The University of Texas M. D. Anderson Cancer Center, Section of Infectious Diseases, 1515 Holcombe Boulevard, Box 47, Houston, TX 77030, USA
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Fluconazole selectively inhibits the activity of cytochrome P450-dependent C-14 lanosterol demethylase (P450 14-DM), a key enzyme in ergosterol biosynthesis in fungi.5 In Saccharomyces cerevisiae, P450 14-DM is the product of the ERG11 gene, a gene essential for the aerobic growth of S. cerevisiae.6 The presumed mechanism of action of fluconazole is the combination of ergosterol depletion and the accumulation of toxic 14-methyl sterols, especially 14-methyl-3,6-diol.7
S. cerevisiae provides an attractive experimental system for the study of azole resistance compared with the closely related, but genetically intractable, C. albicans, and has long served as a model organism for studies in sterol biosynthesis.6,8 Resistance of Saccharomyces spp. to azoles involves accumulation of alternative, less toxic sterols, increased efflux of the drug, and alterations in the target site (P450 14-DM).9 All documented mechanisms of azole resistance in Saccharomyces spp. have also been reported in C. albicans and involve the same gene products.9,10 A well-characterized mechanism of azole resistance in both organisms is conferred by loss-of-function mutations in sterol 5,6-desaturase.7,11 In Saccharomyces spp., this ergosterol biosynthetic enzyme is the product of the ERG3 gene.6 Instead of accumulating the toxic 14
-methyl-3,6-diol when treated with an azole, erg3 mutants accumulate 14
-methylfecosterol, a sterol that allows growth to continue.11
This study provides genetic evidence that mitochondria and oxidative phosphorylation are physiological partners of Erg3p and that mitochondria mediate the conversion of non-toxic 14-methylfecosterol into toxic 14
-methylsterols in the presence of fluconazole.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Standard methods were used to prepare yeast peptone dextrose (YPD), synthetic complete (SC) and yeast peptone glycerol ethanol (YPGE) media and to induce the petite (rho) phenotype.12 All rho strains were derived by inoculation of respiration-proficient strains at a low density (<105 cells/mL) into liquid YPD containing ethidium bromide 10 µg/mL. Cultures were incubated in the dark at 30°C for 48 h; single colonies that were unable to grow on YPGE were then isolated.12 All work was done in the 1278b genetic background. The Tn3::LEU2::lacZ fusion library was used as a disruption mutagen;13 mutants with a Tn3::LEU2::lacZ disruption of ERG3 are resistant to fluconazole.14 The S. cerevisiae strain 10480-6D was used as a wild-type control. S. cerevisiae PDR1-100, a fluconazole-resistant mutant that over-expresses the ABC transporter Pdr5p,15 was used as a resistant control. The yeast strains used in this study are described in the Table
. Fluconazole, a gift from Pfizer, Inc., New York, NY, USA, was kept as a 5 mg/L stock solution in sterile water. Ethidium bromide, 2,4-dinitrophenol (2,4-DNP) and carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP) were purchased from Sigma Chemical Co., St Louis, MO, USA. Stock solutions of ethidium bromide (1 mg/mL in water) and 2,4-DNP (5 mg/mL in 100% ethanol) were wrapped in aluminum foil to protect them from light.
|
Two separate assays were used to test the sensitivity to fluconazole. In the first assay, the growth of each yeast strain, streaked out to form single colonies, was examined; plates of SC medium, which is based on Bacto-yeast nitrogen base with addition of exogenous amino acids,12 containing various concentrations of fluconazole were used. In the second assay, yeast growth was examined by plating 105 cells in the late-logarithmic phase of growth in liquid SC on to SC plates containing fluconazole 500 µg in a paper disc (disc diffusion assay). Plates were incubated aerobically for 3 days at 30°C. SC medium, a defined artificial medium lacking lipid extracts, was used in order to avoid the influence of exogenous lipids on the fluconazole phenotype of the strains tested. The effect of 2,4-DNP was examined by plating approximately 105 cells of each strain in late logarithmic phase on to SC or SC plus fluconazole 128 mg/L plates. A paper disc containing 25 µg of 2,4-DNP was placed on each plate. Plates were then incubated aerobically at 30°C for 3 days.
