5-Fluorocytosine antagonizes the action of sterol biosynthesis inhibitors in Candida glabrata

Hong Siau* and David Kerridge

Department of Biochemistry, University of Cambridge, Cambridge, UK


    Abstract
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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 
The concentration-dependent antagonistic interaction between 5-fluorocytosine and a sterol biosynthesis inhibitor (SBI) was studied using intact cells and cell-free extracts of Candida glabrata. 5-Fluorocytosine promoted incorporation of radioactivity into 4-desmethylsterols (P < 0.01), and enhanced the relative and absolute increases of ergosterol (P< 0.05) in C. glabrata incubated aerobically with an SBI (miconazole or amorolfine). Further aerobic incubation of C. glabrata with combinations of a nucleic acid or protein synthesis inhibitor (rifampicin or chlortetracycline) and an SBI (miconazole) promoted a similar increase in ergosterol biosynthesis. In contrast, 5-fluorocytosine reduced the incorporation of radioactivity into 4,4-dimethylsterols (P < 0.01), but had no obvious effect on the absolute ergosterol level in C. glabrata incubated statically with miconazole. In cell-free extracts of cultures previously incubated with 5-fluorocytosine, ergosterol synthesis was less sensitive to the action of miconazole. Antagonism between 5-fluorocytosine and the SBI is thus mediated by a reversal of inhibition of intracellular ergosterol synthesis. The possible mechanisms underlying antagonism between 5-fluorocytosine and SBIs that inhibit different sites of the sterol biosynthesis pathway, as well as its clinical relevance to combination therapy, are discussed.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
An antagonistic effect of antifungal drugs in combination on the growth of Candida glabrata has been reported recently. 1 This concentration-dependent antagonism was demonstrated using 5-fluorocytosine and a sterol biosynthesis inhibitor (SBI), including terbinafine (an allylamine), amorolfine (a morpholine), miconazole, keto-conazole, clotrimazole, fluconazole or itraconazole (azole derivatives). The growth of C. glabrata incubated with both drugs was twice that of C. glabrata incubated with the SBI alone, in cultures under aerobic or static incubation. Antagonism was observed when rifampicin (a prokaryotic transcription inhibitor) or chlortetracycline (a prokaryotic translation inhibitor) was used in combination with miconazole.

The mode of action of 5-fluorocytosine and SBIs has been studied by several research groups. 5-Fluorocytosine is converted into metabolites of the pyrimidine salvage pathway, which interrupt transcription or translation, 2 while the SBI inhibits the activity of specific enzymes catalysing sterol biosynthesis. 3,4,5 We have shown that 5-fluorocytosine reduces the growth-inhibitory effects of SBIs, 1 and now present the biochemical basis of the underlying antagonistic interactions, as well as a working hypothesis on how conventional nucleic acid or protein synthesis inhibitors (5-fluorocytosine, rifampicin or tetracycline) may interfere with the action of SBIs (terbinafine and miconazole).

The initial experiments were conducted using intact C. glabrata cells. Since miconazole causes an accumulation of lanosterol and a reduction of ergosterol, with a corresponding increased and decreased radioactive labelling of the 4,4-dimethylsterols and 4-desmethylsterols, respectively, 3 the effects of 5-fluorocytosine and miconazole, alone and in combination, on quantitative changes in the incorporation of radioactivity into sterol fractions of cells in aerobic and static incubation, were examined. As synthesis of ergosterol from squalene may proceed via different reaction steps even within the same organism, 6 the identities of individual sterols and their relative abundance were determined next. The resulting data were compared between cells incubated with combinations of nucleic acid or protein synthesis inhibitor (5-fluorocytosine, rifampicin or chlortetracycline) and an SBI (miconazole or amorolfine), and those incubated with the SBI alone. This was followed by measurement of the absolute ergosterol content of C. glabrata grown in the presence or absence of the drug(s).

