Anethole induces apoptotic cell death accompanied by reactive oxygen species production and DNA fragmentation in Aspergillus fumigatus and Saccharomyces cerevisiae Ken-Ichi Fujita1, Miki Tatsumi1, Akira Ogita1,2, Isao Kubo3 and Toshio Tanaka1 1 Graduate School of Science, Osaka City University, Japan 2 Research Center for Urban Health and Sports, Osaka City University, Japan 3 Department of Environmental Science, Policy and Management, University of California, Berkeley, CA, USA

Keywords anethole; apoptosis; DNA fragmentation; fungicidal activity; reactive oxygen species Correspondence K.-I. Fujita, Graduate School of Science, Osaka City University, 3-3-138 Sugimoto, Sumiyoshi-ku, Osaka 558-8585, Japan Fax/Tel: +81 6 6605 2580 E-mail: [email protected] (Received 29 September 2013, revised 17 December 2013, accepted 23 December 2013) doi:10.1111/febs.12706

trans-Anethole (anethole), a major component of anise oil, has a broad antimicrobial spectrum, and antimicrobial activity that is weaker than that of other antibiotics on the market. When combined with polygodial, nagilactone E, and n-dodecanol, anethole has been shown to possess significant synergistic antifungal activity against a budding yeast, Saccharomyces cerevisiae, and a human opportunistic pathogenic yeast, Candida albicans. However, the antifungal mechanism of anethole has not been completely determined. We found that anethole stimulated cell death of a human opportunistic pathogenic fungus, Aspergillus fumigatus, in addition to S. cerevisiae. The anethole-induced cell death was accompanied by reactive oxygen species production, metacaspase activation, and DNA fragmentation. Several mutants of S. cerevisiae, in which genes related to the apoptosis-initiating execution signals from mitochondria were deleted, were resistant to anethole. These results suggest that anethole-induced cell death could be explained by oxidative stress-dependent apoptosis via typical mitochondrial death cascades in fungi, including A. fumigatus and S. cerevisiae.

Introduction An increase in the frequency of opportunistic invasive fungal infections in immunocompromised patients (e.g. owing to AIDS, leukemia, or immunosuppressive therapy after organ transplantation) and the elderly is promoting the development of antifungal antibiotics with novel modes of action [1]. The present targets of antifungals available on the market are confined to structures and functions unique to fungi, namely ergosterol, fungal cell walls, and cytosine deaminase. Polyene macrolide antifungals, represented by amphotericin B [2,3], directly interact with ergosterol in the

cytoplasmic membrane to induce the efflux of potassium ions, owing to the formation of pores in the membrane. These drugs have remarkable antifungal spectra and potencies. However, they can induce severe adverse effects such as nephropathy. Azoles, including miconazole, itraconazole, and fluconazole, block the biosynthetic pathway of ergosterol [4]. Their adverse effects include menstrual abnormalities and liver damage [5]. Selection pressure resulting from continuous exposure to azoles has been reported in Candida spp., which subsequently developed resistance

Abbreviations CFU, colony-forming unit; DAPI, 4′,6-diamidino-2-phenylindole; DCF, 2′,7′-dichlorofluorescein; DCFH, 2′,7′-dichlorodihydrofluorescein; DCFHDA, 2′,7′-dichlorodihydrofluorescein diacetate; FITC, fluorescein isothiocyanate; ME, malt extract; MIC, minimum inhibitory concentration; ROS, reactive oxygen species; TOH, a-tocopherol acetate; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling.

