DIAGN MICROBIOLINFECT DIS 1990;13:31-35
31
MYCOLOGY
Effects of Terconazole and Other Azole Antifungal Agents on the Sterol and Carbohydrate Composition of Candida albicans M.A. Pfaller, J. Riley, and T. Koerner
The effects of terconazole, a triazole antifungal, on the sterol and carbohydrate composition of Candida albicans was compared with that of three imidazoles: clotrimazole, miconazole, and butoconazole. Exposure of C. albicans to terconazole resulted in a profound depletion of ergosterol with a corresponding increase in lanosterol content versus control cells. Carbohydrate analysis revealed a significant (245%) increase in chitin and a minimal effect on glucan and mannan in terconazole-treated cells. Similar effects on sterol and carbohydrate composition were observed zoith clotrimazole and miconazole. Butoconazole had a similar effect on sterol composition
but had no effect on carbohydrate composition. The decreased ergosterol and increased lanosterol content is consistent with 14c~-demethylase inhibition by terconazole and the other azoles. The increase in cell wall chitin is most likely due to deregulation of chitin synthesis secondary to ergosterol depletion in the cell membrane. Because both chitin and ergosterol are critical components of the fungal cell, perturbation of the production and localization of these components by, terconazole is likely to contribute to the selective toxicity of this compound for C. albicans and other fungi.
INTRODUCTION
work has been published on the triazoles, particularly terconazole (Vanden Bossche, 1985; Vanden Bossche et al., 1987; lsaacson et al., 1988). Of particular interest is the effect of these agents on fungal cell membrane and cell wall synthesis and composition, which are obvious points of chemotherapeutic attack. Interference with membrane sterol synthesis resulting in cellular permeability defects and deregulation of cell wall synthesis may serve as a potential means for selective cytotoxicity (Vanden Bossche, 1985; Marichal et al., 1984; Borgers, 1988). Recently, Isaacson et al. (1988) demonstrated that terconazole was a potent inhibitor of C-14 desmethyl sterol synthesis in C. albicans similar to that observed with other azole antifungal agents. Further investigation into the comparative effects of terconazole and other azole derivatives on pathogenic isolates of C. albicans is warranted. In the present study we have investigated the effects of terconazole and three comparable topical azole antifungals, clotrimazole, miconazole, and buto-
Terconazole is a triazole ketal derivative with broadspectrum in vitro and in vivo activities (Van Cutsem et al., 1983; Tolman et al., 1986). Terconazole is particularly active in vitro in preventing the yeast to mycelial transformation in Candida albicans (Tolman et al., 1986). In vivo terconazole has excellent topical activity against vaginal candidiasis and dermatomycoses (Van Cutsem et al., 1983). Although a considerable amount is known about the biochemical effects of the imidazoles, relatively little From the Veterans Administration Medical Center (M.A.P., J.R.), Department of Pathology (M.A.P., T.K.), University of Iowa College of Medicine, Iowa City, Iowa. Address reprint requests to: M. A. Pfaller, M.D., Department of Pathology, University of Iowa College of Medicine, Iowa City, IA 52242. Received June 1, 1989; revised and accepted September 27, 1989. © 1990 Elsevier Science Publishing Co., Inc. 655 Avenue of the Americas, New York, NY 10010 0732-8893190153.50
32
conazole, on the composition of membrane sterols and cell wall carbohydrates of C. albicans in an effort to further define the biochemical effects of the triazole antifungals.
MATERIALS AND M E T H O D S Organisms All experiments were performed with a representative clinical isolate of C. albicans (strain 320-40) that had previously been identified by germ tube formation, morphology on corn meal agar, and assimilation patterns using the API 20C Yeast Identification System (Analytab, Plainview, New York). The isolate was stored in sterile water at room temperature and subcultured on agar medium at monthly intervals to ensure viability.
Antifungal Agents Terconazole and butoconazole were obtained as standard powders from Ortho Pharmaceutical Corporation. Clotrimazole (Miles Pharmaceuticals, West Haven, Connecticut) and miconazole (Janssen Pharmaceutica Inc., Piscataway, New Jersey) were obtained as standard powders from their respective manufacturers. Stock solutions (10,000 ~.g/ml) of each antifungal agent were prepared in DMSO and further diluted in sterile phosphate buffered (pH 7.0) yeast nitrogen base medium (BYNB; Difco Laboratories, Detroit, Michigan) as necessary.
Growth Conditions Identical growth conditions were used before the sterol and carbohydrate analysis experiments. Yeasts were passaged twice on Sabouraud agar plates (Difco Laboratories, Detroit, Michigan) and then grown at 30°C for 48 hr in BYNB broth. Cells from the 48-hr broth culture were inoculated into fresh BYNB and grown at 30°C to mid-log phase. The cells were then harvested by centrifugation (5000 x g for 5 min) and washed in sterile water before use in the various experiments.
