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Antimicrobial Agents and Chemotherapy, June 2001, p. 1660-1670, Vol. 45, No. 6
Department of Advanced Bio-Technologies,
Janssen Research Foundation, B-2340 Beerse,1 and
Steenweg op Gierle 68, B-2300 Turnhout,3
Belgium, and Department of Bio-informatics, R. W. Johnson Pharmaceutical Research Institute, San Diego, California
921212
Received 22 January 2001/Returned for modification 15 February
2001/Accepted 2 March 2001
The application of genome-wide expression profiling to determine
how drugs achieve their therapeutic effect has provided the pharmaceutical industry with an exciting new tool for drug
mode-of-action studies. We used DNA chip technology to study cellular
responses to perturbations of ergosterol biosynthesis caused by the
broad-spectrum antifungal agent itraconazole. Simultaneous examination
of over 6,600 Candida albicans gene transcript levels,
representing the entire genome, upon treatment of cells with 10 µM
itraconazole revealed that 296 genes were responsive. For 116 genes
transcript levels were decreased at least 2.5-fold, while for 180 transcript levels were similarly increased. A global upregulation of
ERG genes in response to azole treatment was observed.
ERG11 and ERG5 were found to be upregulated
approximately 12-fold. In addition, a significant upregulation was
observed for ERG6, ERG1, ERG3, ERG4, ERG10, ERG9, ERG26, ERG25,
ERG2, IDII, HMGS, NCP1, and FEN2, all of which are
genes known to be involved in ergosterol biosynthesis. The effects of
itraconazole on a wide variety of known metabolic processes are
discussed. As over 140 proteins with unknown function were responsive
to itraconazole, our analysis might provide The N-substituted triazole
itraconazole is a broad-spectrum antifungal agent currently available
in the form of oral capsules and an oral solution for the treatment
and/or prophylaxis of aspergillosis, blastomycosis, disseminated and
superficial Candida infections, cryptococcosis,
dermatophytosis, histoplasmosis, paracoccidioidomycosis, sporotrichosis, and some forms of coccidioidomycosis (8,
20).
The antifungal activities of itraconazole and related azole derivatives
arise from a complex multimechanistic process initiated by the
inhibition of two cytochromes P450 involved in the biosynthesis of
ergosterol, namely, the P450 that catalyzes the 14 Ergosterol is an essential component of fungal plasma membranes; it
affects membrane permeability and the activities of membrane-bound enzymes. This sterol is a major component of secretory vesicles and has
an important role in mitochondrial respiration and oxidative phosphorylation (for reviews, see references 3, 27, and
28). It can thus be expected that changes in ergosterol levels
and sterol structure influence the activities of several metabolic pathways.
To examine this further, the effect of itraconazole on the pathogen was
studied by analyzing the level of gene expression from essentially
every gene in the Candida albicans genome prior to and after
treatment of C. albicans with this compound. Transcript profiling with DNA microarrays provides a rapid and systematic method
for the high-throughput analysis of gene expression at the level of the
whole genome (2). The experiments reported here use the
C. albicans Gene Expression Microarray (GEM; Incyte Genomics
Inc., Palo Alto, Calif.), which contains over 6,600 DNA fragments. From
a drug discovery point of view, the expression patterns generated from
the in-parallel analysis of all genes in a microorganism can give clues
to the functions of previously uncharacterized genes (target
identification), as well as provide information about how drugs achieve
their therapeutic effect (mechanism-of-action studies). In addition to
providing a much more complete picture of which genes and pathways are
involved, gene functions other than those currently known might be
discovered (22).
Strains and media.
C. albicans CAI-4
(ura3::imm434/ura3::imm434)
was kindly provided by William Fonzi, Georgetown University
(7). SGM medium contains 0.67% (wt/vol) yeast nitrogen
base without amino acids (Difco), 0.2% (wt/vol) Ura dropout powder
(Bufferad, Inc.), 2% (wt/vol) galactose, and 2% (wt/vol) maltose.
Uridine was added at a final concentration of 20 µg/ml to ensure
growth of CAI-4. Itraconazole (Janssen Pharmaceutica, Beerse, Belgium)
dissolved in dimethyl sulfoxide was added to a final concentration of
10 µM.
Total RNA extraction.
CAI-4 cells were grown overnight at
30°C and 250 rpm to an optical density at 600 nm (OD600)
of ~0.2, the culture was divided in two, and itraconazole was added
to one of the cultures. Cells were grown for an additional 24 h
and harvested (at an OD600 of ~1.25 for the nontreated
cells and an OD600 of ~0.8 for the treated cells). Total
RNA was prepared with the RNeasy midi kit (enzymatic lysis protocol;
Qiagen) according to the manufacturer's instructions. All RNA samples
were treated with DNase I (RNase-free; Boehringer) on RNeasy midi
columns for 15 min according to the manufacturer's instructions. RNA
was precipitated (sodium acetate-ethanol) and the pellet was dissolved
in 20 µl of diethyl pyrocarbonate-treated MilliQ water (Millipore).