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to fluconazole [6]. A synthetic fluorinated analog of cytosine, 5-fluorocytosine, is converted to 5-fluorouracil by cytosolic cytosine deaminase, which human cells do not possess, in susceptible fungal cells [7]. The frequent occurrence of 5-fluorocytosine-resistant fungal strains has been reported in clinical isolates [8]. Essential oils from anise fruits, aniseed, are frequently added to various foods as a spice. transAnethole (anethole), a principal constituent of anise oil, has been reported to have antimicrobial effects on bacteria, yeasts, and filamentous fungi [9,10]. Although the antimicrobial potency of anethole is weaker than that of other antibiotics on the market, anethole was discovered to potentiate the antifungal activity of nagilactone E, polygodial and n-dodecanol against a human opportunistic pathogenic yeast, Candida albicans, and a budding yeast, Saccharomyces cerevisiae [11–13]. In addition, anethole shows less toxic effects on humans. Combination chemotherapy of anethole with other drugs gives the possibility of clinical applications with less adverse effects. In addition, revealing the mechanisms underlying the antifungal action of anethole may be beneficial for the development of antifungal agents that have less adverse effects. In this study, we found that anethole induced the cell death of a human opportunistic pathogenic fungus, Aspergillus fumigatus, accompanied by DNA fragmentation, which indicates apoptotic-like cell death. Although the mechanisms underlying the antimicrobial action of anethole have not yet been elucidated in detail, we recently revealed that anethole-induced growth inhibition with morphological changes in a filamentous fungus, Mucor mucedo, depends on the fragility of cell walls caused by chitin synthase inhibition [14]. Additionally, the inhibition of growth of Mucor spp., including M. mucedo, caused by anethole was accompanied by swollen hyphae resulting from the direct restriction of chitin and chitosan biosynthesis

Anethole-induced apoptotic cell death in fungi

[14]. Chitin and chitosan are principal components of the cell wall in zygomycetes, including Mucor spp. [15]. In A. fumigatus and S. cerevisiae, b-glucan is a main cell wall component [16]. In this study, we investigated the involvement of enzyme inhibition related to the biosynthesis of cell wall b-glucan in the anetholeinduced cell death of A. fumigatus. We also demonstrate the involvement of programmed cell death in the antifungal action of anethole against A. fumigatus and S. cerevisiae.

Results and Discussion Anethole is a major component of essential oils derived from anise and fennel. Recently, this phenylpropanoid has been reported to show a wide variety of biological activities [17–22]. Anethole has a broad spectrum of antimicrobial activity against bacteria and fungi, including human pathogens such as Staphylococcus aureus, Pseudomonas aeruginosa, and C. albicans [13,23]. Although the antimicrobial mechanisms of anethole alone have not been fully clarified, we recently revealed that anethole induced growth inhibition and morphological changes in filamentous fungi (Mucor and Fusarium spp.) via metabolic blockade of cell wall biosynthesis based on the uncompetitive inhibition of chitin synthase [14]. Additionally, the antifungal activity of anethole against a human opportunistic pathogenic fungus, A. fumigatus, has been reported [24], but the primary target of the antifungal activity of anethole is unknown. In the current study, A. fumigatus hyphae treated with anethole were stained with methylene blue dye to determine viability. Anethole-treated hyphae retained the oxidized form of methylene blue dye (Fig. 1). This finding indicated a fungicidal effect of anethole. Conversely, the viable hyphae converted the methylene blue dye to leucomethylene blue (colorless), owing to the retained Control

Anethole

Fig. 1. Anethole-induced cell death confirmed by methylene blue staining. Hyphae of Aspergillus fumigatus IFO 5840 were incubated in ME broth without (Control) or with (Anethole) 625 lM transanethole at 30 °C for 24 h. After incubation, the hyphae were stained with 0.1% methylene blue.

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reducing capacity of the hyphae (Fig. 1). Hyperosmotic conditions have been shown to weaken the antifungal activity of anethole against Mucor and Fusarium spp. [14]. However, hypo-osmotic conditions potentiated the activity against those fungi [14]. In this study, the antifungal activity of anethole against A. fumigatus was not affected by varying the osmotic pressure of the medium (data not shown), indicating that there was no fragility in the A. fumigatus cell wall. Additionally, anethole has been shown to induce the cell death of a budding yeast, S. cerevisiae [25]. We hypothesize that the antifungal activities of anethole against A. fumigatus and S. cerevisiae depend on a similar mechanism. To investigate the fungicidal action of anethole, we first examined whether anethole induced oxidative stress resulting from cellular reactive oxygen species (ROS) production in A. fumigatus by using a ROSsensitive probe, 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA). DCFH-DA is incorporated into Phase contrast

