Experimental Chemotherapy Chemotherapy 2014;60:99–106 DOI: 10.1159/000371413
Received: May 26, 2014 Accepted after revision: December 8, 2014 Published online: February 14, 2015
Effects of Microtubule and Actin Inhibitors on Cryptococcus neoformans Examined by Scanning and Transmission Electron Microscopy Marie Kopecká Department of Biology, Faculty of Medicine, Masaryk University, Brno, Czech Republic
Key Words Yeast · Vincristine · Methyl benzimidazole-2-ylcarbamate · Latrunculin A · Electron microscopy
were dead; only empty cell walls persisted with reduced capsules, shown on SEM. Conclusion: Combined microtubule and actin inhibitors (VIN + LA or BCM + LA), have lethal effects on C. neoformans cells and no resistant cells originate. © 2015 S. Karger AG, Basel
© 2015 S. Karger AG, Basel 0009–3157/15/0602–0099$39.50/0 E-Mail [email protected] www.karger.com/che
Introduction
Cryptococcus neoformans is one of the most important human fungal pathogens [1]. It has a typical fungal ultrastructure [2–4] and capsule – an important pathogenesis factor [5]. It has world-wide distribution, and causes cryptotococcosis, atypical pneumonia and meningoencephalitis, infections of the inner organs, bones and skin and fungaemia in both immune-deficient patients and immune-competent individuals [6–10]. Therapy is difficult, with the frequent development of resistance and considerable mortality. Several anti-fungal strategies have been investigated: (1) polyenes binding to ergosterol of the plasma membrane destabilize it and induce cell lysis (amphotericin B and nystatin), (2) inhibitors of ergosterol synthesis (azoles, e.g. clotrimazole, fluconazole, itraconazole, ketoconazole, miconazole, voriconazole This paper is dedicated to the memory of Miroslav Gabriel, MD, PhD and Associate Professor, with whom we initiated this research in 1999. He died on 7 June, 2008.
Prof. Marie Kopecká, MD, CSc Department of Biology, Faculty of Medicine, Masaryk University, UCB Kamenice 5, A6 CZ–62500 Brno (Czech Republic) E-Mail mkopecka @ med.muni.cz
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Abstract Background: Cryptococcus neoformans is one of the most important human fungal pathogens. Its cells contain rich microtubules required for nuclear division and rich F-actin cytoskeletons for cell division. Disruption of microtubules by a microtubule inhibitor should block nuclear division, and disruption of F-actin by an actin inhibitor should block cell division. We investigated the effects of microtubule and actin inhibitors to find out whether the cytoskeletons of C. neoformans can become a new anti-fungal target for the inhibition of cell division, when examined at the ultrastructural level. Methods: Cells treated with the microtubule inhibitors vincristine (VIN) and methyl benzimidazole-2-ylcarbamate (BCM) and the actin inhibitor latrunculin A (LA), in yeast extract peptone dextrose medium, were examined by scanning (SEM) and transmission electron microscopy (TEM), and the cell number was counted using a Bürker chamber. Results: After 2 days of inhibition with VIN, BCM or LA, the cells did not divide, but later, resistant, proliferating cells appeared in all samples. With combined microtubule and actin inhibitors (VIN + LA or BCM + LA), cells did not divide during 6 or even 14 days, and no resistant cells originated. TEM showed that the inhibited cells were without cytoplasm and
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Materials and Methods Yeast Strain C. neoformans var. neoformans, IFM 41464 (CUH 34, 48–9943, 881, skin, serotype A) from the Medical Mycology Research Centre, Chiba University, Japan [13] was used. Media and Cell Cultivation The strain was maintained on 2.0% (w/v) agar containing yeast extract peptone dextrose (YEPD) medium [1% (w/v) yeast extract, 2% (w/v) peptone and 2% (w/v) glucose] at laboratory temperature. To obtain an exponential culture, cells were cultivated in YEPD medium [1% (w/v) yeast extract, 1% (w/v) peptone and 1% (w/v) glucose] on a shaker overnight at 23 ° C (16 h) and were then diluted to 5 × 105 to 1 × 106 cells/ml by 1% YEPD medium and pipetted in 500-μl volumes to test tubes. They were then used for the application of inhibitors and shaken in the dark in a water bath for 6 or 14 days [12, 14].
Cytoskeleton Inhibitors The microtubule inhibitors used were: methyl benzimidazole2-ylcarbamate (BCM; Chinoin Pharmaceutical Company, Budapest, Hungary) and vincristine (VIN) sulphate salt (Fluka). The actin inhibitor was latrunculin A (LA; Molecular Probes, Eugene, Oreg., USA, and Sigma). All stock solutions were prepared as 10 mM and stored at –20 ° C [12]. LA Treatment. 10 mM of stock solution was prepared by dissolving 100 μg of LA in 25 μl of dimethyl sulfoxide (DMSO), kept at –20 ° C [12, 14]. BCM Treatment. 10 m M of stock solution was prepared by dissolving 2 mg of the drug in 1.0 ml of DMSO, kept at –20 ° C [12]. VIN Treatment. 10 mM of stock solution was prepared by dissolving 5 mg of the drug in 500 μl of DMSO, kept at –20 ° C [12].
Application of Inhibitors A 10-mM stock solution of the inhibitors, dissolved in DMSO, was added to cells inoculated in liquid 1% YEPD to final concentrations of 100 and 200 μM [12]. The cell cultures in the test tubes in 500-μl volumes were shaken in the dark in a water bath and samples for TEM and SEM were taken. TEM Ultrathin sectioning was done as described previously [15, 16]. Cells were fixed in 3% glutaraldehyde for 3 h and contrasted with 1% osmium tetroxide for 1 h. Ultrathin sections were contrasted with 2.5% uranyl acetate for 30 min and lead citrate for 6 min [15]. The ultrathin sections were viewed and photographed with a Morgagni 268D (100 kV) microscope. SEM SEM was done as described [16, 17]. Cells were fixed by 5% paraformaldehyde for 90 min and the further procedure was carried out as described [16, 17]. Preparations were observed and photographed using Vega TS5136 XM (Tescan) with digital microscopy imaging scanning equipment. Image Processing Software Adobe Photoshop CS5 and Adobe InDesign CS5 for Windows were used for the electronic arrangement of figures.