Subsequent work was carried out using cell-free extracts of C. glabrata. An active cell-free system capable of sterol synthesis allows direct study of the inhibitory effects of SBIs on ergosterol formation, as inhibition is unaffected by drug penetration or efflux, as well as growth medium-dependent variations. 7 Hence the ability to synthesize ergosterol in the presence of miconazole was compared between cell-free extracts prepared from cultures incubated with 5-fluorocytosine, and those from cultures grown in drug-free medium. Finally the specific activity of 3ß-hydroxy-3ß-methylglutaryl coenzyme A (HMG-CoA) reductase, in cells incubated with or without 5-fluorocytosine, was determined. HMG-CoA reductase is a primary regulatory site of the sterol biosynthesis pathway. If the reaction catalysed by this enzyme is a rate-limiting step in the synthesis of ergosterol in C. glabrata, variation in its specific activity might explain differences in the degree of inhibition of sterol biosynthesis between cells incubated with 5-fluorocytosine and an SBI, and those incubated with the SBI alone.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Organism and culture methods

Candida glabrata strain 4 is a clinical isolate described previously. 8 The yeast was inoculated in 100 mL Yeast Nitrogen Base (Difco Laboratories, Detroit, MI, USA) supplemented with 1% (w/v) glucose (YNBG, pH 4.9) for aerobic incubation (200 rpm) in an orbital incubator (Gallenkamp, Loughborough, UK) at 37°C. Exponentially growing C. glabrata at 2 x 106 cfu/mL were suspended in 3 mL volumes in test-tubes (150 x 15 mm). Antimicrobial agents and reagents were used as described previously. 1 5-Fluorocytosine (0.025 mg/L; 0.2 µM) and/or miconazole (1.25 mg/L; 3 µM) were added as required. The tubes were incubated aerobically (800 rpm) in a Vxr Ika-Vibrax shaker (Janke & Kunkel GmbH & Co., Staufen, Germany) at 37°C for 18 h. In a separate experiment, 5-fluorocytosine (0.025 mg/L; 0.2 µM) and/or miconazole (15 mg/L; 31 µM) were added to exponentially growing C. glabrata; this set of tubes was incubated at 37°C, without shaking, for 18 h.

For studies on the incorporation of radioactivity into nonsaponifiable lipids, a total of 0.03 Ci of [2-14C]sodium acetate (specific activity, 55.6 mCi/mmol) (Amersham Life Science Ltd., Little Chalfont, UK) was added to each tube.

For studies on the effect of drugs in combination on sterol composition by gas- liquid chromatography (GLC), and measurement of ergosterol content by UV spectroscopy, C. glabrata was also incubated aerobically with other combinations of drugs, such as 5-fluorocytosine (0.0125 mg/L; 0.1 µM) and amorolfine (5 mg/L; 15 µM); rifampicin (10 mg/L; 12 µM) and miconazole (0.46 mg/L; 1 µM); chlortetracycline (10 mg/L; 21 µM) and miconazole (0.23 mg/L; 0.5 µM); or with the respective individual drug alone. At the specified concentration the drugs exhibited an antagonistic effect on the growth of C. glabrata, where cultures incubated with combination of drugs showed a greater growth yield (as measured by absorbance at 600 nm) than that of cultures incubated with the SBI alone.

For cultures required in the preparation of cell-free extracts, exponentially growing C. glabrata was inoculated into 150 mL of YNBG (2 x 106 cfu/mL) with or without 5-fluorocytosine (0.025 mg/L; 0.2 µM) for aerobic incubation at 37°C for 18 h. The stationary phase culture was added to 500 mL of pre-warmed fresh YNBG, either with or without 5-fluorocytosine, and re-incubated as before. When cells of such `released' cultures began growing exponentially, they were harvested.

Extraction of nonsaponifiable lipids

Extraction was carried out at room temperature, unless stated otherwise. After incubation the absorbance of cultures at 600 nm was measured and the cultures were pooled. The yeasts (5 x 108 cfu per sample) were harvested by centrifugation at 1000g for 5 min using a bench-top centrifuge (MSE Scientific Instruments, Crawley, UK), and washed twice with deionized water. Equal volumes (1.5 mL) of water and ethanolic KOH (15% (w/v) KOH in 90% (v/v) ethanol) were added to each sample for saponification at 80°C for 1 h in the dark. The samples were cooled in ice, and the nonsaponifiable lipids were extracted three times with 2 mL of petroleum ether (boiling point 40-60°C) (Sigma Chemical Co., Poole, UK). The nonsaponifiable lipids were dried under a stream of nitrogen before being separated by thin-layer chromatography (TLC).