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cells and then deacetylated to 2′,7′-dichlorodihydrofluorescein (DCFH). ROS facilitate the oxidation of nonfluorescent DCFH to the fluorescent compound 2′,7′dichlorofluorescein (DCF) [26]. ROS production was estimated from the fluorescence intensity of DCF. The fluorescence derived from DCF was examined in hyphae treated with the minimum inhibitory concentration (MIC) of anethole and 2 mM H2O2 for 2 h (Fig. 2A). Fluorescence was observed in the hyphae treated with anethole. This assay was also performed with germinated spores. In the germinated spores, anethole was also found to induce ROS generation (Fig. 2B). The cellular ROS level was also quantified with a fluorescence microplate reader. In this experiment, the germinated spores, rather than the hyphae, were used for the quantitative evaluation. Treatment with the MIC of anethole significantly accelerated ROS generation by approximately four-fold as compared with the control (Fig. 2C). A membrane-permeable lipophilic antioxidant, a-tocopherol acetate

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Fig. 2. Effect of anethole on ROS generation. (A) Hyphae of Aspergillus fumigatus IFO 5840 were incubated in ME broth without or with 625 lM trans-anethole and 2 mM H2O2 at 30 °C for 2 h prior to staining with DCFHDA for the evaluation of ROS generation. (B) Germinated spores were treated for the evaluation of ROS generation under the same conditions as described in (A). (C) Germinated spores (0.1 turbidity at 530 nm) were pretreated with or without 100 lM TOH in ME medium at 30 °C for 1 h. After pretreatment, the spores were further treated with or without 625 lM trans-anethole at 30 °C for 2 h prior to the measurement of ROS generation. (D) Germinated spores (0.1 turbidity at 530 nm) were pretreated with or without 100 lM TOH in ME medium at 30 °C for 4 h. After pretreatment, the spores were treated with or without 625 lM transanethole at 30 °C for 16 h prior to the Alamar blue assay for the evaluation of viability. The viability is indicated as fluorescence intensity. In (C) and (D), data are presented as the mean  standard deviation (n = 3) and were statistically analyzed with Student’s t-test, in which P < 0.05 was considered to be statistically significant (*).

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(TOH), has been reported to restrict fungal ROS generation [26]. Indeed, in the germinated spores pretreated with 100 lM TOH for 1 h, anethole-induced ROS generation was restricted to the level of the control (Fig. 2C). These results indicate that anethole-induced ROS production in A. fumigatus is abolished by pretreatment with TOH. The correlation between cell death and oxidative stress, such as anethole-induced ROS generation, was examined. The ratio of viable hyphae to total hyphae was estimated on the basis of the Alamar blue assay [27]. Although ROS generation was observed in A. fumigatus when treated with anethole for 2 h, a decrease in the viability of A. fumigatus was not detected after anethole treatment (data not shown). This indicates that the viability of the hyphae was not affected by short-term ROS exposure. After 16 h, the MIC of anethole significantly reduced the hyphal viability to 44% of that in the untreated hyphae (Fig. 2D). However, 100 lM TOH was found to enhance the hyphal growth (Fig. 2D). Pretreatment of hyphae with TOH restored viability to the level of the control. Therefore, we conclude that the anethole-induced cell death of A. fumigatus is caused by oxidative stress. In our preliminary experiments, cycloheximide, an inhibitor of protein biosynthesis, restricted the anetholeinduced cell death of a budding yeast, S. cerevisiae (data A