Kopecká
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and posaconazole, allylamines, e.g. thiocarbamates and morpholines), (3) inhibitors of cell-wall synthesis (polyoxins, nikkomycins, papulacandins and echinocandins), (4) inhibitors of synthesis of nucleic acid (5-fluorocytosine, trimethoprim and sulfamethoxazole), (5) inhibitors of protein synthesis (blasticidin and sinefungin), (6) metabolic inhibitors (cispentacin and α-difluoromethylornithine) and (7) inhibitors of nuclear division (griseofulvin and benomyl) [6–10]. The present ‘gold standard therapy’ for cryptococcal meningoencephalitis is amphotericin B (AMB) + 5-flucytosine (5FC), but, unfortunately, AMB-5FC is not available in poor countries due to being expensive, toxic and requiring intravenous application and therapeutic monitoring that are not practical in these regions [9, 10]. Fluconazole (FLU) is the most commonly used agent, but the mortality is as high as 50% as it is much less effective than AMB-5FC; AMB5FC is rapidly fungicidal while FLU is fungistatic only [9, 10]. It is clear that new drugs are needed for efficient treatment of cryptococcosis. Recently, a very interesting approach looking into the anti-cryptococcal activity of previously developed medical drugs, including anti-psychotics, anti-arrhythmics and oncotherapeutics for breast cancer at a final concentration of 50 μM, was initiated to determine whether they are fungicidal to C. neoformans [9]. For example, tamoxifen is an antagonist of estrogen receptors used for breast cancer therapy by targeting calmodulin, and amiodarone is used in cardiology for treating ventricular fibrillations [9, 10]. Interestingly, such drugs appeared to have fungicidal effects on C. neoformans and can be combined with FLU or 5FC [9, 10]. In our basic cell biology research of C. neoformans cells, we discovered the rich cytoskeletons of microtubules and F-actin filaments that mediate key events of nuclear and cell division [2, 3]. Microtubules are required for the nuclear division of C. neoformans, so the disruption of microtubules by specific microtubule inhibitors should block nuclear division. Cell division (cytokinesis) of C. neoformans requires an actin cytoskeleton, so the disruption of F-actin by specific inhibitors should block cell division. We investigate here whether the cytoskeleton can become a new potential anti-fungal target for the inhibition of cell division of C. neoformans by microtubule and actin inhibitors, and whether they can become a ‘magic bullet’ [11] for this human pathogen. We demonstrate the ultrastructural effects of microtubule and actin inhibitors on C. neoformans investigated by scanning (SEM) and transmission electron microscopy (TEM). We recently studied these inhibitors on C. neoformans using phase-contrast and fluorescent microscopy [12].
Results
Fig. 1. a–j SEM of C. neoformans in YEPD medium. a Control cells cultivated with 1% DMSO at the beginning of experiment. b Cells treated with 200 μM BCM for 2 days. c Cells treated with 200 μM VIN for 2 days. d Cells treated with 100 μM of LA for 2 days. e Cells treated with 100 μM BCM for 6 days. f Cells treated with 100 μM VIN for 6 days. g Cells treated with 100 μM LA for 6 days. h Cells treated with combined LA + VIN (100 μM of each) for 6 days. i Cells treated with combined LA + BCM (100 μM of each) for 6 days. j Control cells at the same magnification.
Cytoskeleton Inhibitors and Electron Microscopy of Cryptococcus neoformans
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Ultrastructure of C. neoformans Examined by SEM Cells were inhibited by single microtubule or actin inhibitors for 2 days. Control cells had a spherical shape and reproduced by budding (fig. 1a). The microtubule inhibitor BCM did not block budding; usually three buds developed on the mother cell forming a ‘micro-colony’ of four joined cells (fig. 1b) that did not divide (in comparison with the control cells; table 1). The microtubule inhibitor VIN also did not block budding; usually a ‘micro-colony’ of four joined cells originated (fig. 1c) that did not divide (table 1). Buds originating on inhibited cells were of various sizes and differed in shape, i.e. spherical, triangular and cylindrical, and were potato- or pear-like in appearance (fig. 1b, c). The actin inhibitor LA blocked cell division most efficiently out of all the single inhibitors tested (table 1). The spherical unbudding or budding cells persisted (fig. 1d); some cells developed cylindrical morphology [12]. Some cells originated that were resistant to all the single inhibitors. When we observed the inhibited cells some time later, new, budding, resistant cells had appeared among the BCM-induced ‘micro-colonies’ (fig. 1e) and among the VIN-inhibited cells (fig. 1f). When we looked for resistant cells in LA, only a few cell chains were seen (fig. 1g). The counting of cells showed a gradual increase of resistant cell number (tables 1, 2). The combined effects of the microtubule and actin inhibitors LA + VIN or LA + BCM at 100-μM concentrations disrupted the actin and microtubule cytoskeletons [12] and blocked cell division (table 1). The inhibited cells were similar to the control cells in appearance, but some bud necks were wide (fig. 1h), probably due to the disrupted actin cytokinetic ring by LA, and some cells became cylindrical [12]. No resistant, budding cells appeared during combined inhibition by LA + VIN (fig. 1h) or LA + BCM (fig. 1i).