To study the growth on YPGE medium, the isolates were inoculated on to SC plates and then replica plated with a replica block and velvetine to YPGE and SC plus fluconazole 64 mg/L plates. Growth was examined after aerobic incubation at 30°C for 3 days.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Does the experimental production of cytoplasmic petite (rho) mutants mimic the spontaneous occurrence of fluconazole-resistant petites?
Incubation of wild-type S. cerevisiae in liquid YPD with ethidium bromide 10 mg/L for 2 days at 30°C resulted in the formation of rho mutants that were resistant to fluconazole [Figure 1(a) and (b)]. The erg3 rho mutant was sensitive to fluconazole, whereas the isogenic erg3 rho+ mutant was resistant. This paradoxical sensitivity profile was specific for the erg3 rho fluconazole-resistance mutant, since the PDR1-100 rho mutant was as resistant as its rho+ counterpart [Figure 1
(b)].
|
The pet9 mutant, which requires intact mitochondria for viability,16 did not grow when glycerol and ethanol were used as carbon sources (YPGE plates12), yet it was sensitive to fluconazole (Figure 2). pet9 has a mutation in the adenine nucleotide translocator but, unlike the cytoplasmic petite mutants, it has a functional respiratory chain.16
|
We compared the fluconazole-induced growth inhibition of the wild type when grown aerobically in non-fermentable carbon sources or in glucose. Sensitivity to fluconazole was four times greater in SC with glycerol than in SC with glucose (results not shown).
Is sensitivity to fluconazole dependent on an active oxidative phosphorylation?
As shown in Figure 3, 2,4-DNP, a powerful inhibitor of oxidative phosphorylation,17 suppressed fluconazole toxicity in the wild-type strain. The protective effect of 2,4-DNP was not seen in rho strains, suggesting that the mitochondria were the main target of the 2,4-DNP effect. The protective effect of 2,4-DNP was not specific to the
1278b genetic background and also occurred with FCCP, another inhibitor of oxidative phosphorylation (D. P. Kontoyiannis, unpublished observations). 2,4-DNP had the opposite effect in the erg3 rho+ mutant, rendering it hypersensitive to fluconazole [Figure 3
(e)]. However, the erg3 rho+ mutant was more sensitive to 2,4-DNP [Figure 3
(f)] than the wild type [Figure 3
(c)]. The increased sensitivity of the erg3 mutant to 2,4-DNP in comparison with the wild type has also been demonstrated for a variety of other agents.14 Although the enhanced sensitivity of erg3 rho+ in SC containing fluconazole 128 mg/L and 2,4-DNP could result from a non-specific effect of 2,4-DNP, the finding is also consistent with the paradoxical sensitivity of the erg3 rho mutant to fluconazole (Figure 1
).