Phase contrast

not shown). In the presence of cycloheximide, anethole showed fungistatic rather than fungicidal action. Apoptotic cell death depends on the active participation of the cell to synthesize new proteins [28]; therefore, apoptotic cell death is inhibited by cycloheximide [29–31]. In addition, fungal apoptotic cell death can be induced in S. cerevisiae by oxidative stress, such as the depletion of glutathione or low external doses of H2O2 [31]. We further examined the involvement of the apoptotic process in anethole-induced cell death in fungi. In an S. cerevisiae cell division cycle gene (CDC48) mutant, resulting in a cell cycle arrest, the following typical markers of apoptosis were detected: phosphatidylserine exposure at the outer layer of the cytoplasmic membrane, DNA fragmentation, and chromatin condensation and fragmentation [32]. DNA fragmentation that occurred as a result of apoptosis was detected with the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay in fungi [30,32,33]. The nuclei were also stained with 4′,6-diamidino-2-phenylindole (DAPI). In A. fumigatus hyphae treated with anethole and H2O2, TUNEL staining-positive regions colo-calized with the position of nuclei visualized by DAPI staining (Fig. 3B). The TUNEL-positive nuclei represented 52% of the total nuclei (Fig. 3C). The TUNEL assay was also performed in a budding yeast, S. cerevisiae. DNA

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Fig. 3. Anethole-induced DNA fragmentation in Saccharomyces cerevisiae and Aspergillus fumigatus. Cells of S. cerevisiae ATCC 7754 (A) and hyphae of A. fumigatus IFO 5840 (B) were treated without or with 625 lM trans-anethole and 2 mM H2O2 at 30 °C for 24 h. Nuclei were stained with DAPI. DNA fragmentation was evaluated with the TUNEL assay. In addition, the ratio of TUNEL-positive nuclei in A. fumigatus IFO 5840 is indicated as the mean  standard deviation (n = 3), and were statistically analyzed with Student’s t-test, in which P < 0.05 was considered to be statistically significant (*) (C).

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fragmentation was detected in the anethole-treated cells of the yeast (Fig. 3A). In addition, nuclear fragmentation was observed in the yeast cells. The genome of S. cerevisiae encodes a single metacaspase, Yca1p, which is cleaved like a typical caspase in response to oxidative stress [34]. On the basis of a BLAST search with S. cerevisiae Yca1p as the query, two metacaspase genes, casA and casB, were identified in the A. fumigatus genome [35]. Although metacaspase activation in A. fumigatus contributes to the apoptotic-like loss of membrane phospholipid asymmetry and cell death at the stationary phase [35], both oxidative and amphotericin B-mediated stress also induced apoptotic changes without the induction of caspase-like activity in A. fumigatus [30]. For the in situ detection of active metacaspases in A. fumigatus, fluorescein isothiocyanate (FITC)–VAD-FMK, a membrane-permeable fluorescent probe that irreversibly binds active metacaspases, was employed. In the hyphae treated with anethole and H2O2, FITC–VAD-FMK-derived fluorescence was detected (Fig. 4). The control hyphae Phase Contrast

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Fig. 4. Effect of anethole on metacaspase activity. Hyphae of Aspergillus fumigatus IFO 5840 were treated without or with 625 lM trans-anethole and 2 mM H2O2 at 30 °C for 24 h prior to staining with FITC–VAD-FMK for the evaluation of metacaspase activity.