Table 1. Number of C. neoformans cells before and after 1, 2, 3, 6 and 14 days of inhibition, counted using a Bürker chamber
Objects
Time of inhibition
Control cells (1% DMSO) Cells treated with LA (100 μM) Cells treated with BCM (100 μM) Cells treated with VIN (100 μM) Cells treated with VIN (100 μM) and LA (100 μM) Cells treated with BCM (100 μM) and LA (100 μM)
0h
1 day
2 days
3 days
6 days
14 days
7.5 × 105 7.5 × 105 7.5 × 105 7.5 × 105
5.0 × 106 1.5 × 106 4.25 × 106 2.75 × 106
2.5 × 107 1.87 × 106 6.5 × 106 7.5 × 106
5.0 × 107 2.37 × 106 1.25 × 107 1.5 × 107
1.5 × 108 1.25 × 107 3.75 × 107 1.25 × 108
2.0 × 108 1.75 × 106 1.25 × 108 1.25 × 108
7.5 × 105
1.75 × 106
1.75 × 106
1.87 × 106
1.87 × 106
1.87 × 106
7.5 × 105
2.3 × 106
1.75 × 106
2.12 × 106
2.15 × 106
2.0 × 106
Values represent number of cells per millilitre.
Table 2. Occurrence of resistant C. neoformans cells in samples containing microtubule and actin inhibitors
Inhibitors samples
Time of inhibition
Cells treated with BCM (100 and 200 μM) Cells treated with VIN (100 and 200 μM) Cells treated with LA (100 and 200 μM) Cells treated with VIN (100 μM) + LA (100 μM) Cells treated with BCM (100 μM) + LA (100 μM) b Data
2 days
3 days
6 daysa
14 daysb
− − − − −
− − − − −
+ + + − −
+ + + − −
+ + + − −
from 8 experiments. from 2 experiments.
Ultrastructure of Capsule in Control and Inhibited C. neoformans Cells Control cells had a uniform capsule of ‘curl-headed’ appearance; buds had a fine, denser capsule. The rim of the capsule material was around the bud neck in the dividing cell, and a small circle resembling a bud scar was found on the mother cells (fig. 1a). Capsules on cells inhibited by the single microtubule inhibitors BCM and VIN differed. Each bud contained a capsule of slightly different appearance, probably due to the various stages of bud and capsule development (fig. 1b, c). Capsules on cells inhibited by the actin inhibitor LA were reduced in size, with rare small patches (fig. 1d). Capsules on cells resistant to the single inhibitors BCM, VIN and LA (fig. 1e–g) were similar to those of the control cells (fig. 1a). Capsules on cells inhibited by combined LA + VINC and LA + BCM were reduced in size, with rare small patches (fig. 1h, i). They were similar to the capsules on the LA-inhibited cells (fig. 1d) but different from those on the control cells, when observed at the same magnification (fig. 1h–j). 102
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Effects of Cytoskeleton Inhibitors on C. neoformans Examined by TEM Cells were inhibited by the single microtubule or actin inhibitors VIN, BCM or LA. Control cells had a typical fungal ultrastructure (fig. 2a). Microtubule inhibitors BCM or VIN at 100-μM and 200-μM concentrations did not block budding, but delayed cell division (table 1). The cytoplasm of cells inhibited for 30 h filled with vacuoles gradually disappeared (fig. 2b), suggesting cell death. After 2 days of inhibition by BCM (fig. 2c) or VIN (fig. 2d), ‘micro-colonies’ of joined dead cells were without cytoplasm; the empty cell walls just maintained the shape of the inhibited cells. The disappearance of the cytoplasm was direct evidence of cell death and inhibition by VIN or BCM. Cells inhibited by LA at 200-μM and 100-μM concentrations also contained only a few remnants of cytoplasm and were dead. Their cell walls persisted, maintaining the shape of budding cells (fig. 2e). Kopecká
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a Data
0h
Although TEM of cells inhibited by BCM, VIN or LA showed many dead cells without cytoplasm, a few cells contained cytoplasm. These cells were probably resistant to the inhibitors. After 6 days of inhibition in 100 μM of BCM or VIN (not shown), cells resistant to the single microtubule or actin inhibitors BCM, VIN or LA (table 2) were found; they contained cytoplasm, cell organelles and capsules (fig. 2f). Cells resistant to 100 μM of LA formed chains of budding cells that were almost without cytoplasm following cell death (fig. 2g). Combined Effects of Actin and Microtubule Inhibitors LA + VIN and LA + BCM After 6 days of combined inhibition in LA + VIN (fig. 2h) and LA + BCM (fig. 2i), the dead cells contained only a few remnants of cytoplasm inside almost empty cell walls that retained the shape of the inhibited cells. This explains why combined inhibition cells did not divide and why no resistant cells originated (tables 1, 2) [12]. However, in the combined LA + VIN and LA + BCM inhibition, on TEM, we also noticed very few cells with cytoplasm (not shown) amongst the many empty cell walls of the dead cells. We do not know whether they were artefacts caused by some technical mistake in preparing the samples for ultrathin sectioning, or whether they were very resistant, non-dividing cells or immortal yeast stem cells. We did not identify any resistant, budding cells by cell counting (table 1), phase-contrast microscopy [12] or SEM, so the cause was probably a technical mistake during sectioning. Nevertheless, we cannot exclude the fact that a few non-dividing, resistant cells were present.
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Discussion
SEM and TEM confirmed, at the ultrastructural level, the results of our previous paper [12], i.e. that combined actin + microtubule inhibitors block cell division with
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Fig. 2. TEM of C. neoformans in YEPD medium. a Control cells
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cultivated with 1% DMSO without inhibitor at the beginning of experiment. Cells were treated with 200 μM BCM for 30 h (b) and 2 days (c). d Cells treated with 200 μM VIN for 2 days. e Cells treated with 200 μM LA for 2 days. f Resistant cells were detected after 6 days in BCM. g Chain of resistant dead cells after 6 days in LA. h Cells treated with a combined application of LA + VIN (100 μM of each) for 6 days. i Cells treated with a combined application of LA + BCM (100 μM of each) for 6 days.