Sterol Analysis Flasks containing 250 ml of BYNB and either no drug (control), 0.01 }xmol/L, or 10 ~mol/L of terconazole, clotrimazole, miconazole, or butoconazole were inoculated to an initial cell density of 5 x 106 cells per ml and incubated, with shaking, at 30°C for 18 hr. The cells were then harvested by centrifugation (5000 x g for 5 min) and washed three times in cold (4°C) deionized water. The pellets were placed into pre-
M.A. Pfaller et al.
weighed glass tubes, flash frozen in liquid nitrogen, and lyophilized (in the dark). These samples were weighed and extracted with 80 volumes of chloroform:methanol (1:1), with stirring, for three days (in the dark). Following extraction the mixture was centrifuged (5000 x g, 5 min) and the extract (supernatant) removed. The pellets were washed twice with chloroform:methanol (1:1) and the wash material (supernatant) added to the original extracts. The extracts were then rotoevaporated to dryness, lyophilized, weighed, and saponified for 18 hr at room temperature using 0.1 mol/L methanolic sodium hydroxide. The saponified extracts were rotoevaporated, reconstituted in deionized HPLC-grade water, and dialyzed over a 24-hr period with at least four changes of water. The dialyzed samples were rotoevaporated, lyophilized, and weighed. The nonsaponifiable lipids were then reconstituted in chloroform:methanol (1:1) and applied to a high-performance thin-layer chromatography (HPTLC) plate (Silica gel 60, Merck, West Germany). Standards of ergosterol and ianosterol (Sigma, St. Louis, Missouri) at known concentrations were applied to the same plate. Both samples and standards were run in duplicate. The plates were developed for 20 min using toluene:ethyl acetate (4:1) and visualized with the Lieberman-Burchard reagent plus heat (120°C for 30 min). The ergosterol and lanosterol-containing spots in the test samples were identified by their migration on the HPTLC plates relative to that of the known standards. The plates were optically scanned at 370 nm with a thin-layer plate scanner (Shimadzu Model CS-930). A standard curve was constructed by plotting peak height versus sterol content for the ergosterol and lanosterol standards, and the ergosterol and lanosterol content of the test samples was estimated from the standard curve by linear regression analysis. All analyses were performed in triplicate for each experiment. The standard curves for both ergosterol and langosterol were linear over the range of interest (up to 6 ~g) and the correlation coefficients were ~0.99.
Cell Wall Fractionation and Carbohydrate Analysis The effect of terconazole, clotrimazole, miconazole, and butoconazole on cell wall carbohydrate composition was analyzed according to the methods of Milewski et al. (1986). Flasks containing 250 ml of BYNB and either no drug (control), 0.01 ~mol/L, or 10 ~mol/L of each of the four antifungal agents were inoculated to a density of 5 x 106 cells per ml and incubated with shaking at 30°C for a total of 18 hr. After incubation the cells were harvested by centrifugation (5000 x g for 5 min) and washed twice with cold (4°C) HPLC grade water. A portion of the
Effects of Azoles on C. albicans
cell pellet was retained for cell counts, dry weight determination, and total cell carbohydrate determination. The remainder of the pellet was treated with 2-4 ml cold 10% trichloroacetic acid (TCA) for 20 min. After TCA treatment the suspensions were centrifuged (5000 x g) for 5 min at 4°C and washed twice with cold HPLC grade water. Pellets were suspended in 5 ml of 6% KOH and incubated at 80°C for 90 min. After incubation the suspensions were chilled to 4°C and centrifuged (5000 x g for 5 min) to separate the alkali insoluble precipitates (glucan and chitin) from the supernatants (mannan). The mannan-containing supernatants were treated with 5 ml of Fehlings reagent and incubated overnight at 4°C. The resulting precipitates (mannan fraction) were washed with 2 ml of 0.5 mol/L NaOH, solubilized in 0.1 lal 4N HC1, and finally diluted to 2 ml with HPLC grade water. The alkali insoluble precipitates (glucan and chitin) were washed twice with water and suspended in 2 ml of 0.1 mol/L phosphate buffer (pH 6.0) containing 5 units of chitinase (Sigma, St. Louis, Missouri). The suspension was incubated for 18 hr at 30°C and then centrifuged at 5000 x g for 5 min. The remaining precipitates (insoluble glucan fraction) were washed twice and resuspended in 2 ml HPLC grade water, and the supernatants (chitin fraction) were saved for subsequent carbohydrate analysis. The carbohydrate content of the unfractionated (total cell) material, as well as the glucan and mannan fractions, was determined by the anthrone method of Trevelyan and Harrison (1952). The carbohydrate (N-acetyl glucosamine) content of the chitin fraction was determined by the Morgan-Elson reaction (Dische, 1962). Each determination was performed in triplicate and the results expressed as lag carbohydrate per mg dry weight. The standard curves for the two biochemical assays were linear over the range of interest (0-100 p,g, anthrone; 0-30 tag, Morgan-Elson) and the correlation coeffidents were ~>0.99.
RESULTS A N D D I S C U S S I O N Exposure of C. albicans to subinhibitory (0.01 lamol/ L) concentrations of terconazole, clotrimazole, and miconazole resulted in a substantial decrease in ergosterol content and an increase in lanosterol content relative to control cells (Table 1). At the higher concentration (10 txmol/L), the effect was enhanced with all three antifungal agents, but was most pronounced with terconazole. Although butoconazole did not inhibit the growth of C. albicans at either concentration, it did produce a decrease in ergosterol content and a corresponding increase in lanosterol content following exposure of cells to 10 lamol/ L.