Thin-layer chromatography

Each nonsaponifiable lipid extract was redissolved in 50 µL of petroleum ether (boiling point 40-60°C), and applied on to aluminium-backed silica gel 60 F 254 TLC plates (0.2 mm thickness; Merck, Poole, UK). The sterol fractions were separated in a solvent system of n-heptane:diisopropyl ether:acetic acid (60:40:4, by volume). The TLC plate was further aired at room temperature, and placed in contact with a X-ray film in an autoradiographic cassette. Autoradiograms were left at -80°C for 5 days before being developed. The position of the 4-desmethylsterol fraction was located by visualization under UV light. Radioactive fractions were removed from the TLC plate, and the radioactivity of each fraction was measured using an LS 3801 liquid scintillation counter (Beckman Instruments, High Wycombe, UK).

The radioactive fractions were also identified by reference to previous studies using the same solvent system. 3,9 The relative incorporation of radioactivity into each fraction of the nonsaponifiable lipid extract was expressed as a percentage of the total radioactivity in all three sterol fractions.

Gas- liquid chromatography

The cultures were harvested, washed twice with deionized water and pooled to give 1 x 10 9 cfu per sample. Nonsaponifiable lipids were extracted as described above. The free sterols were acetylated by addition of 0.8 mL of acetic anhydride:pyrimidine mixture (1:1, v/v) to each sample, with incubation at 50°C for 2 h. Samples were washed in 2 mL of deionized water and the sterol acetates were extracted with 2 mL of petroleum ether (boiling point 40-60°C). The process of washing and re-extraction was repeated three times before the extracts were dried under a stream of nitrogen.

The derivatized (sterol acetate) samples were separated using a Durabond Megabore 1 fused silica capillary column (30 m in length, 1.5 µm film thickness) (Jones Chromatography, Hengoed, UK). Each sample was dissolved in 10 µL acetone, and 0.5 µL was loaded on to the column operating isothermally at 260°C in a Pye Series 104 Chromatograph equipped with a flame ionization detector. The flow rate of the carrier gas, helium, was 15 mL/min. Acetate derivatives of commercially available cholesterol, ergosterol and lanosterol (Sigma Chemical Co.) were prepared in acetone at 1 mg/mL and used as standards. The sterols were identified according to their retention time relative to cholesterol and ergosterol, and by reference to published data. 10,11 The relative abundance of a sterol, i.e. the proportion of the sterol to total sterols, was expressed as a percentage of its peak area over total area of the GLC trace.

UV spectroscopy

After saponification (see above) sterols were extracted in 3 mL of n-heptane (Merck). The UV absorption spectrum (330-240 nm) of each extract was determined using a Cary 118 spectrophotometer (Varian Instrument Division, Walton-on-Thames, UK). The concentration of ergosterol was determined by measurement of absorbance at 282 nm and in comparison with commercially available ergosterol (Sigma Chemical Co.) prepared in n-heptane at 20 g/L. The dry weight of C. glabrata in each sample was determined by filtering a known volume of the culture though the Millipore filtration unit where organisms were collected on pre-weighed dried 2.5 cm Whatman glass microfibre discs (Whatman International Ltd., Maidstone, UK). The discs containing C. glabrata were dried at 120°C for 24 h, and reweighed using a Mettler H54 balance (Gallenkamp). Ergosterol content of C. glabrata was expressed as µg ergosterol/mg dry weight.