Color version available online
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and microtubules (green). The capsule size is reduced. d Cells inhibited with combined LA + VIN (or BCM; 100 μM of each) have a nucleus (blue), but do not have microtubules and actin cytoskeletons. Diagrams are based on the combined results of this study and previously submitted data [12]. a = F-actin; mi = microtubules; nu = nucleus.
Color version available online
Fig. 3. Diagram of cells showing actin cytoskeletons, microtubules, nuclei (based on our previous paper) and cell surface covered by capsules. a Control cells have nucleus (blue), actin filaments (red) and microtubules (green) and a capsule. b Cells inhibited with VIN or BCM do not have microtubules (green), but have a nucleus (blue), an actin cytoskeleton (red); three new buds developed, covered with a capsule. c Cells inhibited with LA have a nucleus (blue)
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Fig. 4. a Diagram of control cells showing actin cytoskeletons, mi-
crotubules, nuclei (based on [12]) and cell surfaces covered by capsules. b Cells treated with VIN or BCM are dead and without cytoplasm; only empty cell walls with capsules persist. c Cells treated with LA are dead and without cytoplasm; only empty cell walls
no resistant cells appearing. TEM demonstrated that inhibited cells were dead and without cytoplasm. Their death was induced by microtubule and actin inhibitors. We do not know why C. neoformans cells die after contact with single or combined microtubule and actin inhibitors (figs. 3, 4). Animal cells with disrupted micro104
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with reduced capsules persist. d Cells inhibited with combined LA + VIN or LA + BCM (100 μM of each) are dead, without cytoplasm; only empty cell walls with reduced capsules persist. a = Factin microfilament cables; mi = microtubules; nu = nucleus.
tubules die by apoptosis [18]. The yeast Saccharomyces cerevisiae, with a disrupted single actin gene, dies by lethal mutation [19], and temperature-sensitive yeast actin mutants die at non-permissive temperatures [20, 21] with features of apoptosis [22, 23]. Eukaryotic cells probably cannot exist without actin and microtubule cyKopecká
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toskeletons, and are not viable. While TEM showed the interiors of the inhibited cells, with empty cell walls, no cytoplasm and no cell organelles, in addition to maintaining the original shape of the inhibited cells, SEM showed surface views of the inhibited cells. What we had observed previously by phase-contrast microscopy after 6 days of inhibiting the cells [12] were, in fact, only empty cell walls with capsules but no cytoplasm, that imitated yeast cells. Dead cells without cytoplasm could be observed already after 2 days of inhibition by the single inhibitors. However, resistant, budding cells then appeared. In order to induce cell death of the whole cell population, consisting of about 5 × 105 to 1 × 106 cells/ml, we applied the combined drugs LA + VIN and LA + BCM, each at 100μM concentrations. These disrupted both the microtubules and F-actin [12], and the cells died. No resistant, budding cells originated because the cells were dead, as proven here by TEM. With regard to the capsules, while control mother cells had dense, curl-headed capsules and fine, dense capsules on their buds, the cells inhibited by single BCM or VIN differed; each bud had a different capsule, probably due to the different stages of bud and capsule development. The capsules on the LA-inhibited cells, the LA + VIN-inhibited cells and the LA + BCM-inhibited cells differed in a similar way. Capsules were reduced in size, probably due to the disruption of the actin cytoskeleton, similar to that seen in the related Fellomyces fuzhouensis (inhibited by 100 μM of LA for 4 days), where the capsules completely disappeared [17]. We speculate that the cause may be the disruption of the actin cytoskeleton,
maybe involved in the transport of cytoplasmic vesicles containing capsule polysaccharides or their precursors to the cell cortex. The disruption of microtubules by the microtubule inhibitors BCM or VIN combined with the disruption of actin microfilaments by the actin inhibitor LA causes the most effective blockage of cell division in C. neoformans out of all the inhibitors that we have tested so far. The inhibited cells die, the dead cells cannot divide and resistant cells cannot originate. Our results identified the cytoskeleton of C. neoformans as a new potential target for the inhibition of cell division, the anti-microtubule drugs VIN and BCM and the anti-actin drug LA as new antifungal agents that, when used in combined application, induce cell death.
Conclusion
C. neoformans cells inhibited by combined LA + VINC or LA + BCM die and consist of only empty cell walls with capsules that resemble whole yeast cells on SEM.
Acknowledgement The author thanks Ing. Ladislav Ilkovics, Vladimíra Ramíková, Dobromila Klemová and Jan Šlancar for their excellent technical help. Financial support was provided in 2000–2008 by three grants from the Grant Agency of the Czech Republic: GAP 310/00/0391 and 310/06/0605 (Marie Kopecká), and GAP 310/03/1195 (Miroslav Gabriel). Institutional support by Masaryk University, Brno, in 2009–2014 enabled continuation of this work.
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5 Pierini LM, Doering TL: Spatial and temporal sequence of capsule construction in Cryptococcus neoformans. Mol Microbiol 2001; 41: 105–115. 6 Georgopapadakou NH, Walsh TJ: Human mycoses: drugs and targets for emerging pathogens. Science 1994;264:371–373. 7 Lui G, Lee N, Ip M, Choi KW, Tso YK, Lam E, Chau S, Lai R, Cockram CS: Cryptococcosis in apparently immunocompetent patients. QJM 2006;99:143–151. 8 Sable CA, Strohmaier KM, Chodakewitz JA: Advances in antifungal therapy. Annu Rev Med 2008;59:361–379. 9 Butts A, DiDone L, Koselny K, Baxter BK, Chabrier-Rosello Y, Wellington M, Krysan DJ: A repurposing approach identifies offpatent drugs with fungicidal cryptococcal activity, a common structural chemotype, and
pharmacological properties relevant to the treatment of cryptococcosis. Eukaryot Cell 2013;12:278–287. 10 Butts A, Koselny K, Chabrier-Roselló Y, Semighini CP, Brown JC, Wang X, Annadurai S, DiDone L, Tabroff J, Childers WE Jr, AbouGharbia M, Wellington M, Cardenas ME, Madhani HD, Heitman J, Krysan DJ: Estrogen receptor antagonists are anti-cryptococcal agents that directly bind EF hand proteins and synergize with fluconazole in vivo. MBio 2014;5:e00765–13. 11 Sörgel F: World Conference on Dosing of Antiinfectives: ‘Dosing the Magic Bullets’. Chemotherapy 2004;50:1–5. 12 Kopecká M: Microtubules and actin cytoskeleton of human potentially pathogenic yeast Cryptococcus neoformans as targets for antifungals. Chemotherapy 2014 (under review).
