ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, August 1977, p. 255-260 Copyright © 1977 American Society for Microbiology
Vol. 12, No. 2 Printed in U.S.A.
Morphological Changes in Yeasts as a Result of the Action of 5-Fluorocytosine TADASHI ARAI,* YUZURU MIKAMI, KOJI YOKOYAMA, TOMIO KAWATA, AND KUNIYOSHI MASUDA Department ofAntibiotics, Research Institute for Chemobiodynamics, Chiba University, Narashino, Chiba,*
and Department of Food Microbiology, Tokushima University School of Medicine, Tokushi ma, Japan Received for publication 31 August 1976
The mode of action of 5-fluorocytosine on Candida albicans and Saccharomycerevisiae was studied with special reference to morphology and ultrastructure. Shortly after contact of yeast cells with 10 ,g of 5-fluorocytosine per ml, marked enlargement of the cells occurred. The initial increase in cell size was almost the same with both yeast strains. As incubation proceeded, further increase in cell volume continued with C. albicans, whereas no further change in cell volume was observed with S. cerevisiae. An electron microscope examination of C. albicans cells after exposure to the drug revealed characteristic changes, consisting of an enlarged nucleus and a thin cell wall. The morphological changes were exactly comparable to those reported with inhibitors of deoxyribonucleic acid synthesis in mammalian cells and some bacteria.
ces
Antifungal activity, pharmacological properties, and clinical aspects of 5-fluorocytosine (5FC) have been well documented (20). The mode of action of the compound was also reported by several investigators (3, 5-8, 11). Giege and Weil (8) reported a close relationship between the action mechanism of 5-FC and deamination of the drug to 5-fluorouracil (5-FU) within susceptible cells. Horowitz and Chargaff (10) also observed the incorporation of 5-FU into ribonucleic acid (RNA), where it replaces about 50% of the uracil. Our previous experiments (1, 2, 13) confirmed the above findings and, in addition, the selective incorporation of 5-FC into Candida albicans cells. However, an alternative mechanism of action for 5-FC, consisting of a limitation in deoxyribonucleic acid (DNA) synthesis, cannot be excluded. The inhibition of thymidylate synthetase was established by Heidelberger (9) in the studies of the action mechanism of 5-FU on mammalian cells. On the other hand, Cohen (7) discovered the phenomenon of thymineless death in 1953 in which specific inhibition of DNA synthesis results in unbalanced growth and the subsequent death of the cell. Cohen (6) also showed that n-arabinosylcytosine specifically inhibited DNA synthesis in fibroblasts, which in turn resulted in the enlargement of susceptible cells as their viability falls. In our series of experiments on the mechanism of action of 5-FC, morphological changes characteristic of drugs that selectively inhibit DNA synthesis and that cause thymineless death were observed with C. albicans.
MATERUILS AND METHODS Organisms and growth medium. Clinical isolates of C. albicans strain 7N and Saccharomyces cerevisiae IFM 4060 were used in this study. The cultures were maintained on dextrose Sabouraud agar slants. The medium principally used in the present experiments was a yeast nitrogen base (Difco) supplemented with L-asparagine (0.15%) and dextrose (1.0%). Reagents. 5-FC was kindly supplied by HoffmanLa Roche, Inc., Tokyo, Japan. A stock solution of 5FC (10,000 /g/ml) was prepared in physiological saline and sterilized by filtration. Cell size determination. The test organisms were cultured on 4% dextrose Sabouraud agar slants for 18 h at 37°C and harvested by draining with saline. The cells were inoculated at a concentration of 2 x 105 cells/ml in a yeast nitrogen base (Difco) medium. 5-FC was added to give a final concentration of 10 ,ug/ml. After 3, 6, 12, and 24 h of incubation at 37°C, the cell size distribution was determined by electronic counting with a Coulter counter model ZB-1 (apperture tube of 100 nm and standardized with a paper mulberry pollen). The procedure for sizing was essentially the same as those employed by Orth and Cornwell (14) and Brecher and Stohlman (5). Electron microscopy. Growing cells were suspended in a fresh medium with appropriate dilution and shaken in the presence or absence of the drug at 37°C. The level of 10 ,ug of 5-FC per ml was selected for this experiment, and after the 2-, 6-, and 12-h incubations samples for electron microscopy were withdrawn. These specimens were fixed with 2% glutaraldehyde in 0.1 M phosphate buffer (pH 7.3) for 2 h, washed with the same buffer three times, and postfixed with KMnO4 in distilled water for 20 h at 4°C. Dehydration was carried out in a graded series of ethanol followed by embedding in styrene255
256
ARAI ET AL.
~.