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showed no visible fluorescence (Fig. 4). These results indicate the involvement of metacaspase activation in the anethole-induced cell death of A. fumigatus. Richie et al. [35] indicated that the caspase activity of a ΔcasA/ΔcasB mutant was indistinguishable from that of the wild-type strain, suggesting that other A. fumigatus proteases are responsible for the observed caspase-like activity. To gain further insights into the molecular mechanism underlying the anethole-induced cell death of fungi, we evaluated the anethole susceptibility of the following S. cerevisiae deletion mutants and their association with apoptotic induction: Δaif1, Δcyc1, Δcyc7, Δcyc3, Δyca1, Δfis1, Δdnm1, and Δmdv1. In eukaryotes, many stimuli are transmitted as a cascade of apoptotic execution signals starting from the mitochondria, namely the signals generated by various environmental changes that are first consolidated into the mitochondria. Aif1p translocates from the mitochondria to the nucleus upon apoptosis induction in yeast as well as in mammals [36]. However, the increase in the mRNA expression of aifA, which encodes the AIF-like mitochondrial oxidoreductase, was also observed in farnesol-induced apoptosis of Aspergillus nidulans [37]. Furthermore, the ΔaifA mutant of A. nidulans was more sensitive to farnesol, because of excessive ROS generation [37]. In contrast to the human and yeast homologs, A. nidulans AifAp does not migrate to the nucleus upon farnesol-induced cell death [37]. Cytochrome c is also released from the mitochondria [38]. S. cerevisiae cells possess two isoforms of cytochrome c genes, cyc1 and cyc7. In fungi, the deletion of cyc1 and cyc7 resulted in resistance to manganeseinduced apoptosis [39], indicating that cytochrome c release is also involved in apoptotic cell death [40]. Holocytochrome c synthase (Cyc3p) attaches heme to apocytochrome c, Cyc1p or Cyc7p in the mitochondrial intermembrane space. Δcyc3 mutants are also resistant to hyperosmotic stress-induced metacaspasedependent and mitochondria-dependent apoptosis [41]. The metacaspase Yca1p has been reported to mediate cell death in response to various stimuli, including fungal apoptosis [32,42]. Δyca1 was reported to be resistant to occidiofungin-induced cell death [43]. The mitochondrial fission machinery, which consists of Fis1p, Dmn1p, and Mdv1p, is also involved in metacaspase-dependent apoptosis in yeast [44–46]. In acetic acid-induced and H2O2-induced apoptosis, Fis1p restricts mitochondrial fission mediated by Dnm1p and Mdv1p and subsequent cell death. This indicates a prosurvival function of Fis1p and a proapoptotic function of Dnm1p and Mdv1p during cell death [44,47]. Among the deletion mutants tested in this study, only FEBS Journal 281 (2014) 1304–1313 ª 2014 FEBS

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Δfis1 facilitated anethole-induced cell death (Fig. 5). However, all of the other mutants tested, except Δfis1, were significantly resistant to apoptosis (Fig. 5). In particular, the participation of metacaspase in the fungicidal action of anethole was strongly supported by the results obtained with Δyca1 (Figs 4 and 5). The yeast protein Ybh3p interacts with BCL-X(L) and harbors a functional BH3 domain [48]. Ybh3p translocates to mitochondria and triggers BH3 domaindependent apoptosis [48]. On treatment with H2O2 and acetic acid, Dybh3 cells showed reduced cell death, ROS induction, and apoptotic phosphatidylserine externalization [48]. Thus, Ybh3p regulates apoptotic cell death. Regarding ROS induction and survival rate, similar results were obtained in Dybh3 cells treated with anethole (Figs 5 and 6). A yeast endonuclease G (Nuc1p), when excluded from mitochondria, induces apoptosis independently of metacaspase or of apoptosis-inducing factors [49]. On treatment with H2O2, the overexpression of Nuc1p reduced survival and sensitized cells to apoptotic death [49]. However, on treatment with with H2O2 and acetic acid, Dnuc1 cells showed slightly accelerated cell death and ROS induction [49]. Anethole facilitated ROS induction, although the survival rate was similar to that of the wild-type cells (Figs 5 and 6). In conclusion, our results are in good agreement with other results showing that that apoptotic stimuli induce apoptotic execution signals from the mitochondria. Therefore, anethole most likely causes oxidative stress-dependent apoptosis via typical mitochondrial

death cascades in fungi, including A. fumigatus and S. cerevisiae.

Experimental procedures Chemicals All chemicals were purchased from Sigma-Aldrich (St Louis, MO, USA) unless otherwise stated. Drugs, including anethole, were diluted with N,N-dimethylformamide prior to performance of the experiments.