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13 Nishimura K, Miyaji M, Takeo K, Mikami Y, Kamei K, Yokoyama K, Tanaka R: IFM List of Pathogenic Fungi and Actinomycetes with Photomicrographs, ed 2. Culture Collection of Research Center for Pathogenic Fungi and Microbial Toxicoses, Chiba University. Chiba, Seibunsha, 1998. 14 Kopecká M, Gabriel M: Microtubules and actin cytoskeleton of potentially pathogenic basidiomycetous yeast as targets for antifungals. Chemotherapy 2009;55:278–286. 15 Gabriel M, Kopecká M, Yamaguchi M, Svoboda A, Takeo K, Yoshida S, Ohkusu M, Sugita T, Nakase T: Cytoskeleton in the unique cell reproduction by conidiogenesis of the long neck yeast Fellomyces (Sterigmatomyces) fuzhouensis. Protoplasma 2006; 229: 33– 44.
Received: May 26, 2014 Accepted after revision: December 8, 2014 Published online: February 14, 2015
Effects of Microtubule and Actin Inhibitors on Cryptococcus neoformans Examined by Scanning and Transmission Electron Microscopy Marie Kopecká Department of Biology, Faculty of Medicine, Masaryk University, Brno, Czech Republic
Key Words Yeast · Vincristine · Methyl benzimidazole-2-ylcarbamate · Latrunculin A · Electron microscopy
were dead; only empty cell walls persisted with reduced capsules, shown on SEM. Conclusion: Combined microtubule and actin inhibitors (VIN + LA or BCM + LA), have lethal effects on C. neoformans cells and no resistant cells originate. © 2015 S. Karger AG, Basel
© 2015 S. Karger AG, Basel 0009–3157/15/0602–0099$39.50/0 E-Mail [email protected] www.karger.com/che
Introduction
Cryptococcus neoformans is one of the most important human fungal pathogens [1]. It has a typical fungal ultrastructure [2–4] and capsule – an important pathogenesis factor [5]. It has world-wide distribution, and causes cryptotococcosis, atypical pneumonia and meningoencephalitis, infections of the inner organs, bones and skin and fungaemia in both immune-deficient patients and immune-competent individuals [6–10]. Therapy is difficult, with the frequent development of resistance and considerable mortality. Several anti-fungal strategies have been investigated: (1) polyenes binding to ergosterol of the plasma membrane destabilize it and induce cell lysis (amphotericin B and nystatin), (2) inhibitors of ergosterol synthesis (azoles, e.g. clotrimazole, fluconazole, itraconazole, ketoconazole, miconazole, voriconazole This paper is dedicated to the memory of Miroslav Gabriel, MD, PhD and Associate Professor, with whom we initiated this research in 1999. He died on 7 June, 2008.
Prof. Marie Kopecká, MD, CSc Department of Biology, Faculty of Medicine, Masaryk University, UCB Kamenice 5, A6 CZ–62500 Brno (Czech Republic) E-Mail mkopecka @ med.muni.cz
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Abstract Background: Cryptococcus neoformans is one of the most important human fungal pathogens. Its cells contain rich microtubules required for nuclear division and rich F-actin cytoskeletons for cell division. Disruption of microtubules by a microtubule inhibitor should block nuclear division, and disruption of F-actin by an actin inhibitor should block cell division. We investigated the effects of microtubule and actin inhibitors to find out whether the cytoskeletons of C. neoformans can become a new anti-fungal target for the inhibition of cell division, when examined at the ultrastructural level. Methods: Cells treated with the microtubule inhibitors vincristine (VIN) and methyl benzimidazole-2-ylcarbamate (BCM) and the actin inhibitor latrunculin A (LA), in yeast extract peptone dextrose medium, were examined by scanning (SEM) and transmission electron microscopy (TEM), and the cell number was counted using a Bürker chamber. Results: After 2 days of inhibition with VIN, BCM or LA, the cells did not divide, but later, resistant, proliferating cells appeared in all samples. With combined microtubule and actin inhibitors (VIN + LA or BCM + LA), cells did not divide during 6 or even 14 days, and no resistant cells originated. TEM showed that the inhibited cells were without cytoplasm and
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Materials and Methods Yeast Strain C. neoformans var. neoformans, IFM 41464 (CUH 34, 48–9943, 881, skin, serotype A) from the Medical Mycology Research Centre, Chiba University, Japan [13] was used. Media and Cell Cultivation The strain was maintained on 2.0% (w/v) agar containing yeast extract peptone dextrose (YEPD) medium [1% (w/v) yeast extract, 2% (w/v) peptone and 2% (w/v) glucose] at laboratory temperature. To obtain an exponential culture, cells were cultivated in YEPD medium [1% (w/v) yeast extract, 1% (w/v) peptone and 1% (w/v) glucose] on a shaker overnight at 23 ° C (16 h) and were then diluted to 5 × 105 to 1 × 106 cells/ml by 1% YEPD medium and pipetted in 500-μl volumes to test tubes. They were then used for the application of inhibitors and shaken in the dark in a water bath for 6 or 14 days [12, 14].