Sterol synthesis in cell-free extracts

The experimental procedure was as described by Barrett-Bee et al. 12 When the `released' cultures reached exponential growth phase cells were harvested by centrifugation at 600g at 4°C for 20 min using an RC-5B refrigerated superspeed centrifuge (Sorvall Ltd., Stevenage, UK), and washed twice with ice-cold deionized water. The cells were resuspended in 100 mM potassium phosphate buffer (pH 7.4) containing 30 mM nicotinamide, 5 mM MgCl 2 and 5 mM N-acetyl cysteine at 2 x 109 cfu/mL. The cellular lysate was obtained by shaking the suspension with pre-cooled glass beads (0.45 mm diameter) in a Cell Homogeniser model MSK (B Braun, Melsungen, Germany) operating at low speed for 3 x 1 min with intermittent cooling. Regular checks under a light microscope revealed >=90% breakage of cells. The broken cell preparation was centrifuged at 8000g at 4°C for 20 min. The supernatant fluid was retained. Five microlitres of miconazole solution was added to 940 µL of supernatant fluid, followed by 60 µL of the assay cocktail. The final concentration range of miconazole tested was 0.39-50 ng/mL. The degree of ergosterol synthesis inhibition at each drug concentration was determined in triplicate. The assay cocktail was prepared in 100 mM potassium phosphate buffer (pH 7.4) and contained 1 µmol NADP+, 1 µmol NAD+, 3 µmol reduced glutathione, 3 µmol glucose 6-phosphate, 5 µmol ATP, 2 µmol MnCl 2, 3 µmol MgCl2, 0.7 IU glucose 6-dehydrogenase and 0.25 µCi [2-14C]mevalonic acid (MVA) DBED salt (specific activity, 54 mCi/mL) (Amersham) in 60 µL. Sterol synthesis was allowed to proceed aerobically at 37°C for 2 h. The reaction was stopped by saponification. The radioactive fractions were identified by comparison with published data.3 The protein content of each sample was determined as described below. The incorporation of radioactivity from [2-14C]MVA into each fraction was expressed as a percentage of the total radioactivity detected in all fractions. The IC50 of an SBI is the inhibitory concentration of the SBI required to reduce the relative incorporation of radioactivity into 4-desmethylsterols to half its relative incorporation into the same fraction in the absence of the SBI.

Specific activity of 3ß-hydroxy-3ß-methylglutaryl coenzyme A reductase

` Released' cultures at exponential growth phase were harvested and washed as above, and suspended in 100 mM potassium phosphate buffer (pH 7.0) containing 1 mM EDTA and 1 mM dithiothreitol at 2 x 109 cfu/mL. Breakage of cells was carried out as described above. The specific activity of HMG-CoA reductase (mevalonate:NADP+ oxidoreductase; acetylating coenzyme A; EC 1.1.1.34) of C. glabrata was determined following the method of Quain & Haslam. 13 Triton X-100 (0.5%, v/v) was added to the broken cell preparation and kept in ice for 1 h before centrifugation at 8000g at 4°C for 10 min. An aliquot of the supernatant fluid (200 µL) was added to 700 µL of 100 mM potassium phosphate buffer (pH 7.0) containing 5 µmol of dithiothreitol and 5 µL of Triton X-100, followed by 50 µL of 200 mM sodium carbonate buffer (pH 10.6) with 0.15 µmol of NADPH. After 10 min incubation at 30°C to stabilize the endogenous oxidation of NADPH, 50 µL of 200 mM acetate buffer (pH 4.6) containing 0.15 µmol of HMG-CoA was added to 900 µL of the mixture. The initial change in absorbance at 340 nm due to oxidation of NADPH was recorded. Each sample was assayed in duplicate. The protein content of each sample was determined as described below. The specific activity of HMG-CoA reductase is expressed as nmol NADPH oxidized/mg protein/min.

Protein content

The protein content of each sample was determined by the method of Bradford. 14 The reagent consisted of 42.5 g of orthophosphoric acid and 50 mg of Coomassie Brilliant Blue G250 (Raymond A. Lamb, London, UK) dissolved in 25 mL ethanol and made up to 500 mL with deionized water. Five millilitres of the reagent was added to samples appropriately diluted in 0.15 M NaCl. The samples were left to stand at room temperature for 30 min and the absorbance of each sample at 595 nm was measured. Bovine serum albumin (Sigma Chemical Co.) dissolved in 0.15 M NaCl was used as the standard.

Statistical analysis

The statistical significance of the difference between data of C. glabrata incubated with a nucleic acid or protein synthesis inhibitor (5-fluorocytosine, rifampicin or chlortetracycline) and an SBI (miconazole or amorolfine), and those of the yeast incubated with the SBI alone, was assessed using Student's t-test. A P value of <0.05 was considered significant.