33
Subinhibitory (0.01 lamol/L) concentrations of terconazole, clotrimazole, and miconazole had little effect on the cell wall carbohydrate composition of C. albicans (Table 1); however, exposure of cells to the higher concentration (10 i~mol/L) of each of these agents resulted in a significant increase in chitin content relative to control cells. The effect was most pronounced with terconazole where the chitin content of treated cells was 245% of control. Neither terconazole nor the two imidazoles had any effect on cell wall glucan or mannan content. Butoconazole had no effect on cell wall carbohydrate composition at either concentration. The results of this study confirm the earlier findings of Isaacson et al. (1988) who demonstrated that terconazole inhibited the synthesis of 14-des-methyl sterols, such as ergosterol, in C. albicans. We have extended these findings to demonstrate that terconazole is at least as potent as three other topical azoles, miconazole, clotrimazole, and butoconazole, in producing depletion of ergosterol and a profound increase in lanosterol content in C. albicans. In addition, we have shown that exposure of C. albicans to terconazole, clotrimazole, and miconazole resulted in a marked increase in the chitin content of the cells. The effect of these agents on cell wall carbohydrate appears to be specific for chitin as little or no effect on glucan or mannan content was observed. The effect on chitin appears to be related to the degree of sterol synthesis inhibition. The three azoles that produced significant decreases in ergosterol and increases in lanosterol content also produced increases in chitin content whereas butoconazole, which had the least effect on sterol composition, had no effect on chitin content or the overall carbohydrate composition of treated cells. These findings are similar to those described by Vanden Bossche (1985) in which increases in chitin content were observed in C. albicans after exposure to itraconazole and ketoconazole. Likewise, Hector and Braun (1987) demonstrated that bifonazole had an effect on chitin synthesis in C. albicans and was stimulatory at low concentrations. Given the possibility that ergosterol may be involved in the regulation of chitin synthesis (Chiew et al., 1982; Sekiya and Nozawa, 1983; Vanden Bossche, 1985), one explanation for these observations is that the decreased synthesis of ergosterol and concomitant accumulation of 14-methylsterols such as lanosterol may create unbalanced conditions in the cell resulting in a generalized activation of chitin synthesis (Roberts et al., 1983; Marichal et al., 1984; Vanden Bossche, 1985). Another possibility is suggested by the work of Rast and Bartnicki-Garcia (1981), who noted that low concentrations of nystatin directly stimulated chitin synthase activity. They speculated that there may be specific sterol binding sites on the
M.A. Pfaller et al.
34
TABLE 1.
Effect of Terconazole a n d O t h e r Azoles on Sterol a n d Cell Wall C a r b o h y d r a t e C o m p o s i t i o n of Candida albicans Percent of Control Values (Mean -+ SEM) a'b
Antifungal Agent ~ Terconazole 0.01 10 Clotrimazole 0.01 10 Miconazole 0.01 10 Butoconazole 0.01 10
Growth (Dry wt.)
Ergosterol
Lanosterol
Chitin
Glucan
Mannan
100 +_ 2 71 -+ 4
60 -+ 6 11 +_ 2
193 -+ 25 425 _+ 48
109 -+ 2 245 _+ 7
98 _+ 3 98 +_ 5
98 _+ 4 98 +- 1
100 -+ 5 59 -+ 3
49 -+ 3 37 _+ 2
199 _+ 8 291 -+ 20
134 _+ 15 200 ~ 4
97 _+ 1 89 -+ 11
101 + 2 108 _+ 3
100 _+ 8 75 _-,2 3
71 _+ 6 46 _+ 2
77 -+. 25 520 _+ 32
112 _+ 12 209 = 1
90 + 1 113 _+ 14
87 _+ 2 91 _+ 2
100 + 2 100 +- 1
52 -+ 3 51 _+ 1
110 ~ 7 144 _+ 3
105 _- 1 104 ~ 1
100 _+ 4 109 _+ 3
107 + 6 102 _~ 5
~Results are the Mean _+ SEM of at least six separate determinations. bCalculations based on ~,g sterol per 50 ~.g nonsaponifiable lipid (ergosterol and lanosterol) or ~g carbohydrate per mg dry, weight (chitin, glucan, mannan). "Antifungal concentration in ~mol,'L.
