 μl of a stationary culture of each deletion mutant, grown in YPD in 96-well microplates and diluted in fresh YPD to ∼106 cells/ml, was added to 20 μl of a miconazole dilution series, ranging from 12.5 to 0.025 μg/ml, in 2% Me2SO. A same concentration of Me2SO served as negative control. The 96-well microplates were sealed with plastic EASYseal stickers (Greiner Bio-One) and incubated for 24 h at 30 °C. Growth of the yeast cultures was assessed microscopically, and MIC for each yeast deletion strain was determined. Slow growing yeast mutants were incubated for 48 h till A600 of the corresponding 2% Me2SO control was >0.7 as determined by a microplate reader (VERSAmax, Molecular Devices, Sunnyvale, CA).
      Effect of Endocytosis Inhibitor Compound 5235236, Tryptophan, Tyrosine, and Phenylalanine on Miconazole Action—To test the effect of tryptophan (Trp), tyrosine (Tyr), or phenylalanine (Phe) on the antifungal action of miconazole, an overnight S. cerevisiae W303-1A or Candida albicans culture grown in YPD was 1/100 diluted (∼106 cells/ml) in MM-Trp, MM-Tyr or MM-Phe, respectively, in the presence of miconazole or Me2SO in combination with concentrations of Trp, Tyr, or Phe ranging from 0.1 to 10 mm. To test the effect of C5235236, dissolved in Me2SO, the yeast cultures were 1/100 diluted in phosphate-buffered saline (∼106 cells/ml) in the presence of miconazole or Me2SO in combination with C5235236. After 5 h of incubation in sealed 96-well microplates at 37 °C, viability of the yeast cultures was analyzed by counting the number of colony-forming units/ml on YPD agar plates.
      Fluorescence Methods for Visualization of Actin and Cell Nuclei and for Detection of ROS—Rhodamine-phalloidin staining was performed as previously described for F-actin (
      • Gourlay C.W.
      • Ayscough K.R.
      ,
      • Hagan I.M.
      • Ayscough K.R.
      ). Fixed cells were mounted in medium containing the DNA binding dye 4′6′ diamidino 2-phenylindole dihydrochloride to allow nuclei to be visualized. For co-detection of endogenous ROS levels, miconazole-treated cells were incubated in the presence of 5 μg/ml 2′,7′-dichlorodihydro fluorescein diacetate (Molecular Probes) as described (
      • Gourlay C.W.
      • Carpp L.N.
      • Timpson P.
      • Winder S.J.
      • Ayscough K.R.
      ). Briefly, flow cytometry parameters were set at excitation and emission settings of 304 and 551 nm (filter FL-1), respectively. For each experiment two peaks of fluorescence were observed. The second peak represents cells with high levels of ROS. The summary data plotted in Fig. 3 are the percentage of cells in this second peak. In each case experiments have been repeated at least three times independently. Representative data sets are depicted. Errors are calculated as S.D. from each of these three experiments.
      Figure thumbnail gr3
      FIGURE 3Effect of miconazole on actin in S. cerevisiae. A, wild type S. cerevisiae (BY4741) cells in exponential growth phase were incubated with Me2SO (2%) (upper panels) or 20 μg/ml miconazole (lower panels) for 4 h. Cells were fixed and stained with rhodamine-phalloidin to determine organization of F-actin structures and co-stained with 4′,6-diamidino-2-phenylindole for cell nuclei visualization. Bar,5 μm. B, following miconazole addition, cells were monitored over a period of 4 h for changes in their actin structures and for changes in the level of cellular ROS. The error bars represent the S.D. of three independent experiments.

      RESULTS

      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. Because miconazole susceptibility testing of yeast strains in liquid medium is hampered by trailing growth, i.e. growth of the yeast strain at concentrations above the MIC, selection of an appropriate concentration to analyze the sensitivity phenotype of the yeast mutants is difficult. Therefore, in this study, all the individual yeast mutants were analyzed against 2-fold dilution series of miconazole. Unfortunately, we could not identify miconazole-resistant yeast deletion mutants, i.e. deletion mutants with at least 4-fold increased MIC, indicating that susceptibility of yeast to miconazole in liquid medium is not determined by any of the non-essential genes in S. cerevisiae. Strains with at least 4-fold reduced MIC as compared with MIC for BY4741 wild type, i.e. 1 μg/ml after 24 h of incubation, were designated miconazole-hypersensitive. In this way, we could identify 29 deletion mutants with at least 4-fold increased sensitivity to miconazole. The identified miconazole tolerance genes are listed in Table 1 with their corresponding miconazole hypersensitivity factor (MicoHSF), which was calculated as MIC(WT)/MIC(mutant). 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.