ANTIMICROB. AGENTS CHEMOTHER.
methacrylate resin (12). Thin sectioning was carried out by using a Sorvall Porter-Blum MT-1 ultramicrotome, and the sections were then stained with Reynolds lead citrate for 3 min and observed under a Hitachi HU-11E electron microscope, using an accelerating voltage of 75 kV.
Size distribution curves of C. albicans grow-
ing in the presence and absence of 5-FC (10 ggl ml) are shown in Fig. 2, and that for S. cerevisiae under the same conditions is shown in Fig.
3. A marked increase in cell volume was initiated after the 3-h incubation, and the cell volume had almost doubled after 12 h. After a 24-h incubation, however, the cell sizes began to distribute at random within a wide range of cell volumes, and distribution curves were not obtained. This must be ascribed to the facts that the cells eventually lost their viability and the number of morphologically distorted cells increased. On the other hand, the increase in the
RESULTS The morphology of C. albicans before and after 5-FC treatment was compared by ordinary microscopy. Enlargement of C. albicans cells is evident as shown in Fig. 1. Germ tube formation does not seem to be affected, even in the presence of 5-FC. w,_ f
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(A) Normal control cells
(x1,400);
MODE OF ACTION OF 5-FLUOROCYTOSINE
VOL. 12, 1977 10
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DIAMETER
FIG. 2. Size distribution curves of C. albicans cells growing in the presence of 10 pg of 5-FC per ml.
cell volume of S. cerevisiae with time was not significant, although the initial enlargement was comparable to that of C. albicans. The ultrastructure of a normal control cell of C. albicans obtained by electron microscopy is shown in Fig. 4. Subcellular organelles were displayed as previously reported by Borgers and De Nollin (4). Permanganate fixation has been known to preserve the subcellular organelles well, except for ribosomes. Three distinct layers of the cell wall were observed, and the irregularly shaped nucleus was composed of intermingled fine granular and agranular electron-dense material. In the cytoplasm, many electron-dense granules, possibly glycogen granules (22), were scattered. Under higher magnification, irregular plasmolemmal protrusions were seen, and the nucleus was limited by discontinuous double membranes. The profiles of mitochondria and endoplasmic reticulum were well preserved. Figure 5 shows a Candida cell after a 2-h contact with 10 ug of 5-FC per ml. No significant changes were observed in the structures of the cell wall, plasma membrane, and other intracytoplasmic organelles except for enlargement of the nucleus. After a 12-h incubation with 5-FC, enlargement of the nucleus was prominent, and the cell wall became much thinner, probably as a result of the marked increase in cell volume with the impaired cell wall synthesis, as shown in Fig. 6A and B, respectively. During this period, the nucleus became translucent and its structure was damaged to a considerable extent. Under higher magnification, as shown in Fig. 7, fine filamentous components, sometimes coiled,
257
were observed in the nucleus, and the ground substance was completely lost - these findings are characteristic of almost all kinds of impaired nuclei. The significance of membranous fragments indicated by arrows is still unknown. DISCUSSION Jund and Lacroute have shown that the uptake of both cytosine and 5-FC is mediated by the same permease (11). It is a commonly accepted view that 5-FC is rapidly deaminated in susceptible yeast cells into 5-FU. Arai and Mikami obtained direct evidence of this intracellular conversion of 5-FC with C. albicans (2). Polak and Scholer also studied the fungistatic activity of 5-FC and its reversal by bases and nucleosides of the pyrimidines and purines, as well as uptake and incorporation of 5-FC by C. albicans (18, 19). All pertinent incorporation studies with 5-FC in susceptible fungi showed a constant and fairly quantitative correlation between growth inhibition and incorporation of 5FU into RNA. This incorporation was assumed to be involved in the mode of action of the drug and regarded as a prerequisite for the antifungal activity. Since 5-FC manifests its inhibitory effect after its conversion into 5-FU, however, an additional or alternative mechanism that consists of an inhibition of DNA synthesis may be conceivable, as established in mammalian cells and certain bacteria by Heiderberger (9) and as already pointed out by Polak and
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DIAMETER FIG. 3. Size distribution curves of S. cerevisiae cells growing in the presence of 10 pg of5-FC per ml.
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FIG. 5. C. albicans cell after a 2-h contact with 10 pg of 5-FC per ml. NP, Nuclear pore; V, vacuole. Other abbreviations are the same as in the legend to Fig. 4. 258
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FIG. 6. C. albicans cell after a 12-h contact with 10 ug of 5-FC per ml. (A) Note the enlargement of the nucleus; (B) very thin cell wall. See legend to Fig. 4 for abbreviations. .
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Vol. 12, No. 2 Printed in U.S.A.
