Candidal Vaginitis
19
The Effects of Miconazole on the Ultrastructure of Candida albicans by Dr Sonja De Nollin, Dr M Borgers and Dr H Van Belle (Laboratories of Cell Biology and Biochemistry, Janssen Pharmaceutica, Beerse, Belgium) The use of a new technique for the preparation of Candida albicans for transmission electron microscopy (TEM) makes it possible to obtain good morphology of the control yeast cells (Fig 1) and allows a clear follow up of drug-induced ultrastructural alterations (Borgers & De Nollin 1974). The changes in ultrastructure which occur after miconazole treatment are dose-dependent and time-related (De Nollin & Borgers 1974). After exposing cultures of C. albicans to fungistatic concentrations of miconazole (10-8 to 10-7M) for 24 hours an increase in the volume of the cells was obvious. Morphological changes were seen at the cell periphery and consisted of proliferation of the plasmalemma and thickening of the cell wall. Numerous inclusions of varying size and electron density, limited by a thin osmiophilic membranous structure, were embedded in the walls. Aggregations of these peculiar structures occurred frequently in areas with excessive thickening of the wall or in the vicinity of buds. At these doses the number of peroxisomes had already increased and lipid droplets were more frequent. The other subcellular organelles appeared to be unaltered. With 10-6M miconazole, the volume of the cells increased markedly and the peripheral changes were more pronounced. In the cell interior, peroxisomes became numerous and the central vacuole was filled up with variably sized vesicles and agranular material (Fig 2). With the minimal fungicidal dose (10-5M) most of the cells suffered severe damage. Disruption from partial dissolution to complete loss of the plasmalemma was commonly observed. The same applied to the limiting membrane of the vacuole. Heavily swollen mitochondria, multiple fat deposits, dilated membrane fragments and aggregated ribosomes were noted in the cytoplasm. It was impossible to identify nuclei or nuclear remnants in such injured cells (Fig 3). Of particular interest was the observation that with a total fungicidal dose (10-4M) the remaining cells had a very regular, unaltered cell wall, whereas their interior was completely necrotic (Fig 4). Further evidence for the potent fungicidal action exerted by miconazole was given by studies with the scanning electron microscope (SEM, De Nollin & Borgers 1975). The cells of the control culture showed up separately, having polar buds and bud scars (Fig 5). Exposure to
Dr Sonja De Nollin
miconazole in a fungistatic and minimal fungicidal amount (10-8 to 10-5M) resulted in clusters of interconnected cells. The cell surface was uniformly rough, presenting bud scars which were randomly distributed (Fig 6). The cells that remained after using the fungicidal dose (10-4M) of miconazole had a quite normal appearance when compared with the controls, but they were covered with large amounts of small vesicular material (Fig 7). This material most probably consists of remnants of broken cells. This assumption is strengthened by the frequent occurrence of the walls of broken cells (Fig 7) and also by examination with TEM, which showed that apparently normal-looking cells were completely necrotic inside. One may therefore conclude that the normal size and shape of a cell seen with SEM should not be taken as a criterion of viability. Using this 10-4M dose, completely necrotic aggregations were also seen (Fig 8). In order to gain more insight into the morphologic involutionary processes leading to cell necrosis, taking into account the observation that the number of peroxisomes increased markedly, the distribution of oxidative and peroxidative enzymes was localized cytochemically and the activity quantitated biochemically (De Nollin et al. 1975, De Nollin & Borgers 1976, De Nollin et al. 1977, Borgers et al. 1977). Catalase activity of the peroxisomes (Fig 9) was strongly enhanced after fungistatic treatment (Fig 10), whereas a minimal fungicidal dose resulted in the total lack of this enzyme (Fig 11). Another peroxidative enzyme, cytochrome c-peroxidase, was found in control cells, strong activity being present on the mitochondrial crista
20 Proc. roy. Soc. Med. Volume 70 1977 Supplement 4
:.. .. .:...
. . . . . ..
.................