      TABLE 1Miconazole tolerance genes in S. cerevisiae
      GeneOpen reading frameDescription of gene productMicoHSF
      MicoHSF, miconazole-hypersensitivity factor (MIC (BY4741)/MIC (deletion mutant)).
      Tryptophan biosynthesis (17%)
      ARO2YGL148wBifunctional chorismate synthase and flavin reductase16
      ARO1YDR127wPentafunctional chorismate biosynthesis catalyzing enzyme8
      TRP3YKL211cBifunctional enzyme: indole-3-glycerol-phosphate synthase and anthranilate synthases8
      TRP1YDR007wPhosphoribosylanthranilate isomerase4
      TRP2YER090wAnthranilate synthase catalyzing4
      Membrane trafficking including endocytosis (24%)
      ARV1YLR242cProtein required for normal intracellular sterol distribution and for sphingolipid metabolism16
      GCS1YDL226cADP-ribosylation factor GTPase activating protein, involved in ER-Golgi transport16
      DRS2YAL026cPhospholipids translocase, involved in maintaining lipid bilayer asymmetry8
      RCY1YJL204cF-box protein involved in recycling plasma membrane proteins internalized by endocytosis8
      CDC50YCR094wNoncatalytic subunit of Drs2p, present in the endosomal/trans-Golgi network compartments4
      TVP18YMR071cIntegral membrane protein localized to late Golgi vesicles along with the v-SNARE Tlg2p4
      CLC1YGR167wClathrin light chain, subunit of the coatamer involved in protein transport and endocytosis4
      Regulation of actin cytoskeleton (14%)
      VPS1YKR001cMember of the dynamin family of GTPases, functions in actin cytoskeleton organization8
      HOF1YMR032wProtein required for cytokinesis, regulates actomyosin ring dynamics and septin localization4
      SSD1YDR293cProtein with a role in maintenance of cellular integrity4
      TPD3YAL016wRegulatory subunit A of protein phosphatase 2A, required for cell morphogenesis4
      Regulation of gene expression (28%)
      CBF1YJR060wHelix-loop-helix protein, required for nucleosome positioning at the CACRTG motif4
      FYV6YNL133cProtein that interacts with Rif2, controling telomere length and establishes telomeric silencing4
      GCN5YGR252wHistone acetyltransferase, catalytic subunit of ADA/SAGA complexes4
      HFI1YPL254wAdaptor protein required for structural integrity of the SAGA complex4
      NGG1YDR176wComponent of ADA/SAGA histone acetyltransferase complexes, and the SLIK complex4
      SNT309YPR101wComponent of NineTeen complex (NTC) that is involved in mRNA splicing4
      SPT4YGR063cMediator of activation/inhibition of transcription elongation, pre-mRNA processing4
      THP2YHR167wSubunit of the THA complex, connecting transcription elongation and mitotic recombination4
      Varia (17%)
      PHO88YBR106wMembrane protein involved in phosphate intake16
      RMD8YFR048wProtein required for sporulation and meiotic nuclear division16
      CNB1YKL190wCalcineurinB, regulatory subunit of the Ca2+/calmodulin-regulated phosphatase calcineurin4
      CTR1YPR124wHigh-affinity copper transporter of the plasma membrane4
      YPR089wHypothetical protein4
      a MicoHSF, miconazole-hypersensitivity factor (MIC (BY4741)/MIC (deletion mutant)).
      The fraction of miconazole tolerance genes implicated in gene expression (27.5%) probably represents general stress tolerance mechanisms. In the next paragraphs, the obtained genetic data regarding a putative role of tryptophan biosynthesis, endocytosis, and actin cytoskeleton in miconazole tolerance will be further validated using biochemical approaches.