Cytoskeleton Inhibitors The microtubule inhibitors used were: methyl benzimidazole2-ylcarbamate (BCM; Chinoin Pharmaceutical Company, Budapest, Hungary) and vincristine (VIN) sulphate salt (Fluka). The actin inhibitor was latrunculin A (LA; Molecular Probes, Eugene, Oreg., USA, and Sigma). All stock solutions were prepared as 10 mM and stored at –20 ° C [12]. LA Treatment. 10 mM of stock solution was prepared by dissolving 100 μg of LA in 25 μl of dimethyl sulfoxide (DMSO), kept at –20 ° C [12, 14]. BCM Treatment. 10 m M of stock solution was prepared by dissolving 2 mg of the drug in 1.0 ml of DMSO, kept at –20 ° C [12]. VIN Treatment. 10 mM of stock solution was prepared by dissolving 5 mg of the drug in 500 μl of DMSO, kept at –20 ° C [12].
Application of Inhibitors A 10-mM stock solution of the inhibitors, dissolved in DMSO, was added to cells inoculated in liquid 1% YEPD to final concentrations of 100 and 200 μM [12]. The cell cultures in the test tubes in 500-μl volumes were shaken in the dark in a water bath and samples for TEM and SEM were taken. TEM Ultrathin sectioning was done as described previously [15, 16]. Cells were fixed in 3% glutaraldehyde for 3 h and contrasted with 1% osmium tetroxide for 1 h. Ultrathin sections were contrasted with 2.5% uranyl acetate for 30 min and lead citrate for 6 min [15]. The ultrathin sections were viewed and photographed with a Morgagni 268D (100 kV) microscope. SEM SEM was done as described [16, 17]. Cells were fixed by 5% paraformaldehyde for 90 min and the further procedure was carried out as described [16, 17]. Preparations were observed and photographed using Vega TS5136 XM (Tescan) with digital microscopy imaging scanning equipment. Image Processing Software Adobe Photoshop CS5 and Adobe InDesign CS5 for Windows were used for the electronic arrangement of figures.
Kopecká
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and posaconazole, allylamines, e.g. thiocarbamates and morpholines), (3) inhibitors of cell-wall synthesis (polyoxins, nikkomycins, papulacandins and echinocandins), (4) inhibitors of synthesis of nucleic acid (5-fluorocytosine, trimethoprim and sulfamethoxazole), (5) inhibitors of protein synthesis (blasticidin and sinefungin), (6) metabolic inhibitors (cispentacin and α-difluoromethylornithine) and (7) inhibitors of nuclear division (griseofulvin and benomyl) [6–10]. The present ‘gold standard therapy’ for cryptococcal meningoencephalitis is amphotericin B (AMB) + 5-flucytosine (5FC), but, unfortunately, AMB-5FC is not available in poor countries due to being expensive, toxic and requiring intravenous application and therapeutic monitoring that are not practical in these regions [9, 10]. Fluconazole (FLU) is the most commonly used agent, but the mortality is as high as 50% as it is much less effective than AMB-5FC; AMB5FC is rapidly fungicidal while FLU is fungistatic only [9, 10]. It is clear that new drugs are needed for efficient treatment of cryptococcosis. Recently, a very interesting approach looking into the anti-cryptococcal activity of previously developed medical drugs, including anti-psychotics, anti-arrhythmics and oncotherapeutics for breast cancer at a final concentration of 50 μM, was initiated to determine whether they are fungicidal to C. neoformans [9]. For example, tamoxifen is an antagonist of estrogen receptors used for breast cancer therapy by targeting calmodulin, and amiodarone is used in cardiology for treating ventricular fibrillations [9, 10]. Interestingly, such drugs appeared to have fungicidal effects on C. neoformans and can be combined with FLU or 5FC [9, 10]. In our basic cell biology research of C. neoformans cells, we discovered the rich cytoskeletons of microtubules and F-actin filaments that mediate key events of nuclear and cell division [2, 3]. Microtubules are required for the nuclear division of C. neoformans, so the disruption of microtubules by specific microtubule inhibitors should block nuclear division. Cell division (cytokinesis) of C. neoformans requires an actin cytoskeleton, so the disruption of F-actin by specific inhibitors should block cell division. We investigate here whether the cytoskeleton can become a new potential anti-fungal target for the inhibition of cell division of C. neoformans by microtubule and actin inhibitors, and whether they can become a ‘magic bullet’ [11] for this human pathogen. We demonstrate the ultrastructural effects of microtubule and actin inhibitors on C. neoformans investigated by scanning (SEM) and transmission electron microscopy (TEM). We recently studied these inhibitors on C. neoformans using phase-contrast and fluorescent microscopy [12].
Results
Fig. 1. a–j SEM of C. neoformans in YEPD medium. a Control cells cultivated with 1% DMSO at the beginning of experiment. b Cells treated with 200 μM BCM for 2 days. c Cells treated with 200 μM VIN for 2 days. d Cells treated with 100 μM of LA for 2 days. e Cells treated with 100 μM BCM for 6 days. f Cells treated with 100 μM VIN for 6 days. g Cells treated with 100 μM LA for 6 days. h Cells treated with combined LA + VIN (100 μM of each) for 6 days. i Cells treated with combined LA + BCM (100 μM of each) for 6 days. j Control cells at the same magnification.
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Ultrastructure of C. neoformans Examined by SEM Cells were inhibited by single microtubule or actin inhibitors for 2 days. Control cells had a spherical shape and reproduced by budding (fig. 1a). The microtubule inhibitor BCM did not block budding; usually three buds developed on the mother cell forming a ‘micro-colony’ of four joined cells (fig. 1b) that did not divide (in comparison with the control cells; table 1). The microtubule inhibitor VIN also did not block budding; usually a ‘micro-colony’ of four joined cells originated (fig. 1c) that did not divide (table 1). Buds originating on inhibited cells were of various sizes and differed in shape, i.e. spherical, triangular and cylindrical, and were potato- or pear-like in appearance (fig. 1b, c). The actin inhibitor LA blocked cell division most efficiently out of all the single inhibitors tested (table 1). The spherical unbudding or budding cells persisted (fig. 1d); some cells developed cylindrical morphology [12]. Some cells originated that were resistant to all the single inhibitors. When we observed the inhibited cells some time later, new, budding, resistant cells had appeared among the BCM-induced ‘micro-colonies’ (fig. 1e) and among the VIN-inhibited cells (fig. 1f). When we looked for resistant cells in LA, only a few cell chains were seen (fig. 1g). The counting of cells showed a gradual increase of resistant cell number (tables 1, 2). The combined effects of the microtubule and actin inhibitors LA + VIN or LA + BCM at 100-μM concentrations disrupted the actin and microtubule cytoskeletons [12] and blocked cell division (table 1). The inhibited cells were similar to the control cells in appearance, but some bud necks were wide (fig. 1h), probably due to the disrupted actin cytokinetic ring by LA, and some cells became cylindrical [12]. No resistant, budding cells appeared during combined inhibition by LA + VIN (fig. 1h) or LA + BCM (fig. 1i).