m e m b r a n e s of c h i t o s o m e s a n d that this m a y play a role in the localization, activation, and/or inhibition of chitin s y n t h a s e activity by polyenes, sterols, a n d other factors. The possible i n v o l v e m e n t of a sterol or sterol-like molecule in the operation of chitin synthase is further s u p p o r t e d by the finding of C h i e w et al. (1982), w h o s h o w e d that ergosterol inhibited m e m b r a n e b o u n d chitin s y n t h a s e activity and by Peste et al. (1981), w h o f o u n d that ergosterol deficient, p o l y e n e resistant m u t a n t s of C. albicans had a two-fold higher chitin s y n t h a s e activity than normal cells. Thus, the inhibition of ergosterol synthesis by azoles m a y indirectly stimulate chitin synthesis by depleting cell m e m b r a n e s of ergosterol resulting in activation of chitin synthase. Because chitin synthesis is normally highly regulated a n d localized to the site of fission b e t w e e n the separating d a u g h t e r (bud) and m o t h e r cell in the yeast form a n d to the site of s e p t u m a n d p r i m a r y (apical) wall formation in the h y p h a l form of C. albicans (Braun a n d Calderone, 1978; G o o d a y , 1978; Rogers et al., 1980; V a n d e n Bossche, 1985), deregulation and s u b s e q u e n t increase in chitin content m a y be detrimental to the cell a n d contribute to the selective cytotoxicity of the azoles for fungal cells. This is s u p p o r t e d in the present s t u d y by the microscopic observation of aberrant b u d d i n g patterns a n d inhibition of h y p h a l formation by a p p r o x i m a t e l y 30% (data not shown) in terconazole-treated versus control cells. The in vivo significance of azole-induced disturbances in ergosterol and chitin content is u n k n o w n . The results of the p r e s e n t study, as well as those of
Hector and Braun (1987) and V a n d e n Bossche (1985), indicate that such effects take place at relatively low concentrations of azoles. Given the fact that at the higher concentrations achieved in topical applications of agents such as terconazole, miconazole, a n d clotrimazole the m e c h a n i s m of action is likely to be a direct lytic effect (Iwata et al., 1973; Cope, 1980; Beggs, 1983), the m o s t subtle effects on m e m b r a n e a n d cell wall c o m p o s i t i o n m a y be of little consequence. These effects m a y be of greater i m p o r t a n c e in the setting of systemic t h e r a p y w h e r e l o w e r concentrations of azole antifungal agents are achieved. In s u m m a r y we h a v e d e m o n s t r a t e d effects of terconazole a n d three c o m p a r a t i v e azole antifungal agents on both m e m b r a n e sterols a n d cell wall carboh y d r a t e s of C. albicans. Terconazole was as p o t e n t as miconazole, clotrimazole, a n d b u t o c o n a z o l e in altering the p r o d u c t i o n of chitin a n d ergosterol in the fungal cell. Because both ergosterol and chitin are critical c o m p o n e n t s of the fungal cell, p e r t u r b a t i o n of the p r o d u c t i o n a n d localization of these c o m p o n e n t s by terconazole is likely to contribute to the selective toxicity of this c o m p o u n d for C. albicans a n d other fungi. These findings further d o c u m e n t the p o t e n t anticandidal activity of terconazole a n d indicate that additional in vitro a n d in vivo studies are w a r r a n t e d .
This work was supported in part by the Veterans Administration and by a grant from the Ortho Pharmaceutical Corporation. The excellent secretarial skills of Ms. Ruth Kjaer are appreciated. Ms. Kathy Walker provided technical assistance.
Effects of Azoles on C. albicans
35
REFERENCES Beggs WH (1983) Comparison of miconazole and ketoconazole-induced release of K* from Candida species. J Antimicrob Chemother 11:381. Borgers M (1988) Ultrastructural correlates of antimycotic treatment. In Current Topics in Medical Mycology, Vol 2, Ed., MR McGinnis. New York: Springer-Verlag, pp 139. Braun PC, Calderone RA (1978) Chitin synthesis in Candida albicans: comparison of yeast and hyphal forms. J Bacteriol 133:1472. Chiew YY, Sullivan PA, Shepherd MG (1982) The effects of ergosterol and alcohols on germ-tube formation and chitin synthase in Candida albicans. Can ] Biochem 60:15. Cope JE (1980) Mode of action of miconazole on Candida albicans: effect on growth, viability and K* release. J Gen Microbiol 119:245. Dische Z (1962) Color reactions of hexosamines. In Methods in Carbohydrate Chemistry, Vol 1, Eds., RL Whistler and MZ Wolfrom. New York: Academic Press, pp 507-512. Gooday GW (1978) The enzymology of hyphal growth. In The Filamentous Fungi: Developmental Biology, Vol 3, Eds., JE Smith and DR Berry. London: Edward Arnold, pp 51-77. Hector RF, Braun PC (1987) The effects of bifonazole on chitin synthesis in Candida albicans. In Recent Trends in the Discovery, Development and Evaluation of Antifungal Agents, Ed., RA Fromtling. S.A.: JR Prous Science Publishers, pp 369-382. Isaacson DM, Tolman EL, Tobia AJ, Rosenthale ME, McGuire JL, Vanden Bossche H, Janssen PAJ (1988) Selective inhibition of 14 a-desmethyl sterol synthesis in Candida albicans by terconazole, a new triazole antimycotic. ] Antimicrob Chemother 21:333. lwata K, Yamaguchi H, Hirantani H (1973) Mode of action of clotrimazole. Sabouraudia 11:158. Marichal P, Gorrens J, Vanden Bossche H (1984) The action of itraconazole and ketoconazole on growth and sterol