      Miconazole Tolerance Genes Implicated in Tryptophan Metabolism—We identified five miconazole tolerance genes involved in Trp biosynthesis (i.e. ARO1, ARO2, TRP1, TRP2, and TRP3), pointing to a protective effect of Trp against miconazole action. Indeed, incubation of S. cerevisiae W303-1A wild type with 0.1 to 5 mm Trp antagonizes the activity of 5 μg/ml miconazole (Fig. 1). Survival of the yeast culture after 5 h of incubation with 5 μg/ml miconazole was 44%, whereas addition of 0.1–5 mm Trp increased the yeast cell survival to up to 100%. These data corroborate earlier findings regarding alleviation of the growth inhibitory effect of an antifungal compound by Trp (
      • Rodriguez-Hernandez C.J.
      • Sanchez-Perez I.
      • Gil-Mascarell R.
      • Rodriguez-Afonso A.
      • Torres A.
      • Perona R.
      • Murguia J.R.
      ,
      • Schmidt A.
      • Hall M.N.
      • Koller A.
      ). In these studies, either 0.4 or 5 mm Trp was used. The observed antagonism between Trp and miconazole is lost when incubating the yeast cells with 10 mm Trp. In contrast, incubation of yeast cells with 0.5–10 mm of the other aromatic amino acids tyrosine and phenylalanine, in combination with 5 μg/ml miconazole, did not result in protection of yeast cells against miconazole antifungal action (data not shown), pointing to a Trp-specific antagonizing effect of miconazole activity.
      Figure thumbnail gr1
      FIGURE 1Effect of Trp on miconazole antifungal action. An overnight culture of S. cerevisiae, diluted 1/100 in fresh MM-Trp supplemented with various concentrations of Trp, was incubated with 5 μg/ml miconazole in sealed microtiter plates. After 5 h of incubation, viability of the yeast cultures was analyzed by counting numbers of colony-forming units/ml and % viability was determined relative to viability of Me2SO-treated yeast culture. Each bar represents the mean of three independent replicates. This figure is representative of three experiments.
      Miconazole Tolerance Genes Implicated in Membrane Trafficking including Endocytosis—We identified seven miconazole tolerance genes involved in membrane trafficking including endocytosis (i.e. ARV1, GCS1, DRS2, CDC50, TVP18, CLC1, and RCY1) and further assessed the effect of compound 5235236 (C5235236), which inhibits endocytosis in yeast (
      • Toenjes K.A.
      • Munsee S.M.
      • Ibrahim A.S.
      • Jeffrey R.
      • Edwards J.E.
      • Johnson D.I.
      ), on miconazole antifungal action. We observed a synergy between C5235236 and miconazole on S. cerevisiae cells starting from 6.3 μg/ml miconazole and 25 μg/ml C5235236 (Fig. 2A); incubation of yeast cells with either 25 μg/ml C5235236 or 6.3 μg/ml miconazole resulted in 20 or 10% survival, respectively, whereas the combined action resulted in less than 0.02% survival, pointing to a synergy of at least a factor 100.
      Figure thumbnail gr2
      FIGURE 2Effect of the endocytosis inhibitor C5235236 on miconazole antifungal action on S. cerevisiae W303 (A) and C. albicans (B). Overnight cultures were diluted 1/100 in phosphate-buffered saline and incubated with various concentrations of miconazole and either 0 μg/ml C5235236 (black circles), 6.25 μg/ml C5235236 (black squares), 12.5 μg/ml C5235236 (black triangles), 25 μg/ml C5235236 (open squares), or 50 μg/ml C5235236 (open triangles) in sealed microtiter plates. After 5 h of incubation, viability of the yeast cultures was analyzed by counting number of colony-forming units/ml and % viability was determined relative to viability of Me2SO-treated yeast culture. This figure is representative of three experiments.