Morphological Changes in Yeasts as a Result of the Action of 5-Fluorocytosine TADASHI ARAI,* YUZURU MIKAMI, KOJI YOKOYAMA, TOMIO KAWATA, AND KUNIYOSHI MASUDA Department ofAntibiotics, Research Institute for Chemobiodynamics, Chiba University, Narashino, Chiba,*
and Department of Food Microbiology, Tokushima University School of Medicine, Tokushi ma, Japan Received for publication 31 August 1976
The mode of action of 5-fluorocytosine on Candida albicans and Saccharomycerevisiae was studied with special reference to morphology and ultrastructure. Shortly after contact of yeast cells with 10 ,g of 5-fluorocytosine per ml, marked enlargement of the cells occurred. The initial increase in cell size was almost the same with both yeast strains. As incubation proceeded, further increase in cell volume continued with C. albicans, whereas no further change in cell volume was observed with S. cerevisiae. An electron microscope examination of C. albicans cells after exposure to the drug revealed characteristic changes, consisting of an enlarged nucleus and a thin cell wall. The morphological changes were exactly comparable to those reported with inhibitors of deoxyribonucleic acid synthesis in mammalian cells and some bacteria.
ces
Antifungal activity, pharmacological properties, and clinical aspects of 5-fluorocytosine (5FC) have been well documented (20). The mode of action of the compound was also reported by several investigators (3, 5-8, 11). Giege and Weil (8) reported a close relationship between the action mechanism of 5-FC and deamination of the drug to 5-fluorouracil (5-FU) within susceptible cells. Horowitz and Chargaff (10) also observed the incorporation of 5-FU into ribonucleic acid (RNA), where it replaces about 50% of the uracil. Our previous experiments (1, 2, 13) confirmed the above findings and, in addition, the selective incorporation of 5-FC into Candida albicans cells. However, an alternative mechanism of action for 5-FC, consisting of a limitation in deoxyribonucleic acid (DNA) synthesis, cannot be excluded. The inhibition of thymidylate synthetase was established by Heidelberger (9) in the studies of the action mechanism of 5-FU on mammalian cells. On the other hand, Cohen (7) discovered the phenomenon of thymineless death in 1953 in which specific inhibition of DNA synthesis results in unbalanced growth and the subsequent death of the cell. Cohen (6) also showed that n-arabinosylcytosine specifically inhibited DNA synthesis in fibroblasts, which in turn resulted in the enlargement of susceptible cells as their viability falls. In our series of experiments on the mechanism of action of 5-FC, morphological changes characteristic of drugs that selectively inhibit DNA synthesis and that cause thymineless death were observed with C. albicans.
MATERUILS AND METHODS Organisms and growth medium. Clinical isolates of C. albicans strain 7N and Saccharomyces cerevisiae IFM 4060 were used in this study. The cultures were maintained on dextrose Sabouraud agar slants. The medium principally used in the present experiments was a yeast nitrogen base (Difco) supplemented with L-asparagine (0.15%) and dextrose (1.0%). Reagents. 5-FC was kindly supplied by HoffmanLa Roche, Inc., Tokyo, Japan. A stock solution of 5FC (10,000 /g/ml) was prepared in physiological saline and sterilized by filtration. Cell size determination. The test organisms were cultured on 4% dextrose Sabouraud agar slants for 18 h at 37°C and harvested by draining with saline. The cells were inoculated at a concentration of 2 x 105 cells/ml in a yeast nitrogen base (Difco) medium. 5-FC was added to give a final concentration of 10 ,ug/ml. After 3, 6, 12, and 24 h of incubation at 37°C, the cell size distribution was determined by electronic counting with a Coulter counter model ZB-1 (apperture tube of 100 nm and standardized with a paper mulberry pollen). The procedure for sizing was essentially the same as those employed by Orth and Cornwell (14) and Brecher and Stohlman (5). Electron microscopy. Growing cells were suspended in a fresh medium with appropriate dilution and shaken in the presence or absence of the drug at 37°C. The level of 10 ,ug of 5-FC per ml was selected for this experiment, and after the 2-, 6-, and 12-h incubations samples for electron microscopy were withdrawn. These specimens were fixed with 2% glutaraldehyde in 0.1 M phosphate buffer (pH 7.3) for 2 h, washed with the same buffer three times, and postfixed with KMnO4 in distilled water for 20 h at 4°C. Dehydration was carried out in a graded series of ethanol followed by embedding in styrene255
256
ARAI ET AL.
~.
ANTIMICROB. AGENTS CHEMOTHER.
methacrylate resin (12). Thin sectioning was carried out by using a Sorvall Porter-Blum MT-1 ultramicrotome, and the sections were then stained with Reynolds lead citrate for 3 min and observed under a Hitachi HU-11E electron microscope, using an accelerating voltage of 75 kV.