Table 1. Number of C. neoformans cells before and after 1, 2, 3, 6 and 14 days of inhibition, counted using a Bürker chamber
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Time of inhibition
Control cells (1% DMSO) Cells treated with LA (100 μM) Cells treated with BCM (100 μM) Cells treated with VIN (100 μM) Cells treated with VIN (100 μM) and LA (100 μM) Cells treated with BCM (100 μM) and LA (100 μM)
0h
1 day
2 days
3 days
6 days
14 days
7.5 × 105 7.5 × 105 7.5 × 105 7.5 × 105
5.0 × 106 1.5 × 106 4.25 × 106 2.75 × 106
2.5 × 107 1.87 × 106 6.5 × 106 7.5 × 106
5.0 × 107 2.37 × 106 1.25 × 107 1.5 × 107
1.5 × 108 1.25 × 107 3.75 × 107 1.25 × 108
2.0 × 108 1.75 × 106 1.25 × 108 1.25 × 108
7.5 × 105
1.75 × 106
1.75 × 106
1.87 × 106
1.87 × 106
1.87 × 106
7.5 × 105
2.3 × 106
1.75 × 106
2.12 × 106
2.15 × 106
2.0 × 106
Values represent number of cells per millilitre.
Table 2. Occurrence of resistant C. neoformans cells in samples containing microtubule and actin inhibitors
Inhibitors samples
Time of inhibition
Cells treated with BCM (100 and 200 μM) Cells treated with VIN (100 and 200 μM) Cells treated with LA (100 and 200 μM) Cells treated with VIN (100 μM) + LA (100 μM) Cells treated with BCM (100 μM) + LA (100 μM) b Data
2 days
3 days
6 daysa
14 daysb
− − − − −
− − − − −
+ + + − −
+ + + − −
+ + + − −
from 8 experiments. from 2 experiments.
Ultrastructure of Capsule in Control and Inhibited C. neoformans Cells Control cells had a uniform capsule of ‘curl-headed’ appearance; buds had a fine, denser capsule. The rim of the capsule material was around the bud neck in the dividing cell, and a small circle resembling a bud scar was found on the mother cells (fig. 1a). Capsules on cells inhibited by the single microtubule inhibitors BCM and VIN differed. Each bud contained a capsule of slightly different appearance, probably due to the various stages of bud and capsule development (fig. 1b, c). Capsules on cells inhibited by the actin inhibitor LA were reduced in size, with rare small patches (fig. 1d). Capsules on cells resistant to the single inhibitors BCM, VIN and LA (fig. 1e–g) were similar to those of the control cells (fig. 1a). Capsules on cells inhibited by combined LA + VINC and LA + BCM were reduced in size, with rare small patches (fig. 1h, i). They were similar to the capsules on the LA-inhibited cells (fig. 1d) but different from those on the control cells, when observed at the same magnification (fig. 1h–j). 102
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Effects of Cytoskeleton Inhibitors on C. neoformans Examined by TEM Cells were inhibited by the single microtubule or actin inhibitors VIN, BCM or LA. Control cells had a typical fungal ultrastructure (fig. 2a). Microtubule inhibitors BCM or VIN at 100-μM and 200-μM concentrations did not block budding, but delayed cell division (table 1). The cytoplasm of cells inhibited for 30 h filled with vacuoles gradually disappeared (fig. 2b), suggesting cell death. After 2 days of inhibition by BCM (fig. 2c) or VIN (fig. 2d), ‘micro-colonies’ of joined dead cells were without cytoplasm; the empty cell walls just maintained the shape of the inhibited cells. The disappearance of the cytoplasm was direct evidence of cell death and inhibition by VIN or BCM. Cells inhibited by LA at 200-μM and 100-μM concentrations also contained only a few remnants of cytoplasm and were dead. Their cell walls persisted, maintaining the shape of budding cells (fig. 2e). Kopecká
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a Data
0h
Although TEM of cells inhibited by BCM, VIN or LA showed many dead cells without cytoplasm, a few cells contained cytoplasm. These cells were probably resistant to the inhibitors. After 6 days of inhibition in 100 μM of BCM or VIN (not shown), cells resistant to the single microtubule or actin inhibitors BCM, VIN or LA (table 2) were found; they contained cytoplasm, cell organelles and capsules (fig. 2f). Cells resistant to 100 μM of LA formed chains of budding cells that were almost without cytoplasm following cell death (fig. 2g). Combined Effects of Actin and Microtubule Inhibitors LA + VIN and LA + BCM After 6 days of combined inhibition in LA + VIN (fig. 2h) and LA + BCM (fig. 2i), the dead cells contained only a few remnants of cytoplasm inside almost empty cell walls that retained the shape of the inhibited cells. This explains why combined inhibition cells did not divide and why no resistant cells originated (tables 1, 2) [12]. However, in the combined LA + VIN and LA + BCM inhibition, on TEM, we also noticed very few cells with cytoplasm (not shown) amongst the many empty cell walls of the dead cells. We do not know whether they were artefacts caused by some technical mistake in preparing the samples for ultrathin sectioning, or whether they were very resistant, non-dividing cells or immortal yeast stem cells. We did not identify any resistant, budding cells by cell counting (table 1), phase-contrast microscopy [12] or SEM, so the cause was probably a technical mistake during sectioning. Nevertheless, we cannot exclude the fact that a few non-dividing, resistant cells were present.