      The obtained data with S. cerevisiae implicate that efficient antifungal therapy could consist of miconazole in combination with C5235236. Therefore, we assessed the effect of C5235236 on miconazole antifungal action against C. albicans. We observed a similar synergy between C5235236 and miconazole starting from 25 μg/ml C5235236 and 6.3 μg/ml miconazole on C. albicans (Fig. 2B); incubation of yeast cells with either 25 μg/ml C5235236 or 6.3 μg/ml miconazole resulted in 70 or 40% survival, respectively, whereas the combined action resulted in 0.4% survival, pointing to a synergy of a factor 70. These data point to a functional endocytotic pathway as a prerequisite for miconazole tolerance in both yeast species.
      Effect of Miconazole on Actin Cytoskeleton—We identified four miconazole tolerance genes involved in regulation of the actin cytoskeleton (i.e. TPD3, SSD1, HOF1, and VPS1). Because miconazole induces ROS generation in susceptible fungi, and actin cytoskeleton has been implicated in mitochondrial activity and regulation of endogenous ROS levels (
      • Gourlay C.W.
      • Ayscough K.R.
      ), we further focused on a putative effect of miconazole on actin cytoskeleton organization. To this end, miconazole (20 μg/ml) or Me2SO was added to wild type yeast cells (BY4741) during exponential growth phase. Samples were taken hourly to determine the effects of the drug on the actin cytoskeleton, using the actin visualization dye rhodamine-phalloidin. In untreated cells, the normal organization of cortical actin patches and polarized actin cables were clearly observed at all time points (Fig. 3A). Addition of 20 μg/ml miconazole resulted in changes in the actin cytoskeleton such that 66% of cells contained F-actin aggregates after 3 h of miconazole treatment; this increased to 82% after 4 h of miconazole treatment (Fig. 3, A and B). Experiments that were conducted in parallel to determine the level of ROS in cells after 2, 3, and 4 h of miconazole incubation indicated that the proportion of cells with high levels of ROS was less than 1% after 2 h, rising to 12% after 4 h (Fig. 3B). Analysis of these cells by flow cytometry indicated no levels of autofluorescence in the samples in the absence of the ROS detection dye (data not shown). After 2 h of miconazole incubation, cell nuclei and mitochondria as visualized by 4′,6-diamidino-2-phenylindole staining appeared the same as in untreated cells with no observable condensation or fragmentation detected (data not shown). After 4 h of miconazole treatment, some nuclear fragmentation could be detected (Fig. 3A). In addition, loss of mitochondrial morphology was observed as faint 4′,6-diamidino-2-phenylindole-positive spots in miconazole-treated cells (Fig. 3A). This is not surprising because mitochondria align along actin cables, which are disrupted upon miconazole treatment. All these data indicate that the changes to the actin cytoskeleton precede the induction of ROS and are in agreement with the model that increased actin stability can act as a trigger for ROS induction and programmed cell death pathway in yeast (
      • Gourlay C.W.
      • Ayscough K.R.
      ,
      • Gourlay C.W.
      • Carpp L.N.
      • Timpson P.
      • Winder S.J.
      • Ayscough K.R.
      ).
      Moreover, we assessed the actin cytoskeleton organization in the identified four miconazole-hypersensitive yeast mutants (Fig. 4). Interestingly, the actin cytoskeleton of these mutants closely resembles the actin cytoskeleton of wild type yeast cells after miconazole treatment, pointing to the importance for the actin cytoskeleton in miconazole toxicity in yeast.
      Figure thumbnail gr4
      FIGURE 4Actin organization in miconazole-sensitive mutants. Wild type cells and Δtpd3, Δvps1, Δhof1, and Δssd1 mutant strains were grown to late logarithmic phase overnight. Cells were then fixed and processed for rhodamine-phalloidin visualization of F-actin structures. Bar,5 μm.

      DISCUSSION

      To uncover tolerance mechanisms of yeast against the imidazole miconazole and gain more insight in its mode of action, we screened the complete set of haploid deletion mutants of S. cerevisiae for increased sensitivity and resistance toward miconazole. No miconazole-resistant yeast deletion mutants could be identified, indicating that susceptibility of yeast to miconazole in liquid medium is not determined by any of the non-essential genes in S. cerevisiae. However, we identified 29 genes in yeast that upon deletion result in at least 4-fold hyper-sensitivity toward miconazole and hence are implicated in miconazole tolerance. Major functional groups of miconazole tolerance genes are implicated in (i) tryptophan biosynthesis, (ii) membrane trafficking including endocytosis, (iii) regulation of actin cytoskeleton, and (iv) regulation of gene expression. Using haplo-insufficiency profiling data, Flaherty et al. (
      • Flaherty P.