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Sterol biosynthesis in intact cells

In C. glabrata incubated aerobically or statically with 5- fluorocytosine or drug-free media, approximately 90% of the total radioactivity was present in the 4-desmethysterols(Table I). When C. glabrata was incubated aerobically with miconazole, radioactivity in the 4-desmethylsterols was reduced to 30% of the total radioactivity detected, and radioactivity in the 4,4-dimethylsterols and 4-methylsterols increased to 54% and 17%, respectively. When C. glabrata was incubated aerobically with both 5-fluorocytosine and miconazole, 46% of total radioactivity was detected in 4-desmethylsterols, which was significantly higher than that of cultures incubated with miconazole alone (P< 0.01). In addition there was a significant reduction in the proportion of radioactivity in the 4,4-dimethylsterols (39%) (P < 0.01).


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Table I. Effect of 5-fluorocytosine and miconazole on the incorporation of radioactivity from [2-14C]acetate into sterol fractions of C. glabrata in aerobic and static incubation
 
In C. glabrata incubated statically with 5-fluorocytosine and miconazole, the proportion of radioactivity in the 4,4-dimethylsterols (50%) was also significantly lower than that of cultures incubated with miconazole (58%) alone (P< 0.01) (Table I). However, the relative incorporation of radioactivity into 4-desmethylsterols in cultures incubated with both drugs, and those with SBI alone, were similar.

Sterol composition of intact cells

Ergosterol was the major sterol in C. glabrata grown aerobically in drug-free medium, as well as in the presence of 5-fluorocytosine (0.0125 or 0.025 mg/L), rifampicin (10 mg/L) or chlortetracycline (10 mg/L) (Table II). The relative abundance of ergosterol in C. glabrata incubated aerobically with the various antagonistic combinations of drugs was significantly higher (P < 0.05) than that of cultures incubated with the SBI alone.


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Table II. Effect of drug(s) on the sterol composition of C. glabrata in aerobic incubation
 
Ergosterol precursors accumulated in aerobic cultures incubated with an SBI. There was a substantial proportion of lanosterol (54%) or ignosterol (49%) in C. glabrata incubated with miconazole (1.25 mg/L) or amorolfine, respectively (Table II). Zymosterol was also present in C. glabrata incubated with amorolfine, at 15% of the total sterols. In the presence of a second drug, the proportion of lanosterol in cultures incubated with 5-fluorocytosine and miconazole (38%) was significantly lower (P< 0.01) than that of cultures incubated with miconazole alone. There was also a significant reduction (P< 0.01) in the proportion of zymosterol in cultures incubated with 5-fluorocytosine and amorolfine (7%), although the proportions of ignosterol in cultures containing both drugs, or amorolfine alone, were similar.

Ergosterol content of intact cells

The ergosterol content of C. glabrata incubated aerobically with 5-fluorocytosine (0.0125 and 0.025 mg/L), rifampicin (10 mg/L) or chlortetracycline (10 mg/L) was similar to that of cultures incubated in drug-free medium (Table III). The mean ergosterol content of C. glabrata incubated aerobically with various antagonistic combinations of drugs was significantly higher (P< 0.05) than those incubated with the SBI alone. The ergosterol content of C. glabrata incubated statically in drug-free medium (3.0 mg/g dry weight) was approximately half the ergosterol content of cultures incubated aerobically (Table III) 5-Fluorocytosine did not affect the ergosterol level, as cells incubated statically with the fluoropyrimidine had similar ergosterol content to those in drug-free medium. The ergosterol content of C. glabrata incubated statically with 5-fluorocytosine and miconazole (1.2 mg/g dry weight) was similar to that of cultures incubated with miconazole alone. This result is consistent with the similar proportions of radioactivity incorporated in the 4-desmethylsterol fraction of C. glabrata incubated with miconazole, with or without 5-fluorocytosine. The absence of an increase in ergosterol content of C. glabrata in static cultures with both drugs relative to those with miconazole alone was in contrast to the results obtained from aerobic cultures.