synthesis in Aspergillus fumigatus and Aspergillus niger. Sabouraudia: J Med Vet Mycol 22:13. Milewski S, Chmara H, Borowski E (1986) Antibiotic tetaine: a selective inhibitor of chitin and mannoprotein biosynthesis in Candida albicans. Arch Microbiol 145:234. Peste M, Campbell JM, Peberdy JF (1981) Alteration of ergosterol content and chitin synthase activity in Candida albicans. Curr Microbiol 5:187. Rast DM, Bartnicki-Garcia S (1981) Effects of amphotericin B, nystatin, and other polyene antibiotics on chitin synthase. Proc Natl Acad Sci 78:1233. Roberts RL, Blowers B, Slater ML, Cabib E (1983) Chitin synthesis and localization in cell division cycle mutants of Saccharomyces cerevisiae. Mol Cell Biol 3:922. Rogers HJ, Perkins HR, Ward JB (1980) Biosynthesis of Cell Walls and Membranes. London: Chapman and Hall, pp 478-507. Sekiya T, Nozawa Y (1983) Reorganization of membrane ergosterol during cell fission events of Candida albicans: a freeze-fracture study of distribution of filipin-ergosterol complexes. ] Ultrastruct Res 83:48. Tolman EL, Isaacson DM, Rosenthale ME, McGuire JL, Van Cutsem J, Borgers M, Vanden Bossche H (1986) Anticandidal activities of terconazole, a broad-spectrum antimycotic. Antimicrob Agents Chemother 29:986. Trevelyan WE, Harrison JS (1952) Studies on yeast metabolism. Biochem J 50:298. Vanden Bossche H (1985) Biochemical targets for antifungal azole derivatives: hypothesis on the mode of action. In Current Topics on Medical Mycology, Vol 1, Ed., MR McGinnis. New York: Springer-Verlag, pp 313-351. Vanden Bossche H, Willemsens G, Marichal P (1987) AntiCandida drugs: the biochemical basis for their activity. CRC Crit Rev MicrobioI 15:57. Van Cutsem J, Van Gerven F, Zaman R, Janssen PAJ (1983) Terconazole: a new broad-spectrum antifungal. Chemotherapy 29:322.
31
MYCOLOGY
Effects of Terconazole and Other Azole Antifungal Agents on the Sterol and Carbohydrate Composition of Candida albicans M.A. Pfaller, J. Riley, and T. Koerner
The effects of terconazole, a triazole antifungal, on the sterol and carbohydrate composition of Candida albicans was compared with that of three imidazoles: clotrimazole, miconazole, and butoconazole. Exposure of C. albicans to terconazole resulted in a profound depletion of ergosterol with a corresponding increase in lanosterol content versus control cells. Carbohydrate analysis revealed a significant (245%) increase in chitin and a minimal effect on glucan and mannan in terconazole-treated cells. Similar effects on sterol and carbohydrate composition were observed zoith clotrimazole and miconazole. Butoconazole had a similar effect on sterol composition
but had no effect on carbohydrate composition. The decreased ergosterol and increased lanosterol content is consistent with 14c~-demethylase inhibition by terconazole and the other azoles. The increase in cell wall chitin is most likely due to deregulation of chitin synthesis secondary to ergosterol depletion in the cell membrane. Because both chitin and ergosterol are critical components of the fungal cell, perturbation of the production and localization of these components by, terconazole is likely to contribute to the selective toxicity of this compound for C. albicans and other fungi.
INTRODUCTION
work has been published on the triazoles, particularly terconazole (Vanden Bossche, 1985; Vanden Bossche et al., 1987; lsaacson et al., 1988). Of particular interest is the effect of these agents on fungal cell membrane and cell wall synthesis and composition, which are obvious points of chemotherapeutic attack. Interference with membrane sterol synthesis resulting in cellular permeability defects and deregulation of cell wall synthesis may serve as a potential means for selective cytotoxicity (Vanden Bossche, 1985; Marichal et al., 1984; Borgers, 1988). Recently, Isaacson et al. (1988) demonstrated that terconazole was a potent inhibitor of C-14 desmethyl sterol synthesis in C. albicans similar to that observed with other azole antifungal agents. Further investigation into the comparative effects of terconazole and other azole derivatives on pathogenic isolates of C. albicans is warranted. In the present study we have investigated the effects of terconazole and three comparable topical azole antifungals, clotrimazole, miconazole, and buto-
Terconazole is a triazole ketal derivative with broadspectrum in vitro and in vivo activities (Van Cutsem et al., 1983; Tolman et al., 1986). Terconazole is particularly active in vitro in preventing the yeast to mycelial transformation in Candida albicans (Tolman et al., 1986). In vivo terconazole has excellent topical activity against vaginal candidiasis and dermatomycoses (Van Cutsem et al., 1983). Although a considerable amount is known about the biochemical effects of the imidazoles, relatively little From the Veterans Administration Medical Center (M.A.P., J.R.), Department of Pathology (M.A.P., T.K.), University of Iowa College of Medicine, Iowa City, Iowa. Address reprint requests to: M. A. Pfaller, M.D., Department of Pathology, University of Iowa College of Medicine, Iowa City, IA 52242. Received June 1, 1989; revised and accepted September 27, 1989. © 1990 Elsevier Science Publishing Co., Inc. 655 Avenue of the Americas, New York, NY 10010 0732-8893190153.50
32
conazole, on the composition of membrane sterols and cell wall carbohydrates of C. albicans in an effort to further define the biochemical effects of the triazole antifungals.