      • Giaever G.
      • Kumm J.
      • Jordan M.I.
      • Arkin A.P.
      ) recently identified various genes not previously known to confer sensitivity to azoles. Among these were the membrane trafficking-related TVP18, HFI1 involved in gene expression and YPR090w encoding a hypothetical protein (
      • Flaherty P.
      • Giaever G.
      • Kumm J.
      • Jordan M.I.
      • Arkin A.P.
      ), which were also identified as miconazole tolerance genes in this study.
      Because miconazole induces ROS in susceptible fungi, it is surprising that none of the genes encoding proteins involved in (i) antioxidant functions, such as peroxidases, catalases, and glutaredoxin, (ii) mitochondrial function, including protein synthesis, respiration, and mitochondrial genome maintenance, or (iii) specific oxidative stress transcription factors were identified as miconazole tolerance genes. However, we recently performed a similar screen on solid medium for miconazole hypersensitivity in yeast.
      I. E. J. A. François, unpublished data.
      In this genome-wide screen, yeast mutants with at least 4-fold increased miconazole hypersensitivity were retained. Interestingly, in this screen, 30% of the identified miconazole tolerance genes were involved in mitochondrial function. Whether differences in oxygen tension between screening of yeast mutants on solid and liquid media account for the different sets of miconazole tolerance genes is currently under investigation.
      The fraction of miconazole tolerance genes implicated in gene expression (27.5%) probably represents general stress tolerance mechanisms. In this respect, yeast mutants affected in genes involved in DNA synthesis and repair, transcription, and chromatin structure (including ADA/Spt-Ada-Gcn5-acetyltransferase (SAGA) histone acetyltransferase complexes or SWI/SNF nucleosome remodeling complex) were previously identified as hypersensitive to a variety of stresses, including oxidative and chemical stress (
      • Jain P.
      • Akula I.
      • Edlind T.
      ,
      • Thorpe G.W.
      • Fong C.S.
      • Alic N.
      • Higgins V.J.
      • Dawes I.W.
      ,
      • Tucker C.L.
      • Fields S.
      ). This finding highlights the requirement for de novo transcription in response to environmental stress. In accordance with the above genetic data, histone deacetylase inhibitors like trichostatin A have been shown to enhance C. albicans sensitivity to azoles (
      • Smith W.L.
      • Edlind T.D.
      ).
      Regarding the importance of tryptophan biosynthesis for miconazole tolerance, we could demonstrate a specific antagonism between Trp and miconazole action. Other aromatic amino acids like tyrosine and phenylalanine are not able to protect yeast cells against miconazole antifungal action. Various studies have postulated a general role for Trp in antifungal resistance. Trp was shown to mitigate the antifungal activity of ibuprofen, an anti-inflammatory agent with antifungal action, and of the calcineurin inhibitor FK506, an immunosuppressant with antifungal activity (
      • Tucker C.L.
      • Fields S.
      ,
      • Heitman J.
      • Koller A.
      • Kunz J.
      • Henriquez R.
      • Schmidt A.
      • Movva N.R.
      • Hall M.N.
      ). Interestingly, we also found the calcineurin cnb1 deletion mutant to be hypersensitive to miconazole, as was previously demonstrated by Edlind et al. (
      • Edlind T.
      • Smith L.
      • Henry K.
      • Katiyar S.
      • Nickels J.
      ). However, it is not clear how Trp can protect yeast cells against various stresses.
      In accordance with the observed importance for membrane trafficking and regulation of actin cytoskeleton in miconazole tolerance, we could demonstrate a synergy between miconazole and an inhibitor of endocytosis (C5235236) in yeast. The obtained data with S. cerevisiae implicate that efficient antifungal therapy could consist of miconazole in combination with an endocytosis inhibitor. Indeed, our observations with S. cerevisiae appear to have direct relevance to the human pathogen C. albicans; we demonstrate a synergy between miconazole and C5235236 on this opportunistic pathogen. These data corroborate the very recent findings of Cornet et al. (
      • Cornet M.