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Table III. Effect of drug(s) on the ergosterol content of C. glabrata in aerobic and static incubation
 
Sterol synthesis in cell-free extracts

Sterol synthesis in cell-free extracts was studied using `released' cultures growing at exponential phase. The overnight stationary phase cultures previously incubated with or without drug(s) were `released' into fresh drug-free medium for further incubation until the exponential phase of growth was attained: C. glabrata incubated overnight in drug-free medium or with 5-fluorocytosine required 2-5 h of re- incubation in fresh medium,while those incubated overnight with both miconazole and 5-fluorocytosine, or miconazole alone, required at least 14 h of re-incubation (range 14-24 h). Hence the study of sterol synthesis in cell-free extracts was conducted using the former cultures only, where the effect of miconazole on the incorporation of radioactivity from [2-14C]MVA into sterol fractions of cell-free extracts prepared from drug-free cultures (i.e. drug-free controls), and those previously incubated with 5-fluorocytosine (i.e. 5-fluorocytosine-treated), were compared.

The IC 50 of miconazole on sterol synthesis in cell-free extracts of 5-fluorocytosine-treated (n = 2) and drug-free (n = 2) samples was 25 and 6.25 ng/mL, respectively. Protein contents were similar, with 6.3 g/L in the former and 6.1 g/L in the latter cell-free extracts. Thus ergosterol synthesis in cell-free extracts prepared from C. glabrata previously incubated with 5-fluorocytosine was less sensitive to the action of miconazole.

Specific activity of HMG-CoA reductase

The specific activities of HMG-CoA reductase obtained from cell-free extracts of 5-fluorocytosine-treated (n = 3) and drug-free (n = 3) samples were similar, with a respective mean ± S.D. of 6.2 ± 1.2 and 5.0 ± 1.0 nmol NADPH oxidized/mg protein/min. Thus 5-fluorocytosine (0.025 mg/L) did not affect the specific activity of the regulatory enzyme in C. glabrata.


    Discussion
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5-Fluorocytosine antagonizes the action of SBIs on sterol biosynthesis in C. glabrata. During aerobic incubation there were significant increases in the relative and absolute levels of the 4-desmethylsterol ergosterol, in cells incubated with 5-fluorocytosine and an SBI, compared with those of cells incubated with the SBI alone (Tables I-III). However, this was not observed during static incubation (Tables I and III), although under both aerobic and static incubation there was a significant decrease in the relative incorporation of radioactivity into 4,4-dimethylsterols, the ergosterol precursors (Table I) In addition the synthesis of ergosterol in cell-free extracts prepared from C. glabrata incubated previously with 5-fluorocytosine was less susceptible to the action of miconazole, with a higher IC 50 than those obtained from cells incubated in drug-free medium. Antagonism was also observed when rifampicin or chlortetracycline was tested in combination with miconazole (Tables II and III).

The increase in ergosterol formation in intact cells, as well as in cell-free extracts, showed that antagonism did not occur at the permeability barrier involving reduced drug accumulation or increased efflux mediated by multidrug transporters. 15,16 The sterol intermediates that accumulated in cells incubated in the presence of an SBI with or without the second drug were identical (Table II), and therefore there was no bypass of inhibited steps via another sterol biosynthetic pathway. The specific activity of HMG-CoA reductase remained the same whether cells were incubated with 5-fluorocytosine or not, ruling out the possibility of an increased rate of sterol biosynthesis due to increased catalysis by the main regulatory enzyme. Miconazole reduced ergosterol synthesis in cell-free extracts of C. glabrata previously incubated with 5-fluorocytosine, albeit at a higher concentration, which suggests that antagonism is not likely to result from a change in enzyme configuration. 17,18 When effects of 5-fluorocytosine (25 ng/mL) and miconazole (3- 50 ng/mL) on the incorporation of radioactivity into sterol fractions were studied using cell-free extracts prepared from drug-free controls, the distribution of radioactivity amongst the sterol fractions of cell-free extracts incubated with both drugs did not differ from those of extracts incubated with the SBI alone (data not shown). This indicated that 5-fluorocytosine is unlikely to bind allosterically to the target enzyme of miconazole, cytochrome P450-dependent lanosterol 14{alpha}-demethylase (cyt P450 14DM). It is also not likely that the lipophilic rifampicin or the hydrophilic chlortetracycline would interact with cyt P450 14DM in a similar fashion to promote the synthesis of ergosterol.