MATERIALS AND M E T H O D S Organisms All experiments were performed with a representative clinical isolate of C. albicans (strain 320-40) that had previously been identified by germ tube formation, morphology on corn meal agar, and assimilation patterns using the API 20C Yeast Identification System (Analytab, Plainview, New York). The isolate was stored in sterile water at room temperature and subcultured on agar medium at monthly intervals to ensure viability.
Antifungal Agents Terconazole and butoconazole were obtained as standard powders from Ortho Pharmaceutical Corporation. Clotrimazole (Miles Pharmaceuticals, West Haven, Connecticut) and miconazole (Janssen Pharmaceutica Inc., Piscataway, New Jersey) were obtained as standard powders from their respective manufacturers. Stock solutions (10,000 ~.g/ml) of each antifungal agent were prepared in DMSO and further diluted in sterile phosphate buffered (pH 7.0) yeast nitrogen base medium (BYNB; Difco Laboratories, Detroit, Michigan) as necessary.
Growth Conditions Identical growth conditions were used before the sterol and carbohydrate analysis experiments. Yeasts were passaged twice on Sabouraud agar plates (Difco Laboratories, Detroit, Michigan) and then grown at 30°C for 48 hr in BYNB broth. Cells from the 48-hr broth culture were inoculated into fresh BYNB and grown at 30°C to mid-log phase. The cells were then harvested by centrifugation (5000 x g for 5 min) and washed in sterile water before use in the various experiments.
Sterol Analysis Flasks containing 250 ml of BYNB and either no drug (control), 0.01 }xmol/L, or 10 ~mol/L of terconazole, clotrimazole, miconazole, or butoconazole were inoculated to an initial cell density of 5 x 106 cells per ml and incubated, with shaking, at 30°C for 18 hr. The cells were then harvested by centrifugation (5000 x g for 5 min) and washed three times in cold (4°C) deionized water. The pellets were placed into pre-
M.A. Pfaller et al.
weighed glass tubes, flash frozen in liquid nitrogen, and lyophilized (in the dark). These samples were weighed and extracted with 80 volumes of chloroform:methanol (1:1), with stirring, for three days (in the dark). Following extraction the mixture was centrifuged (5000 x g, 5 min) and the extract (supernatant) removed. The pellets were washed twice with chloroform:methanol (1:1) and the wash material (supernatant) added to the original extracts. The extracts were then rotoevaporated to dryness, lyophilized, weighed, and saponified for 18 hr at room temperature using 0.1 mol/L methanolic sodium hydroxide. The saponified extracts were rotoevaporated, reconstituted in deionized HPLC-grade water, and dialyzed over a 24-hr period with at least four changes of water. The dialyzed samples were rotoevaporated, lyophilized, and weighed. The nonsaponifiable lipids were then reconstituted in chloroform:methanol (1:1) and applied to a high-performance thin-layer chromatography (HPTLC) plate (Silica gel 60, Merck, West Germany). Standards of ergosterol and ianosterol (Sigma, St. Louis, Missouri) at known concentrations were applied to the same plate. Both samples and standards were run in duplicate. The plates were developed for 20 min using toluene:ethyl acetate (4:1) and visualized with the Lieberman-Burchard reagent plus heat (120°C for 30 min). The ergosterol and lanosterol-containing spots in the test samples were identified by their migration on the HPTLC plates relative to that of the known standards. The plates were optically scanned at 370 nm with a thin-layer plate scanner (Shimadzu Model CS-930). A standard curve was constructed by plotting peak height versus sterol content for the ergosterol and lanosterol standards, and the ergosterol and lanosterol content of the test samples was estimated from the standard curve by linear regression analysis. All analyses were performed in triplicate for each experiment. The standard curves for both ergosterol and langosterol were linear over the range of interest (up to 6 ~g) and the correlation coefficients were ~0.99.