      • Gaillardin C.
      • Richard M.L.
      ), who demonstrate that deletion of two endocytic components in C. albicans lead to azole hypersensitivity. Although C5235236 (
      • Toenjes K.A.
      • Munsee S.M.
      • Ibrahim A.S.
      • Jeffrey R.
      • Edwards J.E.
      • Johnson D.I.
      ) is cytotoxic, it should be possible to identify derivatives that are acceptable in this respect. Whether such (micon)azole combinatorial antifungal therapy is efficient in vivo needs to be investigated further.
      Because miconazole induces ROS generation in susceptible fungi and actin cytoskeleton has been implicated in mitochondrial activity and regulation of endogenous ROS levels and apoptosis in yeast (
      • Gourlay C.W.
      • Ayscough K.R.
      ,
      • Gourlay C.W.
      • Du W.
      • Ayscough K.R.
      ), we further focused on a putative effect of miconazole on actin cytoskeleton organization. Interestingly, we could demonstrate that miconazole induces changes in actin cytoskeleton indicative of increased filament stability. Similar actin cytoskeleton changes were observed in miconazole-hypersensitive mutants impaired in the regulation of actin cytoskeleton, pointing to the importance for the actin cytoskeleton in miconazole toxicity. The actin filament stabilization was induced by miconazole prior to its ROS induction. This finding is in agreement with the model that increased actin stability can act as a trigger for ROS induction and apoptosis in yeast (
      • Gourlay C.W.
      • Ayscough K.R.
      ,
      • Gourlay C.W.
      • Carpp L.N.
      • Timpson P.
      • Winder S.J.
      • Ayscough K.R.
      ). In this respect, the observed nuclear fragmentation in some miconazole-treated yeast cells further points to apoptosis induction by miconazole. Assays such as terminal deoxynucleotidyl transferase biotin-dUTP nick-end labeling (TUNEL) or annexin V staining (for detection of DNA fragmentation phosphatidylserine flip-flop at the plasma membrane, respectively) (
      • Madeo F.
      • Frohlich E.
      • Frohlich K.U.
      ) could now be used to substantiate our findings further. The induction of apoptosis in yeast by antifungal azoles has never been described before. Moreover, fluconazole toxicity was previously found not to depend on oxidative stress or apoptotic effector mechanisms in yeast (
      • Kontoyiannis D.P.
      • Murray P.J.
      ). There is only one other report on an antifungal drug affecting the organization of the actin cytoskeleton, namely jasplakinolide (
      • Ayscough K.R.
      ). Jasplakinolide is an antifungal cyclodepsipeptide that induces F-actin stability and prevents turnover such that all actin becomes incorporated in a single large F-actin clump. Jasplakinolide has also been observed to induce ROS (and apoptosis) in S. cerevisiae (
      • Gourlay C.W.
      • Carpp L.N.
      • Timpson P.
      • Winder S.J.
      • Ayscough K.R.
      ).
      In conclusion, this is the first report describing an effect of an azole on the organization of the actin cytoskeleton in yeast, resulting in actin clumping prior to induction of endogenous ROS and cell death. This ROS accumulation is most likely the result of dysfunctional mitochondria. Such actin-mediated ROS production possibly involves Ras activation (
      • Gourlay C.W.
      • Du W.
      • Ayscough K.R.
      ). In yeast, Ras activation leads to cAMP production and activation of protein kinase A. A link between cAMP-PKA signaling pathway and miconazole sensitivity in yeast has previously been reported (
      • Jain P.
      • Akula I.
      • Edlind T.
      ); it was demonstrated that yeast mutants affected in cAMP-PKA signaling were hypersensitive toward miconazole and the observed miconazole hypersensitivity of these mutants could be partially restored by exogenous addition of cAMP (
      • Jain P.
      • Akula I.
      • Edlind T.
      ). Whether miconazole activates Ras signaling in yeast, resulting in elevated cAMP levels and ROS levels via actin-mediated mitochondrial dysfunction, needs to be investigated further.

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