We propose that the antagonism may arise from an increased quantity of enzymes catalysing sterol biosynthesis, due to an increase in either transcription or translation. Our hypothesis is based on data obtained from Saccharomyces cerevisiae, in which the regulation and expression of genes encoding enzymes of the sterol biosynthesis pathway have been studied in detail. The derived amino acid sequence of C. glabrata cyt P45014DM is more homologous to that of S. cerevisiae, with an 84.5% identity, than to that of Candida albicans, with which it has a 65% identity. 19 In addition, the putative haem-binding domain H2 region of cyt P45014DM in C. glabrata and that of S. cerevisiae are 95% identical. Reduced susceptibility to SBIs due to quantitative increase of target enzymes has been reported previously. The decreased susceptibility of two strains of C. glabrata to allylamines and/or azole derivatives was partly due to an increased amount of cyt P45014DM ,20,21 and reduced susceptibility of S. cerevisiae to morpholines was due to overproduction of sterol {Delta}14-reductase.22

The antibiotics rifampicin and chlortetracycline have been suggested to interfere with the initiation of transcription and protein synthesis, respectively, in eukaryotes. 23,24 However, the concentrations of 5-fluorocytosine, rifampicin and chlortetracycline used (0.025, 10 and 10 mg/L, respectively) did not affect the growth of C. glabrata substantially. 1 Moreover, when these compounds were combined with an SBI, the growth yield (measured as absorbance at 600 nm) and ergosterol content of C. glabrata were higher than those of cultures incubated with the SBI alone (Table III).

Hence the effect of these nucleic acid or protein synthesis inhibitors used at such low concentrations on yeast is thus not known. Instead of functioning as nucleic acid or protein synthesis inhibitors, they may act as direct or indirect `stimulators' of ergosterol synthesis. Rifampicin is known to induce microsomal cytochrome P450. 25,26,27 Therefore we postulate that at low concentrations these conventional nucleic acid or protein synthesis inhibitors stimulate haem synthesis and lead to the cascade of events resulting in the induction of mRNA for the specific enzymes involved in the sterol biosynthesis pathway in C. glabrata under aerobic incubation (see Figure). Haem and squalene epoxidase syntheses are induced in the presence of oxygen. 28 An increase in haem facilitates the binding of the trans-acting factor haem-activating protein (HAP1) to the upstream activating sequence (UAS1) of the gene ERG11, which encodes cyt P450 14DM, 29 thereby inducing its transcription. Haem also activates the expression of the gene (HMG1) encoding HMG-CoA reductase. 30 Although genes encoding the target sites of amorolfine, sterol {Delta}8-{Delta}7 isomerase (ERG2) and sterol {Delta}14-reductase (ERG24), have been cloned, 31,32,33,34 insufficient information about their regulation and expression prevent their inclusion in the model.



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Figure. Proposed transcriptional regulation of enzymes involved in sterol biosythesis in C. glabrata incubated with antagonistic combinations of drugs. Drawn with reference to reports by M' Baya & Karst,28 Thorsness et al.30 and Turi & Loper. 35 5-Flucytosine or one of its metabolites, rifampicin or chlortetracycline, simulates the synthesis of haem in the presence of oxygen directly or indirectly. This in turn activates transcription of enzymes involved in the sterol biosynthesis pathway. Abbreviations: ERG1, gene encoding squalene epoxidase; HMG, gene encoding 3ß-hydroxy-3ß-methylglutaryl coenzyme A (HMG CoA) reductase; ROX1, regulatory protein involved in repression of genes expressed under anaerobic condition; HAP1, haem activating protein; ERG11, gene encoding cytochrome P450-dependent lanosterol 14{alpha}-demethylase (cyt P450 14DM); UAS, upstream activating sequence.