RNA concentrations were determined spectrophotometrically by measuring
the absorption at 260 nm in a UV-1601 UV-visible spectrophotometer (Shimadzu).
Microarray preparation.
DNA fragments for microarray
fabrication were generated by PCR
(http://www.incytegenomics.com/gem/technology/index.shtml). PCR
products were purified by gel filtration with Sephacryl-400 (Amersham
Pharmacia Biotech, Inc., Piscataway, N.J.) equilibrated in 0.2× SSC
(1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate). The filtrate was
dried and rehydrated in 1/10 volume distilled H2O
(dH2O) for arraying of the DNA solutions. The DNA solutions were arrayed by robotics on modified glass slides. After arraying of
the DNA solutions, the slides were processed to fix the DNA to the
prepared glass surface and washed three times in dH2O at ambient temperature. The slides were then treated with 0.2% I-Block (Tropix, Bedford, Mass.) dissolved in 1× Dulbecco's
phosphate-buffered saline (Life Technologies, Gaithersburg, Md.) at
60°C for 30 min. GEMs were rinsed in 0.2% sodium dodecyl sulfate for
2 min, followed by three 1-min washes in dH2O.
Fluorescent labeling of probe for Microarray hybridization.
Only one set of RNAs (treated and non-treated) was used for microarray
hybridization experiments. Isolated total RNAs were reverse transcribed
with 5' Cy3- or Cy5-labeled random 9-mers (Operon Technologies, Inc.,
Alameda, Calif.). The reaction mixtures were incubated for 2 h at
37°C with 1 µg of total RNA, 200 U of Moloney murine leukemia virus
reverse transcriptase (Life Technologies, Gaithersburg, Md.), 4 mM
dithiothreitol, 1 U RNase inhibitor (Ambion, Austin, Tex.), 0.5 mM
deoxynucleoside triphosphates, and 2 µg of labeled 9-mers in a
25-µl volume with enzyme buffer supplied by the manufacturer. The
reaction was terminated by incubation at 85°C for 5 min. The paired
reactions were combined, and the labeled nucleic acid was purified with
a TE-30 column. (Clontech, Palo Alto, Calif.). The eluate was brought
to 90 µl with dH2O and precipitated with 2 µl of 1 mg
of glycogen per ml-60 µl of 5 M ammonium acetate-300 µl of ethyl
alcohol. After centrifugation the supernatant was decanted and the
pellet was dissolved in 24 µl of hybridization buffer: 5× SSC, 0.2%
sodium dodecyl sulfate, and 1 mM dithiothreitol.
Microarray hybridization and scanning.
Before use, probe
solutions were incubated at 65°C for 5 min with mixing. The probe
solution was applied to the array, which was covered with a
22-mm2 glass coverslip and placed in a sealed humidified
chamber to prevent evaporation. After hybridization at 60°C for 6.5 h
the slides were washed in three consecutive washes of decreasing ionic strength.
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.6.1660-1670.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Genomic Profiling of the Response of Candida
albicans to Itraconazole Treatment Using a DNA
Microarray
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
in combination with
phenotypic datafirst hints of their potential function. The present
report is the first to describe the application of DNA chip technology
to study the response of a major human fungal pathogen to drug treatment.
INTRODUCTION
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Abstract
Introduction
Materials and Methods
Results and Discussion
References
-demethylation of
lanosterol or eburicol, encoded by ERG11 (CYP51),
and 
22-desaturase, encoded by ERG5
(CYP61) (12, 31, 32, 33). The
sterol-14-demethylase is the major fungal target for all azole
derivatives studied so far. Interaction with CYP51 results in a decreased availability of ergosterol and accumulation of 14-methylsterols and 3-ketosteroids (17, 27, 28, 29, 31, 34,
36).
MATERIALS AND METHODS
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Abstract
Introduction
Materials and Methods
Results and Discussion
References
Microarray data processing and analysis. GEMtools software (Incyte Genomics Inc.) was used for image analysis and data visualization. Each element area was determined with a gridding and detection algorithm. The area surrounding each element was used to calculate the local background value, which was subtracted from the total element signal value. Signals from which the background value was subtracted were used to determine differential expression (Cy3/Cy5 ratio) for each element. For these calculations GEM elements were selected according to the following acceptance criteria: (i) each element must have a minimum area of 40% of the average area calculated for all elements on the GEM, and (ii) the element's signal-to-background ratio must be at least 2.5. To be accepted an element must pass both criteria in at least one channel.