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Discussion
SEM and TEM confirmed, at the ultrastructural level, the results of our previous paper [12], i.e. that combined actin + microtubule inhibitors block cell division with
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Fig. 2. TEM of C. neoformans in YEPD medium. a Control cells
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cultivated with 1% DMSO without inhibitor at the beginning of experiment. Cells were treated with 200 μM BCM for 30 h (b) and 2 days (c). d Cells treated with 200 μM VIN for 2 days. e Cells treated with 200 μM LA for 2 days. f Resistant cells were detected after 6 days in BCM. g Chain of resistant dead cells after 6 days in LA. h Cells treated with a combined application of LA + VIN (100 μM of each) for 6 days. i Cells treated with a combined application of LA + BCM (100 μM of each) for 6 days.
Color version available online
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and microtubules (green). The capsule size is reduced. d Cells inhibited with combined LA + VIN (or BCM; 100 μM of each) have a nucleus (blue), but do not have microtubules and actin cytoskeletons. Diagrams are based on the combined results of this study and previously submitted data [12]. a = F-actin; mi = microtubules; nu = nucleus.
Color version available online
Fig. 3. Diagram of cells showing actin cytoskeletons, microtubules, nuclei (based on our previous paper) and cell surface covered by capsules. a Control cells have nucleus (blue), actin filaments (red) and microtubules (green) and a capsule. b Cells inhibited with VIN or BCM do not have microtubules (green), but have a nucleus (blue), an actin cytoskeleton (red); three new buds developed, covered with a capsule. c Cells inhibited with LA have a nucleus (blue)
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Fig. 4. a Diagram of control cells showing actin cytoskeletons, mi-
crotubules, nuclei (based on [12]) and cell surfaces covered by capsules. b Cells treated with VIN or BCM are dead and without cytoplasm; only empty cell walls with capsules persist. c Cells treated with LA are dead and without cytoplasm; only empty cell walls
no resistant cells appearing. TEM demonstrated that inhibited cells were dead and without cytoplasm. Their death was induced by microtubule and actin inhibitors. We do not know why C. neoformans cells die after contact with single or combined microtubule and actin inhibitors (figs. 3, 4). Animal cells with disrupted micro104
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with reduced capsules persist. d Cells inhibited with combined LA + VIN or LA + BCM (100 μM of each) are dead, without cytoplasm; only empty cell walls with reduced capsules persist. a = Factin microfilament cables; mi = microtubules; nu = nucleus.
tubules die by apoptosis [18]. The yeast Saccharomyces cerevisiae, with a disrupted single actin gene, dies by lethal mutation [19], and temperature-sensitive yeast actin mutants die at non-permissive temperatures [20, 21] with features of apoptosis [22, 23]. Eukaryotic cells probably cannot exist without actin and microtubule cyKopecká
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toskeletons, and are not viable. While TEM showed the interiors of the inhibited cells, with empty cell walls, no cytoplasm and no cell organelles, in addition to maintaining the original shape of the inhibited cells, SEM showed surface views of the inhibited cells. What we had observed previously by phase-contrast microscopy after 6 days of inhibiting the cells [12] were, in fact, only empty cell walls with capsules but no cytoplasm, that imitated yeast cells. Dead cells without cytoplasm could be observed already after 2 days of inhibition by the single inhibitors. However, resistant, budding cells then appeared. In order to induce cell death of the whole cell population, consisting of about 5 × 105 to 1 × 106 cells/ml, we applied the combined drugs LA + VIN and LA + BCM, each at 100μM concentrations. These disrupted both the microtubules and F-actin [12], and the cells died. No resistant, budding cells originated because the cells were dead, as proven here by TEM. With regard to the capsules, while control mother cells had dense, curl-headed capsules and fine, dense capsules on their buds, the cells inhibited by single BCM or VIN differed; each bud had a different capsule, probably due to the different stages of bud and capsule development. The capsules on the LA-inhibited cells, the LA + VIN-inhibited cells and the LA + BCM-inhibited cells differed in a similar way. Capsules were reduced in size, probably due to the disruption of the actin cytoskeleton, similar to that seen in the related Fellomyces fuzhouensis (inhibited by 100 μM of LA for 4 days), where the capsules completely disappeared [17]. We speculate that the cause may be the disruption of the actin cytoskeleton,
maybe involved in the transport of cytoplasmic vesicles containing capsule polysaccharides or their precursors to the cell cortex. The disruption of microtubules by the microtubule inhibitors BCM or VIN combined with the disruption of actin microfilaments by the actin inhibitor LA causes the most effective blockage of cell division in C. neoformans out of all the inhibitors that we have tested so far. The inhibited cells die, the dead cells cannot divide and resistant cells cannot originate. Our results identified the cytoskeleton of C. neoformans as a new potential target for the inhibition of cell division, the anti-microtubule drugs VIN and BCM and the anti-actin drug LA as new antifungal agents that, when used in combined application, induce cell death.
Conclusion
C. neoformans cells inhibited by combined LA + VINC or LA + BCM die and consist of only empty cell walls with capsules that resemble whole yeast cells on SEM.
Acknowledgement The author thanks Ing. Ladislav Ilkovics, Vladimíra Ramíková, Dobromila Klemová and Jan Šlancar for their excellent technical help. Financial support was provided in 2000–2008 by three grants from the Grant Agency of the Czech Republic: GAP 310/00/0391 and 310/06/0605 (Marie Kopecká), and GAP 310/03/1195 (Miroslav Gabriel). Institutional support by Masaryk University, Brno, in 2009–2014 enabled continuation of this work.
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