Fig 1 Candida albicans control cell grown for 24 hours. The preservation of subcellular organelles is shown for comparison: cell wall (cw), a convoluted plasmalemma (pl), mitochondria (m), vacuole (vac). x 21 600 Fig 2 C. albicans exposed to 10-6M miconazole for 24 hours. Note the presence of lomasomal bodies (arrow) and vesicles (i) between the cell membrane (pl) and the cell wall (cw). Dark, membrane-limited inclusions (i) in the cell wall are randomly distributed. A general thickening of the cell wall is seen. The other cell organelles appear unchanged. Nucleus (n) peroxisomes (p) mitochondria (m) budformation (b), vacuole (vac). x 13 475
Fig 3 C. albicans exposed to 1O-5M miconazole for 24 hours. A complete necrotic cell is shown; the cell wall (cw) is thickened and contains several inclusions (i the plasmalemma is fragmentary, in the cytoplasm mitochondrial remnants (m), lipid globules (1) and membrane fragments are present. The cell shape has drastically changed. x 15 975 Fig 4 C. albicans exposed to 10-4M micanazolefor 24 hours. The cell exhibits a regular outlined cell wall (cw), whereas the plasmalemma is only fragmentary (arrows) and the cytoplasm is entirely necrotic. Multiple lipid deposits (1) are observed, mostly on the cellperiphery. x 15 850
Candidal Vaginitis
21
I
I Fig 5 Scanning electron micrograph. Untreated C. albicans culture. The cells appear as isolated yeast forms showing theformation ofpolar buds. x 2075 Fig 6 Scanning electron micrograph. C. albicans culture exposed tofungistatic dose of miconazole. After budding, the cells continue to adhere to one another. Their surface is wrinkled and randomly distributed bud scars are visible over the surface of the cells. x 6000
Fig 7 Scanning electron micrograph. C. albicans culture exposed to afungicidal dose of miconazole. Thefew remaining cells are isolated. Their surfaces are smooth but are covered with vesicules of broken cells. TEM examination reveals that the interior of these cells is completely lysed. x 2675 Fig 8 Scanning electron micrograph. C. albicans culture exposed to afungicidal dose of miconazole. The picture shows the remains ofa necrotic cell. x 2675
22 Proc. roy. Soc. Med. Volume 701977 Supplement 4
Figs 9-11 C. albicans Catalase and perioxidase activity Fig 9 Control cell. The catalase positive peroxisome (p) is situated near a mitochondrion (m) showing intefering cytochrome c-peroxidase activity. x 69 950 Fig 10 Exposure to 10--6M miconazolefor 24 hours. All the peroxisomes (p) present on this section are strongly catalase positive. Cell wall (cw) plasmalemma (pl) inclusions (i). The peroxidase activity on the mitochondrion (m) is visibly lowered. x 50 375 Fig 11 Exposure to 10-5M miconazole for 24 hours. No activity remains on the peroxisomes (p). The cytochrome c-peroxidase activity on the mitochondria (m) has also disappeared. x 74 625
Figs 12-14 C. albicans NADHoxidase activity Fig 12 Control cell. The granular precipitate is very weak on the mitochondrial cristae (m) and is present in moderate amounts in the vacuole (arrow). x 45 775 Fig 13 Exposure to 10-6M miconazolefor 24 hours. Strong reaction product is deposited in the vacuole (vac). The activity on the mitochondrial cristae is also enhanced as compared with that in Fig 12. x 38 700 Fig 14 Exposure to 10-6M miconazolefor 24 hours. A necrotic cell shows abundant precipitate distributed in clumps in the mitochondrial matrices (arrows). x 42 525
Candidal Vaginitis (Fig 9). Fungistatic treatment induced a marked decrease of the peroxidase (Fig 1 1). Cells exposed to a minimal fungicidal dose showed complete lack of peroxidase activity (Fig 12). This observation suggested to us that hydrogen peroxide (H202) was involved in this cellular necrosis. A candidate for the enzyme taking part- in this metabolic oxidative system is NADH oxidase. This enzyme was present on the mitochondrial criste and on the central vacuole of control cells (Fig 12). The NADH oxidase activity increased markedly in the central vacuoles and their contents and in the mitochondria after treatment with fungistatic doses (Fig 13). Exposure to the minimal fungicidal dose resulted in an increase of NADH oxidase present in clumps in mitochondria and in most cases distributed all over the cytoplasm (Fig 14). These cytochemical observations were confirmed by quantitative biochemical measurements of the enzyme activities. From the present ultrastructural data on C. albicans exposed to fungistatic doses of miconazole, it appears that this drug provokes changes on the plasmalemma and the cell wall. Simultaneously, the number of peroxisomes increases. The other cell constituents are involved when higher doses are imposed. The changes in cell shape are probably due to an osmotic imbalance provoked by a drug-induced alteration in cell membrane permeability (De Nollin & Borgers 1974, Sreedhara et al. 1974, Van den Bossche 1974). On the other hand, with total fungicidal doses no such changes at the periphery seem to occur. Because of this discrepancy in response towards fungistatic and fungicidal doses, it was thought that the mechanisms by which miconazole exerts its lethal effect on C. albicans might be different from that suggested by the morphologic changes at the cell periphery after fungistatic doses. This assumption was based on data on the enzymatic behaviour of oxidative and peroxidative enzymes. These data are interpreted as follows. Control cells are equipped with a series of oxidative