Microbial strains and culture A. fumigatus IFO 5840 was obtained from the Institute for Fermentation (Osaka, Japan). The wild-type strain of S. cerevisiae ATCC 7754 was obtained from the American Type Culture Collection (Manassas, VA, USA). The parental strain of S. cerevisiae BY4741 (MATa, ura3D0, leu2D0, met15D0, and his3D1) and its deletion mutants were purchased from Open Biosystems (Lafayette, CO, USA). Prior to performance of the experiments, the spores of A. fumigatus harvested from potato dextrose agar plates were suspended in 3 mL of 2.5% malt extract (ME; Oriental Yeast, Tokyo, Japan) broth to achieve 0.05 turbidity at 530 nm. Additionally, exponentially growing cells of S. cerevisiae were suspended in 3 mL of ME broth to achieve 106 CFUmL 1. Yeast cell viability was determined by measuring the viable cell numbers as colony-forming units (CFUs).

Antimicrobial susceptibility test The MICs were determined with a serial broth dilution method. After the addition of 30 lL of drug solution to 3 mL of ME broth containing fungal spores or yeast cells in test tubes (10 mm in diameter), the culture was incubated statically at 30 °C for 48 h. After incubation, the MIC was defined as the lowest concentration of the test compound with which no visible growth was seen.

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Strain Fig. 5. Effect of anethole on the viability of Saccharomyces cerevisiae deletion mutants. The parent and deletion mutant cells of S. cerevisiae BY 4741 (106 cellsmL 1) were incubated in YPD broth with or without 625 lM anethole at 30 °C for 4 h. After incubation, the viability was estimated according to CFUs. Data are presented as the mean  standard deviation (n = 3). The cell viability of each deletion mutant was significantly different from that of the parent cells (P < 0.05).

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The spores of A. fumigatus that were suspended in ME broth to achieve 0.05 turbidity at 530 nm were incubated at 30 °C for 24 h. After incubation, 40 lM DCFH-DA was added to the culture, which was then incubated at 30 °C for 1 h. Anethole or H2O2 was added to the culture, which was then incubated at 30 °C for 2 h. The hyphae were washed with NaCl/ Pi twice on a membrane filter (Ultrafree-MC; Millipore, Billerica, MA, USA), and then suspended in 200 lL of NaCl/Pi. The stained hyphae were observed under a fluorescence microscope (excitation wavelength of 485 nm; emission wavelength of 535 nm). ROS generation was also detected in

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Fig. 6. Effect of anethole on ROS generation by Saccharomyces cerevisiae deletion mutants. After treatment with DCFH-DA, the parent and deletion mutant cells of S. cerevisiae BY 4741 (107 cellsmL 1) were incubated in YPD broth with or without 625 lM anethole at 30 °C for 1 h. The cells were observed by the use of phase-contrast microscopy and fluorescence microscopy (A–C). ROS generation was estimated on the basis of DCFD-DA staining (D). Data are presented as the mean  standard deviation (n = 3), and were statistically analyzed with Student’s t-test, in which P < 0.05 was considered to be statistically significant (*).

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germinated spores with a Tecan GENios Fluorescence Detector (MTX Lab Systems, Vienna, VA, USA) at an excitation wavelength of 485 nm and an emission wavelength of 535 nm. S. cerevisiae cells (107 cellsmL 1) were incubated in YPD (1% yeast extract, 2% peptone, 2% dextrose) broth with 40 lM DCFH-DA at 30 °C for 1 h. Then, anethole was added to the culture, which was further incubated at 30 °C for 1 h. After washing with NaCl/Pi, the cells were observed under a fluorescence microscope (excitation wavelength of 485 nm; emission wavelength of 535 nm), and the fluorescence was quantified with the Tecan GENios Fluorescence Detector (MTX Lab Systems) as described above.