Cell Wall Fractionation and Carbohydrate Analysis The effect of terconazole, clotrimazole, miconazole, and butoconazole on cell wall carbohydrate composition was analyzed according to the methods of Milewski et al. (1986). Flasks containing 250 ml of BYNB and either no drug (control), 0.01 ~mol/L, or 10 ~mol/L of each of the four antifungal agents were inoculated to a density of 5 x 106 cells per ml and incubated with shaking at 30°C for a total of 18 hr. After incubation the cells were harvested by centrifugation (5000 x g for 5 min) and washed twice with cold (4°C) HPLC grade water. A portion of the
Effects of Azoles on C. albicans
cell pellet was retained for cell counts, dry weight determination, and total cell carbohydrate determination. The remainder of the pellet was treated with 2-4 ml cold 10% trichloroacetic acid (TCA) for 20 min. After TCA treatment the suspensions were centrifuged (5000 x g) for 5 min at 4°C and washed twice with cold HPLC grade water. Pellets were suspended in 5 ml of 6% KOH and incubated at 80°C for 90 min. After incubation the suspensions were chilled to 4°C and centrifuged (5000 x g for 5 min) to separate the alkali insoluble precipitates (glucan and chitin) from the supernatants (mannan). The mannan-containing supernatants were treated with 5 ml of Fehlings reagent and incubated overnight at 4°C. The resulting precipitates (mannan fraction) were washed with 2 ml of 0.5 mol/L NaOH, solubilized in 0.1 lal 4N HC1, and finally diluted to 2 ml with HPLC grade water. The alkali insoluble precipitates (glucan and chitin) were washed twice with water and suspended in 2 ml of 0.1 mol/L phosphate buffer (pH 6.0) containing 5 units of chitinase (Sigma, St. Louis, Missouri). The suspension was incubated for 18 hr at 30°C and then centrifuged at 5000 x g for 5 min. The remaining precipitates (insoluble glucan fraction) were washed twice and resuspended in 2 ml HPLC grade water, and the supernatants (chitin fraction) were saved for subsequent carbohydrate analysis. The carbohydrate content of the unfractionated (total cell) material, as well as the glucan and mannan fractions, was determined by the anthrone method of Trevelyan and Harrison (1952). The carbohydrate (N-acetyl glucosamine) content of the chitin fraction was determined by the Morgan-Elson reaction (Dische, 1962). Each determination was performed in triplicate and the results expressed as lag carbohydrate per mg dry weight. The standard curves for the two biochemical assays were linear over the range of interest (0-100 p,g, anthrone; 0-30 tag, Morgan-Elson) and the correlation coeffidents were ~>0.99.
RESULTS A N D D I S C U S S I O N Exposure of C. albicans to subinhibitory (0.01 lamol/ L) concentrations of terconazole, clotrimazole, and miconazole resulted in a substantial decrease in ergosterol content and an increase in lanosterol content relative to control cells (Table 1). At the higher concentration (10 txmol/L), the effect was enhanced with all three antifungal agents, but was most pronounced with terconazole. Although butoconazole did not inhibit the growth of C. albicans at either concentration, it did produce a decrease in ergosterol content and a corresponding increase in lanosterol content following exposure of cells to 10 lamol/ L.
33
Subinhibitory (0.01 lamol/L) concentrations of terconazole, clotrimazole, and miconazole had little effect on the cell wall carbohydrate composition of C. albicans (Table 1); however, exposure of cells to the higher concentration (10 i~mol/L) of each of these agents resulted in a significant increase in chitin content relative to control cells. The effect was most pronounced with terconazole where the chitin content of treated cells was 245% of control. Neither terconazole nor the two imidazoles had any effect on cell wall glucan or mannan content. Butoconazole had no effect on cell wall carbohydrate composition at either concentration. The results of this study confirm the earlier findings of Isaacson et al. (1988) who demonstrated that terconazole inhibited the synthesis of 14-des-methyl sterols, such as ergosterol, in C. albicans. We have extended these findings to demonstrate that terconazole is at least as potent as three other topical azoles, miconazole, clotrimazole, and butoconazole, in producing depletion of ergosterol and a profound increase in lanosterol content in C. albicans. In addition, we have shown that exposure of C. albicans to terconazole, clotrimazole, and miconazole resulted in a marked increase in the chitin content of the cells. The effect of these agents on cell wall carbohydrate appears to be specific for chitin as little or no effect on glucan or mannan content was observed. The effect on chitin appears to be related to the degree of sterol synthesis inhibition. The three azoles that produced significant decreases in ergosterol and increases in lanosterol content also produced increases in chitin content whereas butoconazole, which had the least effect on sterol composition, had no effect on chitin content or the overall carbohydrate composition of treated cells. These findings are similar to those described by Vanden Bossche (1985) in which increases in chitin content were observed in C. albicans after exposure to itraconazole and ketoconazole. Likewise, Hector and Braun (1987) demonstrated that bifonazole had an effect on chitin synthesis in C. albicans and was stimulatory at low concentrations. Given the possibility that ergosterol may be involved in the regulation of chitin synthesis (Chiew et al., 1982; Sekiya and Nozawa, 1983; Vanden Bossche, 1985), one explanation for these observations is that the decreased synthesis of ergosterol and concomitant accumulation of 14-methylsterols such as lanosterol may create unbalanced conditions in the cell resulting in a generalized activation of chitin synthesis (Roberts et al., 1983; Marichal et al., 1984; Vanden Bossche, 1985). Another possibility is suggested by the work of Rast and Bartnicki-Garcia (1981), who noted that low concentrations of nystatin directly stimulated chitin synthase activity. They speculated that there may be specific sterol binding sites on the
M.A. Pfaller et al.
34
TABLE 1.
Effect of Terconazole a n d O t h e r Azoles on Sterol a n d Cell Wall C a r b o h y d r a t e C o m p o s i t i o n of Candida albicans Percent of Control Values (Mean -+ SEM) a'b
Antifungal Agent ~ Terconazole 0.01 10 Clotrimazole 0.01 10 Miconazole 0.01 10 Butoconazole 0.01 10
Growth (Dry wt.)