23
enzymes (e.g. NADH oxidase) necessary for energy requirements and metabolism and further, with peroxidative enzymes (cytochrome c-peroxidase and catalase) to avoid accumulation of the H202 which is formed during oxidative processes. It is well known that H202 is highly toxic to the cells. Exposure of cultures to fungistatic doses of miconazole results in a marked decrease of cytochrome c-peroxidase activity and a simultaneous strong increase of catalase activity. This increase might be indicative of an enhanced rate of production of H202 and probably represents a cellular defence reaction designed to rescue the cell from H202 intoxication. The increased NADH oxidase activity and hence a larger amount of intracellular H202 could induce catalase activity. The complete lack of both peroxidase and catalase activities after treatment with the minimal fungicidal dose, while NADH oxidase is still active, indicates that H202 intoxication may occur and that this is probably the cause of the cellular necrosis. REFERENCES Borgers M & De Nollin S (1974) Journal of Cell Biology 62, 574 Borgers M, De Nollin S, Thone F & Van Belle H (1977) Journal ofHistochemistry and Cytochemistry (in press) De Nollins S & Borgers M (1974) Sabouraudia 12, 341 (1975) Antimicrobial Agents and Chemotherapy 7, 704 (1977) Mykosen (in press) De Nollin S, Thone F & Borgers M (1975) Journal of Histochemistry and Cytochemistry 23, 758 De Nollin S, Van Belle H, Goosens F, Thome F & Borgers M (1977) Antimicrobial Agents and Chemotherapy (in press) Sreedhara Swamy K M, Sirsi M & Ramananda Rao G (1974) Antimicrobial Agents and Chemotherapy 5, 420 Van den Bossche H (1974) Biochemical Pharmacology 23, 887
DISCUSSION
Professor J R Hobbs (London) said that the killing of candida by the white cells used similar enzymes, catalase and oxidase; he wondered whether the drug inhibited this process, although if it did such an effect would not interfere with its usefulness.
19
The Effects of Miconazole on the Ultrastructure of Candida albicans by Dr Sonja De Nollin, Dr M Borgers and Dr H Van Belle (Laboratories of Cell Biology and Biochemistry, Janssen Pharmaceutica, Beerse, Belgium) The use of a new technique for the preparation of Candida albicans for transmission electron microscopy (TEM) makes it possible to obtain good morphology of the control yeast cells (Fig 1) and allows a clear follow up of drug-induced ultrastructural alterations (Borgers & De Nollin 1974). The changes in ultrastructure which occur after miconazole treatment are dose-dependent and time-related (De Nollin & Borgers 1974). After exposing cultures of C. albicans to fungistatic concentrations of miconazole (10-8 to 10-7M) for 24 hours an increase in the volume of the cells was obvious. Morphological changes were seen at the cell periphery and consisted of proliferation of the plasmalemma and thickening of the cell wall. Numerous inclusions of varying size and electron density, limited by a thin osmiophilic membranous structure, were embedded in the walls. Aggregations of these peculiar structures occurred frequently in areas with excessive thickening of the wall or in the vicinity of buds. At these doses the number of peroxisomes had already increased and lipid droplets were more frequent. The other subcellular organelles appeared to be unaltered. With 10-6M miconazole, the volume of the cells increased markedly and the peripheral changes were more pronounced. In the cell interior, peroxisomes became numerous and the central vacuole was filled up with variably sized vesicles and agranular material (Fig 2). With the minimal fungicidal dose (10-5M) most of the cells suffered severe damage. Disruption from partial dissolution to complete loss of the plasmalemma was commonly observed. The same applied to the limiting membrane of the vacuole. Heavily swollen mitochondria, multiple fat deposits, dilated membrane fragments and aggregated ribosomes were noted in the cytoplasm. It was impossible to identify nuclei or nuclear remnants in such injured cells (Fig 3). Of particular interest was the observation that with a total fungicidal dose (10-4M) the remaining cells had a very regular, unaltered cell wall, whereas their interior was completely necrotic (Fig 4). Further evidence for the potent fungicidal action exerted by miconazole was given by studies with the scanning electron microscope (SEM, De Nollin & Borgers 1975). The cells of the control culture showed up separately, having polar buds and bud scars (Fig 5). Exposure to