Size distribution curves of C. albicans grow-
ing in the presence and absence of 5-FC (10 ggl ml) are shown in Fig. 2, and that for S. cerevisiae under the same conditions is shown in Fig.
3. A marked increase in cell volume was initiated after the 3-h incubation, and the cell volume had almost doubled after 12 h. After a 24-h incubation, however, the cell sizes began to distribute at random within a wide range of cell volumes, and distribution curves were not obtained. This must be ascribed to the facts that the cells eventually lost their viability and the number of morphologically distorted cells increased. On the other hand, the increase in the
RESULTS The morphology of C. albicans before and after 5-FC treatment was compared by ordinary microscopy. Enlargement of C. albicans cells is evident as shown in Fig. 1. Germ tube formation does not seem to be affected, even in the presence of 5-FC. w,_ f
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B FIG. 1. Enlargement of C. albicans cells induced by 5-FC treatment. (B) after 12-h contact with 10 pg of 5-FC per ml (xl,400).
(A) Normal control cells
(x1,400);
MODE OF ACTION OF 5-FLUOROCYTOSINE
VOL. 12, 1977 10
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0
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DIAMETER
FIG. 2. Size distribution curves of C. albicans cells growing in the presence of 10 pg of 5-FC per ml.
cell volume of S. cerevisiae with time was not significant, although the initial enlargement was comparable to that of C. albicans. The ultrastructure of a normal control cell of C. albicans obtained by electron microscopy is shown in Fig. 4. Subcellular organelles were displayed as previously reported by Borgers and De Nollin (4). Permanganate fixation has been known to preserve the subcellular organelles well, except for ribosomes. Three distinct layers of the cell wall were observed, and the irregularly shaped nucleus was composed of intermingled fine granular and agranular electron-dense material. In the cytoplasm, many electron-dense granules, possibly glycogen granules (22), were scattered. Under higher magnification, irregular plasmolemmal protrusions were seen, and the nucleus was limited by discontinuous double membranes. The profiles of mitochondria and endoplasmic reticulum were well preserved. Figure 5 shows a Candida cell after a 2-h contact with 10 ug of 5-FC per ml. No significant changes were observed in the structures of the cell wall, plasma membrane, and other intracytoplasmic organelles except for enlargement of the nucleus. After a 12-h incubation with 5-FC, enlargement of the nucleus was prominent, and the cell wall became much thinner, probably as a result of the marked increase in cell volume with the impaired cell wall synthesis, as shown in Fig. 6A and B, respectively. During this period, the nucleus became translucent and its structure was damaged to a considerable extent. Under higher magnification, as shown in Fig. 7, fine filamentous components, sometimes coiled,
257
were observed in the nucleus, and the ground substance was completely lost - these findings are characteristic of almost all kinds of impaired nuclei. The significance of membranous fragments indicated by arrows is still unknown. DISCUSSION Jund and Lacroute have shown that the uptake of both cytosine and 5-FC is mediated by the same permease (11). It is a commonly accepted view that 5-FC is rapidly deaminated in susceptible yeast cells into 5-FU. Arai and Mikami obtained direct evidence of this intracellular conversion of 5-FC with C. albicans (2). Polak and Scholer also studied the fungistatic activity of 5-FC and its reversal by bases and nucleosides of the pyrimidines and purines, as well as uptake and incorporation of 5-FC by C. albicans (18, 19). All pertinent incorporation studies with 5-FC in susceptible fungi showed a constant and fairly quantitative correlation between growth inhibition and incorporation of 5FU into RNA. This incorporation was assumed to be involved in the mode of action of the drug and regarded as a prerequisite for the antifungal activity. Since 5-FC manifests its inhibitory effect after its conversion into 5-FU, however, an additional or alternative mechanism that consists of an inhibition of DNA synthesis may be conceivable, as established in mammalian cells and certain bacteria by Heiderberger (9) and as already pointed out by Polak and
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DIAMETER FIG. 3. Size distribution curves of S. cerevisiae cells growing in the presence of 10 pg of5-FC per ml.
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''h4 FIG. 4. Ultrastructure of control C. albicans cells after 12-h incubation. Cell wall (CW), plasma membrane (PM), nucleus (N), nuclear membrane (NM), endoplasmic reticulum (ER), and mitochondria (M) are clearly seen. Bars equal 0.5 jum.
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FIG. 5. C. albicans cell after a 2-h contact with 10 pg of 5-FC per ml. NP, Nuclear pore; V, vacuole. Other abbreviations are the same as in the legend to Fig. 4. 258
'W
FIG. 6. C. albicans cell after a 12-h contact with 10 ug of 5-FC per ml. (A) Note the enlargement of the nucleus; (B) very thin cell wall. See legend to Fig. 4 for abbreviations. .
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