Ergosterol
Lanosterol
Chitin
Glucan
Mannan
100 +_ 2 71 -+ 4
60 -+ 6 11 +_ 2
193 -+ 25 425 _+ 48
109 -+ 2 245 _+ 7
98 _+ 3 98 +_ 5
98 _+ 4 98 +- 1
100 -+ 5 59 -+ 3
49 -+ 3 37 _+ 2
199 _+ 8 291 -+ 20
134 _+ 15 200 ~ 4
97 _+ 1 89 -+ 11
101 + 2 108 _+ 3
100 _+ 8 75 _-,2 3
71 _+ 6 46 _+ 2
77 -+. 25 520 _+ 32
112 _+ 12 209 = 1
90 + 1 113 _+ 14
87 _+ 2 91 _+ 2
100 + 2 100 +- 1
52 -+ 3 51 _+ 1
110 ~ 7 144 _+ 3
105 _- 1 104 ~ 1
100 _+ 4 109 _+ 3
107 + 6 102 _~ 5
~Results are the Mean _+ SEM of at least six separate determinations. bCalculations based on ~,g sterol per 50 ~.g nonsaponifiable lipid (ergosterol and lanosterol) or ~g carbohydrate per mg dry, weight (chitin, glucan, mannan). "Antifungal concentration in ~mol,'L.
m e m b r a n e s of c h i t o s o m e s a n d that this m a y play a role in the localization, activation, and/or inhibition of chitin s y n t h a s e activity by polyenes, sterols, a n d other factors. The possible i n v o l v e m e n t of a sterol or sterol-like molecule in the operation of chitin synthase is further s u p p o r t e d by the finding of C h i e w et al. (1982), w h o s h o w e d that ergosterol inhibited m e m b r a n e b o u n d chitin s y n t h a s e activity and by Peste et al. (1981), w h o f o u n d that ergosterol deficient, p o l y e n e resistant m u t a n t s of C. albicans had a two-fold higher chitin s y n t h a s e activity than normal cells. Thus, the inhibition of ergosterol synthesis by azoles m a y indirectly stimulate chitin synthesis by depleting cell m e m b r a n e s of ergosterol resulting in activation of chitin synthase. Because chitin synthesis is normally highly regulated a n d localized to the site of fission b e t w e e n the separating d a u g h t e r (bud) and m o t h e r cell in the yeast form a n d to the site of s e p t u m a n d p r i m a r y (apical) wall formation in the h y p h a l form of C. albicans (Braun a n d Calderone, 1978; G o o d a y , 1978; Rogers et al., 1980; V a n d e n Bossche, 1985), deregulation and s u b s e q u e n t increase in chitin content m a y be detrimental to the cell a n d contribute to the selective cytotoxicity of the azoles for fungal cells. This is s u p p o r t e d in the present s t u d y by the microscopic observation of aberrant b u d d i n g patterns a n d inhibition of h y p h a l formation by a p p r o x i m a t e l y 30% (data not shown) in terconazole-treated versus control cells. The in vivo significance of azole-induced disturbances in ergosterol and chitin content is u n k n o w n . The results of the p r e s e n t study, as well as those of
Hector and Braun (1987) and V a n d e n Bossche (1985), indicate that such effects take place at relatively low concentrations of azoles. Given the fact that at the higher concentrations achieved in topical applications of agents such as terconazole, miconazole, a n d clotrimazole the m e c h a n i s m of action is likely to be a direct lytic effect (Iwata et al., 1973; Cope, 1980; Beggs, 1983), the m o s t subtle effects on m e m b r a n e a n d cell wall c o m p o s i t i o n m a y be of little consequence. These effects m a y be of greater i m p o r t a n c e in the setting of systemic t h e r a p y w h e r e l o w e r concentrations of azole antifungal agents are achieved. In s u m m a r y we h a v e d e m o n s t r a t e d effects of terconazole a n d three c o m p a r a t i v e azole antifungal agents on both m e m b r a n e sterols a n d cell wall carboh y d r a t e s of C. albicans. Terconazole was as p o t e n t as miconazole, clotrimazole, a n d b u t o c o n a z o l e in altering the p r o d u c t i o n of chitin a n d ergosterol in the fungal cell. Because both ergosterol and chitin are critical c o m p o n e n t s of the fungal cell, p e r t u r b a t i o n of the p r o d u c t i o n a n d localization of these c o m p o n e n t s by terconazole is likely to contribute to the selective toxicity of this c o m p o u n d for C. albicans a n d other fungi. These findings further d o c u m e n t the p o t e n t anticandidal activity of terconazole a n d indicate that additional in vitro a n d in vivo studies are w a r r a n t e d .
This work was supported in part by the Veterans Administration and by a grant from the Ortho Pharmaceutical Corporation. The excellent secretarial skills of Ms. Ruth Kjaer are appreciated. Ms. Kathy Walker provided technical assistance.
Effects of Azoles on C. albicans
35
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