Dr Sonja De Nollin
miconazole in a fungistatic and minimal fungicidal amount (10-8 to 10-5M) resulted in clusters of interconnected cells. The cell surface was uniformly rough, presenting bud scars which were randomly distributed (Fig 6). The cells that remained after using the fungicidal dose (10-4M) of miconazole had a quite normal appearance when compared with the controls, but they were covered with large amounts of small vesicular material (Fig 7). This material most probably consists of remnants of broken cells. This assumption is strengthened by the frequent occurrence of the walls of broken cells (Fig 7) and also by examination with TEM, which showed that apparently normal-looking cells were completely necrotic inside. One may therefore conclude that the normal size and shape of a cell seen with SEM should not be taken as a criterion of viability. Using this 10-4M dose, completely necrotic aggregations were also seen (Fig 8). In order to gain more insight into the morphologic involutionary processes leading to cell necrosis, taking into account the observation that the number of peroxisomes increased markedly, the distribution of oxidative and peroxidative enzymes was localized cytochemically and the activity quantitated biochemically (De Nollin et al. 1975, De Nollin & Borgers 1976, De Nollin et al. 1977, Borgers et al. 1977). Catalase activity of the peroxisomes (Fig 9) was strongly enhanced after fungistatic treatment (Fig 10), whereas a minimal fungicidal dose resulted in the total lack of this enzyme (Fig 11). Another peroxidative enzyme, cytochrome c-peroxidase, was found in control cells, strong activity being present on the mitochondrial crista
20 Proc. roy. Soc. Med. Volume 70 1977 Supplement 4
:.. .. .:...
. . . . . ..
.................
Fig 1 Candida albicans control cell grown for 24 hours. The preservation of subcellular organelles is shown for comparison: cell wall (cw), a convoluted plasmalemma (pl), mitochondria (m), vacuole (vac). x 21 600 Fig 2 C. albicans exposed to 10-6M miconazole for 24 hours. Note the presence of lomasomal bodies (arrow) and vesicles (i) between the cell membrane (pl) and the cell wall (cw). Dark, membrane-limited inclusions (i) in the cell wall are randomly distributed. A general thickening of the cell wall is seen. The other cell organelles appear unchanged. Nucleus (n) peroxisomes (p) mitochondria (m) budformation (b), vacuole (vac). x 13 475
Fig 3 C. albicans exposed to 1O-5M miconazole for 24 hours. A complete necrotic cell is shown; the cell wall (cw) is thickened and contains several inclusions (i the plasmalemma is fragmentary, in the cytoplasm mitochondrial remnants (m), lipid globules (1) and membrane fragments are present. The cell shape has drastically changed. x 15 975 Fig 4 C. albicans exposed to 10-4M micanazolefor 24 hours. The cell exhibits a regular outlined cell wall (cw), whereas the plasmalemma is only fragmentary (arrows) and the cytoplasm is entirely necrotic. Multiple lipid deposits (1) are observed, mostly on the cellperiphery. x 15 850
Candidal Vaginitis
21
I
I Fig 5 Scanning electron micrograph. Untreated C. albicans culture. The cells appear as isolated yeast forms showing theformation ofpolar buds. x 2075 Fig 6 Scanning electron micrograph. C. albicans culture exposed tofungistatic dose of miconazole. After budding, the cells continue to adhere to one another. Their surface is wrinkled and randomly distributed bud scars are visible over the surface of the cells. x 6000
Fig 7 Scanning electron micrograph. C. albicans culture exposed to afungicidal dose of miconazole. Thefew remaining cells are isolated. Their surfaces are smooth but are covered with vesicules of broken cells. TEM examination reveals that the interior of these cells is completely lysed. x 2675 Fig 8 Scanning electron micrograph. C. albicans culture exposed to afungicidal dose of miconazole. The picture shows the remains ofa necrotic cell. x 2675
22 Proc. roy. Soc. Med. Volume 701977 Supplement 4
Figs 9-11 C. albicans Catalase and perioxidase activity Fig 9 Control cell. The catalase positive peroxisome (p) is situated near a mitochondrion (m) showing intefering cytochrome c-peroxidase activity. x 69 950 Fig 10 Exposure to 10--6M miconazolefor 24 hours. All the peroxisomes (p) present on this section are strongly catalase positive. Cell wall (cw) plasmalemma (pl) inclusions (i). The peroxidase activity on the mitochondrion (m) is visibly lowered. x 50 375 Fig 11 Exposure to 10-5M miconazole for 24 hours. No activity remains on the peroxisomes (p). The cytochrome c-peroxidase activity on the mitochondria (m) has also disappeared. x 74 625