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Sterol biosynthesis is an aerobic process requiring haem and molecular oxygen, and it involves enzymes located both in the cytoplasm and in mitochondria.6,18 There is biochemical evidence that haem-dependent cytochrome derivatives are co-factors in the desaturation at C5.19 However, the physiological role of these derivatives in that reaction is poorly understood. Here we show that fluconazole requires the combination of an intact Erg3p and mitochondria in order to exert its toxicity. Respiration-deficient erg3 mutants that are unable to grow in non-fermentable carbon sources in the presence of a concomitant hem1 mutation have been described.20 Thus, it is possible that the absence of mitochondrial function in erg3 rho mutants increases the degree of dysfunction of 5,6-desaturase and further diminishes the conversion of 14
-methylfecosterol to 14
-methylsterols. However, because mitochondria could regulate the balance between 14
-fecosterol and a toxic intermediate in a parallel pathway, the erg3 rho mutant is paradoxically sensitive to azoles. Our data can be accommodated by the genetic model depicted in Figure 4
. In wild-type cells, inhibition of mitochondria leads to a decrease in the conversion of 14
-methylfecosterol to 14-methylsterols and a concomitant increase in the conversion of the former to episterol, which leads to the increased formation (through Erg3p) and more preservation of ergosterol. In contrast, in erg3 mutants lacking mitochondria, a toxic sterol (e.g. episterol) cannot be converted to ergosterol but accumulates and, in combination with the decreased formation of 14
-methylfecosterol, causes toxicity. More specifically, the preservation of ergosterol in this model could be the combined result of both the overexpression of cytochrome P450 lanosterol demethylase (Erg11p) in the setting of mitochondrial inhibition as previously described21 and the de-repression of another enzyme later in the ergosterol pathway (Figure 4
). Further biochemical studies to measure the fungal membrane sterol content of these mutants in the presence or absence of fluconazole are needed to test our hypothesis. Finally, because resistance to fluconazole and cross-resistance to amphotericin B caused by defective
5,6-desaturation has also been found in clinical isolates of C. albicans,22 it would be of interest to see whether this phenotypic change is reversed by mitochondrial inhibitors in this species.
|
This work has several implications. Conservation of the role of mitochondria in azole toxicity in some pathogenic fungi could lead to the development of compounds that enhance drug activity by interfering with mitochondrial activation. A relationship between mitochondrial DNA deficiency and resistance to azoles has been demonstrated recently in azole-resistant isolates of Candida glabrata.26 It is also possible that some fungi may be tolerant to azoles in vivo in conditions where their mitochondrial activity is diminished (e.g. abscesses). Finally, azoles could serve as interesting probes in further increasing our knowledge about respiratory control of oxidative phosphorylation.
![]() |
Acknowledgments |
---|
![]() |
Notes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2
.
Georgopapadakou, N. H. & Walsh, T. J. (1996). Antifungal agents: chemotherapeutic targets and immunologic strategies. Antimicrobial Agents and Chemotherapy 40, 27991.
3 . Bodey, G. P. (1992). Azole antifungal agents. Clinical Infectious Diseases 14, Suppl. 1, 1619.
4
.
Rex, J. H., Rinaldi, M. G. & Pfaller, M. A. (1995). Resistance of Candida species to fluconazole. Antimicrobial Agents and Chemotherapy 39, 18
5 . Vanden Bossche, H., Koymans, L. & Moereels, H. (1995). P450 inhibitors of use in medical treatment: focus on mechanisms of action. Pharmacology and Therapeutics 67, 79100.[Medline]
6 . Henri, S. A. (1982). Membrane lipids of yeast: biochemical and genetic studies. In The Molecular and Cellular Biology of the Yeast Saccharomyces, (Strathern, J., Jones, E. W. & Broach, J. Eds), pp. 10158. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
7 . Kelly, S. L., Lamb, D. C., Corran, A. J., Balwin, B. C. & Kelly, D. E. (1995). Mode of action and resistance to azole antifungals associated with the formation of 14 alpha-methylergosta-8,24 (28)-dien-3 beta, 6 alpha-diol. Biochemical and Biophysical Research Communications 207, 9105.[ISI][Medline]
8 . Parks, L. W., Smith, S. J. & Crowley, J. H. (1995). Biochemical and physiological effects of sterol alterations in yeasta review. Lipids 30, 22730.[ISI][Medline]
9 . Joseph-Horne, T. & Hollomon, D. W. (1997). Molecular mechanisms of azole resistance in fungi. FEMS Microbiology Letters 149, 1419.[ISI][Medline]
10
.
White, T. C., Marr, K. A. & Bowden, R. A. (1998). Clinical, cellular and molecular factors that contribute to antifungal drug resistance. Clinical Microbiology Reviews 11, 382402.