TUNEL assay The TUNEL assay was performed with the method of Kitagaki et al. [33], with modifications. The spores of A. fumigatus that were suspended in ME broth to achieve 0.05 turbidity at 530 nm were incubated in ME broth at

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30 °C for 24 h. After incubation, anethole or H2O2 was added to the culture, and the culture was then incubated statically at 30 °C for 24 h. Hyphae were washed with NaCl/Pi twice on a membrane filter, and then suspended in 100 lL of NaCl/Pi. The hyphal suspensions were dropped onto round glass slides coated with poly(L-lysine) and thoroughly dried. The hyphae were washed with NaCl/Pi three times, and then fixed with 3.7% formaldehyde in NaCl/Pi at room temperature for 30 min. After fixation, the hyphae were washed once with NaCl/Pi, and then with 1.2 M sorbitol in NaCl/Pi. The hyphae were incubated with gentle shaking in 50 mM trisodium citrate buffer (pH 5.8) containing 16 mg mL 1 b-D-glucanase, 81.4 units mL 1 lyticase, 10 mg mL 1 driselase and 10% egg white at room temperature for 1 h to lyse the cell wall. Prior to use, 200 mg mL 1 b-D-glucanase was preincubated in 50 mM trisodium citrate buffer (pH 4.5) at 55 °C for 5 min to deactivate the proteases. After incubation, the hyphae were washed twice with 1.2 M sorbitol in NaCl/Pi. The hyphae were treated with 0.1% sodium cit-









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rate containing 0.1% Triton X-100 on ice for 15 min to permeabilize the cell membrane. After permeabilization, the hyphae were washed with 1.2 M sorbitol in NaCl/Pi, and then incubated in the solution provided in the In Situ Cell Death Detection Kit, Fluorescein (Roche, Basel, Switzerland). After incubation, the solution was removed, and the hyphae were washed three times with 1.2 M sorbitol in NaCl/Pi. The hyphae were stained with 5 lg mL 1 DAPI at room temprerature for 5 min. The hyphae were observed under a fluorescence microscope (excitation wavelength of 485 nm and emission wavelength of 535 nm for TUNEL staining; excitation wavelength of 350 nm and emission wavelength of 420 nm for DAPI staining). The TUNEL assay was also performed with S. cerevisiae, on the basis of the method of Kitagaki et al. [33]. Exponentially growing S. cerevisiae cells suspended in 100 mL of ME broth to achieve 0.1 turbidity at 660 nm were incubated with anethole and H2O2 at 30 °C for 2 and 4 h.



Detection of metacaspase activity The spores of A. fumigatus that were suspended in ME broth to achieve 0.05 turbidity at 530 nm were incubated at 30 °C for 24 h. After incubation, anethole or H2O2 was added to the culture, and the culture was then incubated at 30 °C for 24 h. The hyphae were washed twice with NaCl/Pi on a membrane filter (Ultrafree-MC; Millipore), and then suspended in 100 lL of NaCl/Pi. The hyphal suspensions were dropped onto round glass slides coated with poly(L-lysine) and thoroughly dried. The hyphae were incubated on the glass at room temperature for 30 min with 2 lM CaspACE FITC–VAD-FMK In Situ Marker (Promega, Madison, WI, USA) as a substrate for metacaspase. After incubation, the substrate solution was removed by aspiration. The hyphae were washed with NaCl/Pi twice, and then fixed with 10% formaldehyde in NaCl/Pi. The hyphae were observed under a fluorescence microscope (excitation wavelength of 485 nm; emission wavelength of 535 nm).

Statistical analysis The statistical analyses were performed with Student’s t-test, in which P < 0.05 was considered to be statistically significant.

Acknowledgements We are grateful to H. Kitagaki at Saga University for technical help with TUNEL staining. This work was partly funded by Grants-in-Aid for Scientific Research (C) 20580113 and 25460128.

FEBS Journal 281 (2014) 1304–1313 ª 2014 FEBS

Anethole-induced apoptotic cell death in fungi

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FEBS Journal 281 (2014) 1304–1313 ª 2014 FEBS

Anethole-induced apoptotic cell death in fungi

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Anethole induces apoptotic cell death accompanied by reactive oxygen species production and DNA fragmentation in Aspergillus fumigatus and Saccharomyces cerevisiae.

trans-Anethole (anethole), a major component of anise oil, has a broad antimicrobial spectrum, and antimicrobial activity that is weaker than that of ...
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