Figs 12-14 C. albicans NADHoxidase activity Fig 12 Control cell. The granular precipitate is very weak on the mitochondrial cristae (m) and is present in moderate amounts in the vacuole (arrow). x 45 775 Fig 13 Exposure to 10-6M miconazolefor 24 hours. Strong reaction product is deposited in the vacuole (vac). The activity on the mitochondrial cristae is also enhanced as compared with that in Fig 12. x 38 700 Fig 14 Exposure to 10-6M miconazolefor 24 hours. A necrotic cell shows abundant precipitate distributed in clumps in the mitochondrial matrices (arrows). x 42 525
Candidal Vaginitis (Fig 9). Fungistatic treatment induced a marked decrease of the peroxidase (Fig 1 1). Cells exposed to a minimal fungicidal dose showed complete lack of peroxidase activity (Fig 12). This observation suggested to us that hydrogen peroxide (H202) was involved in this cellular necrosis. A candidate for the enzyme taking part- in this metabolic oxidative system is NADH oxidase. This enzyme was present on the mitochondrial criste and on the central vacuole of control cells (Fig 12). The NADH oxidase activity increased markedly in the central vacuoles and their contents and in the mitochondria after treatment with fungistatic doses (Fig 13). Exposure to the minimal fungicidal dose resulted in an increase of NADH oxidase present in clumps in mitochondria and in most cases distributed all over the cytoplasm (Fig 14). These cytochemical observations were confirmed by quantitative biochemical measurements of the enzyme activities. From the present ultrastructural data on C. albicans exposed to fungistatic doses of miconazole, it appears that this drug provokes changes on the plasmalemma and the cell wall. Simultaneously, the number of peroxisomes increases. The other cell constituents are involved when higher doses are imposed. The changes in cell shape are probably due to an osmotic imbalance provoked by a drug-induced alteration in cell membrane permeability (De Nollin & Borgers 1974, Sreedhara et al. 1974, Van den Bossche 1974). On the other hand, with total fungicidal doses no such changes at the periphery seem to occur. Because of this discrepancy in response towards fungistatic and fungicidal doses, it was thought that the mechanisms by which miconazole exerts its lethal effect on C. albicans might be different from that suggested by the morphologic changes at the cell periphery after fungistatic doses. This assumption was based on data on the enzymatic behaviour of oxidative and peroxidative enzymes. These data are interpreted as follows. Control cells are equipped with a series of oxidative
23
enzymes (e.g. NADH oxidase) necessary for energy requirements and metabolism and further, with peroxidative enzymes (cytochrome c-peroxidase and catalase) to avoid accumulation of the H202 which is formed during oxidative processes. It is well known that H202 is highly toxic to the cells. Exposure of cultures to fungistatic doses of miconazole results in a marked decrease of cytochrome c-peroxidase activity and a simultaneous strong increase of catalase activity. This increase might be indicative of an enhanced rate of production of H202 and probably represents a cellular defence reaction designed to rescue the cell from H202 intoxication. The increased NADH oxidase activity and hence a larger amount of intracellular H202 could induce catalase activity. The complete lack of both peroxidase and catalase activities after treatment with the minimal fungicidal dose, while NADH oxidase is still active, indicates that H202 intoxication may occur and that this is probably the cause of the cellular necrosis. REFERENCES Borgers M & De Nollin S (1974) Journal of Cell Biology 62, 574 Borgers M, De Nollin S, Thone F & Van Belle H (1977) Journal ofHistochemistry and Cytochemistry (in press) De Nollins S & Borgers M (1974) Sabouraudia 12, 341 (1975) Antimicrobial Agents and Chemotherapy 7, 704 (1977) Mykosen (in press) De Nollin S, Thone F & Borgers M (1975) Journal of Histochemistry and Cytochemistry 23, 758 De Nollin S, Van Belle H, Goosens F, Thome F & Borgers M (1977) Antimicrobial Agents and Chemotherapy (in press) Sreedhara Swamy K M, Sirsi M & Ramananda Rao G (1974) Antimicrobial Agents and Chemotherapy 5, 420 Van den Bossche H (1974) Biochemical Pharmacology 23, 887
DISCUSSION
Professor J R Hobbs (London) said that the killing of candida by the white cells used similar enzymes, catalase and oxidase; he wondered whether the drug inhibited this process, although if it did such an effect would not interfere with its usefulness.