11 . Watson, P. F., Rose, M. E., Ellis, S. W., England, H. & Kelly, S. L. (1989). Defective sterol C56 desaturation and azole resistance: a new hypothesis for the mode of action of azole antifungals. Biochemical and Biophysical Research Communications 164, 11705.[ISI][Medline]
12 . Guthrie, C. & Fink, G. R. (1991). Guide to Yeast Genetics and Molecular Biology. Academic Press, San Diego, CA.
13 . Burns, N. B., Grimwade, P. B., Ross-Macdonald, P. B., Choi, E. Y., Finberg, K., Roeder, G. S. et al. (1994). Large-scale analysis of gene expression, protein localization, and gene disruption in Saccharomyces cerevisiae. Genes and Development 8, 1087105.[Abstract]
14
.
Kontoyiannis, D. P. (1999). Genetic analysis of azole resistance by transposon mutagenesis in Saccharomyces cerevisiae. Antimicrobial Agents and Chemotherapy 43, 27315.
15
.
Kontoyiannis, D. P. (2000). Efflux-mediated resistance to fluconazole could be modulated by sterol homeostasis in Saccharomyces cerevisiae. Journal of Antimicrobial Chemotherapy 46, 199203.
16
.
Lawson, J. E., Gawaz, M., Klingenberg, M. & Douglas, M. G. (1990). Structurefunction studies of adenine nucleotide transport in mitochondria. I. Construction and genetic analysis of yeast mutants encoding the ADP/ATP carrier protein of mitochondria. Journal of Biological Chemistry 265, 14195201.
17 . Stryer, L. (1995). Biochemistry, 4th edn, pp. 52958. W. H. Freeman, New York.
18 . Parks, L. W. & Casey, W. M. (1995). Physiological implications of sterol biosynthesis in yeast. Annual Review of Microbiology 49, 95116.[ISI][Medline]
19 . Osumi, T., Nishino, T. & Katsuki, H. (1979). Studies on the delta-5 desaturation in ergosterol biosynthesis in yeast. Journal of Biochemistry 85, 81926.[Abstract]
20 . Smith, S. J. & Parks, L. W. (1993). The ERG3 gene in Saccharomyces cerevisiae is required for the utilization of respiratory substrates and in heme-deficient cells. Yeast 9, 117787.[ISI][Medline]
21 . Wiseman, A. & King, D. J. (1982). In Topics in Enzyme and Fermentation Biotechnology, (Wiseman, A., Ed.), pp. 151206. Ellis Horwood, Chichester.
22
.
Kelly, S. L., Lamb, D. C., Kelly, D. E., Manning, N. J., Loeffler, J., Hebart, H. et al. (1997). Resistance to fluconazole and cross-resistance to amphotericin B in Candida albicans from AIDS patients caused by defective sterol 5,6-desaturation. FEBS Letters 400, 802.[ISI][Medline]
23 . Portillo, F. & Gancedo, C. (1984). Mode of action of miconazole in yeast: inhibition of the mitochondrial ATPase. European Journal of Biochemistry 143, 2736.[Abstract]
24 . Bammert, G. F. & Fostel, J. M. (1999). Gene expression patterns in Saccharomyces cerevisiae: comparing response to drug treatment with mutations in ergosterol biosynthetic genes. In Program and Abstracts of the Thirty-Ninth Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco, CA, 1999. Abstract 36J, p. 448. American Society for Microbiology, Washington, DC.
25 . Astin, A. M. & Haslam, J. M. (1977). The effects of altered membrane sterol composition on oxidative phosphorylation in a haem mutant of Saccharomyces cerevisiae. Biochemical Journal 166, 28798.[ISI][Medline]
26 . Defontaine, A., Bouchara, J.-P., Declerk, P., Planchenault, C., Chabasse, D. & Hallett, J.-N. (1999). In-vitro resistance to azoles associated with mitochondrial DNA deficiency in Candida glabrata. Journal of Medical Microbiology 48, 66370.[Abstract]
Received 19 July 1999; returned 29 November 1999; revised 7 January 2000; accepted 23 March 2000