 
In cells under static incubation, the lower dissolved oxygen concentration may stimulate ERG11 mRNA level, via ROX1 on UAS2 of ERG11, 35 and thus increase the quantity of cyt P450 14DM catalysing the demethylation of lanosterol, a 4,4-dimethyl sterol. Hence there was a relative reduction of the 4,4-dimethyl ergosterol precursors (Table I). However, the enzyme may function at suboptimal level since it requires molecular oxygen and haem for optimal catalysis. Thus there was no significant difference in the absolute ergosterol content between cultures incubated statically with both drugs, and those incubated with the SBI alone (Table III). This is in line with the report by Stanfield et al. 36 who observed that a reduced oxygen level in cultures of S. cerevisiae prevents the oxidative steps of the late stage of sterol biosynthesis from proceeding at the rates possible in cultures incubated aerobically, despite an increase in the mRNA for cyt P45014DM. On the other hand, the factors that contributed to a relatively higher growth yield in static cultures are not apparent. We suspect that the significant decrease in the accumulation of 4,4-dimethylsterols in C. glabrata incubated with 5-fluorocytosine and miconazole, compared with that of cells incubated with miconazole alone (Table I), is likely to contribute to a reduction in membrane fluidity and therefore the higher growth yield.

There may be more than one mechanism by which 5- fluorocytosine, rifampicin and chlortetracycline reduce the inhibitory effect of an SBI. An increase in enzymes could also arise from a greater stability of the transcribed mRNA, and modifications at post-transcriptional or post-translational level.

Further experiments to measure the cyt P450 14DM content, determine the affinity of microsomal P450s for SBIs and quantify specific mRNA in C. glabrata incubated with 5-fluorocytosine and SBIs, would facilitate our understanding of the molecular basis of the antagonism.

The main aim of this communication is to present the biochemical evidence of potential mechanisms behind the in-vitro antagonism between agents that inhibit nucleic acid or protein synthesis, and those that block sterol biosynthesis, using C. glabrata strain 4 as the test organism. Aspects pertaining to the rationale of the experimental design, and the clinical significance of this work, should now be addressed. Firstly, would antagonism be observed using other species and genera of yeast? Preliminary work suggests that the antagonistic effect was strain-specific, occurring in C. glabrata strain 4, S. cerevisiae strain 237 and C. albicans strain 72R, but not with C. albicans strain 6406 or C. parapsilosis strain 3104. 1 Secondly, would antagonism be demonstrated if 5-fluorocytosine were combined with triazole derivatives, such as fluconazole and itraconazole, which are used for treatment of systemic mycosis, instead of the imidazole derivative miconazole, a topical agent for superficial mycosis? The antagonistic effect on C. glabrata has been observed when 5-fluorocytosine was used in combination with fluconazole or itraconazole. 1 We chose miconazole as the representative SBI in the present work, as C. glabrata strain 4 was most susceptible to this azole derivative. 1 Thirdly, would antagonism be exhibited whenever 5-fluorocytosine was used in combination with an SBI? Antagonism has been observed in a concentration-dependent manner. 1 Different miconazole concentrations (0.23- 1.25 mg/L) were required when the azole derivative was combined with 5-fluorocytosine, rifampicin or chlortetracycline (Table II). The drug concentrations were determined empirically. Each pair of drugs was combined in a range of concentrations in a chequerboard fashion, to determine the optimal drug concentration and/or ratio that gives rise to a relative increase in the growth of C. glabrata. Consequently the concentration of 5-fluorocytosine chosen in this manner was well below levels considered to be therapeutic. Lastly, would the in vitro antagonism be observed in vivo? If the critical parameters that influence experimental outcome in vitro could be maintained in vivoover a sufficient period of time (e.g. exponentially budding yeasts in the presence of good aeration and antifungal drugs at a constant drug concentration and/or ratio), it is possible to observe antagonism. Further work is necessary to ascertain if usage of 5-fluorocytosine in combination with SBIs is contraindicated in vivo.


    Acknowledgments
 
We thank Professor K. Y. Yuen for critical reading of the manuscript. This work was presented in part at the Eleventh Congress of the International Society for Human and Animal Mycology, Montreal, Canada, June 1991 (Abstract PS3.41).


    Notes
 
* Correspondence address. Department of Microbilogy, Univesity of Hong Kong, University Pathology Building, Queen Mary Hospital Compound, Pokfalum Road, Hong Kong. Tel: +852-2855-4891; Fax: +852-2855-1241; E-mail: hongsiau{at}hkusua.hku.hk Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Received 5 August 1998; returned 22 December 1998; revised 12 January 1999; accepted 29 January 1999