A correction factor was applied to account for systematic differences in the probe labels. The ratio of the average Cy3 and Cy5 signals from which background values were subtracted was used to balance the Cy5 signals. In the present study only elements with balanced differential expression (Cy3/balanced Cy5 ratio) higher than 2 were considered in further analyses. Elements for which the homogeneity of the spotted DNA was not confirmed by PCR analysis were excluded. DNA sequences were annotated on the basis of the results of BLAST (n and x) searches against PathoSeq (release, March 2000; Incyte Genomics Inc.) EMBL, SwissProt, and C. albicans genome (release, July 1999; Stanford University) databases. Information on gene functions was also retrieved from the Bioknowledge library (Proteome Inc., Beverly, Mass.). The correlation coefficient between two different hybridization experiments was determined essentially as described by Eisen et al. (6).| |
RESULTS AND DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
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Global gene expression monitoring in C. albicans upon
itraconazole treatment: experimental design.
The use of microarray
techniques in combination with the C. albicans genome
sequence now permits investigation of global changes in gene expression
associated with drug treatment. We examined the level of gene
expression from essentially every gene in the C. albicans
genome simultaneously using the GEM technology offered by Incyte
Genomics Inc. Over 6,600 unique open reading frames (ORFs) are
represented on a single glass slide. Total RNA was isolated from both
nontreated C. albicans CAI-4 cells and cells treated with 10 µM itraconazole as described above. DNA probes for hybridization were
prepared by reverse transcription with either Cy3- or Cy5-labeled
random nonamers. Experiments were performed in duplicate, and
fluorescent signals were detected by scanning. Normalization and ratio
determination were performed with GEMTools software. The correlation
coefficient between (normalized) fluorescent signals of untreated and
treated CAI-4 cells was found to be 0.93 (6) (Fig.
1). A total of 296 genes were identified
as responsive, including the majority of genes from the ergosterol
biosynthetic pathway: 116 genes showed a more than 2.5-fold decrease in
expression upon itraconazole treatment, while 180 showed a more than
2.5-fold increase (Table 1).
In-house
comparison with other microarray experimental data sets (obtained upon
treatment of C. albicans with other compounds or upon
comparison of the non-ergosterol-related genes of parental strain CAI-4
and genetic knockouts strains) revealed that the majority of these
responses were specific (data not shown). However, the moderate
upregulation of many genes involved in protein biosynthesis was found
to be a rather unspecific response. To facilitate downstream analysis,
differentially expressed genes were divided over known functional
classes, as shown in Fig. 2.
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Effect on genes in or linked to the ergosterol biosynthetic
pathway.
Transcript profiles revealed a global upregulation of
ERG genes in response to azole treatment. This is in
agreement with previous studies showing that this pathway is the target
of azoles and is responsive to modulations in ergosterol levels. A
similar observation has recently been reported for Saccharomyces
cerevisiae, in which nine ERG genes were found to be
responsive upon azole treatment (1). The number and
characteristics of the responsive Candida genes. are shown
in Table 1. We found intermediate (+3.9- to 5.1-fold) increases in
expression for ERG2, ERG9, ERG25 (which depends on
CYB5), ERG26, and CYB5 genes and very
strong (+9.2 to +50.3 fold) upregulation of the ERG11, ERG5,
ERG3, ERG4, and ERG6 genes (all of which act downstream
of ERG11) and of the ERG10, ID11, HMGS, and
ERG1 genes (all of which act upstream of ERG11) (Fig. 3). In addition, NCP1,
which encodes NADP-cytochrome P450 reductase and which is the electron
donor for squalene epoxidase (the product of the ERG1 gene),
lanosterol 14-demethylase (ERG11), and sterol C-22
desaturase (ERG5), was upregulated 6.7-fold. Previous studies with S. cerevisiae (1, 26) had shown
consistent and coordinated increases in the transcript levels of
ERG11 and NCP1. In addition, the FEN2
(for fenpropimorph resistance 2) gene was found to be upregulated about
fivefold. In S. cerevisiae the product of this gene may be
involved in sensing ergosterol levels in the plasma membrane to allow
adjustment of ergosterol biosynthesis to match the growth conditions
(15). Inhibition of sterol biosynthesis results in
increased levels of transcription of some of the genes linked to this
pathway, presumably as a result of feedback control (5).
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Effect on other genes. In addition to its role in the yeast plasma membrane, ergosterol is a major component in secretory vesicles and plays a role in mitochondrial respiration and oxygen sensing. Depletion of ergosterol and subsequent accumulation of 14-methylsterols can result in alterations of membrane functions, synthesis and activity of membrane-bound enzymes, and mitochondrial activities, as well as in an uncoordinated behavior of the yeast cell (21, 27, 28). Apart from enzymes involved in ergosterol and lipid biosynthesis, several other enzymes including ATPases, cytochrome c peroxidase, and chitin synthase(s) have been shown to change expression upon azole treatment (for reviews, see references 10 and 27).
Chitin is an important component of the primary septa in yeast. It has been proven that high ergosterol levels inhibit chitin synthases, whereas mutants of C. albicans with low ergosterol contents show increased levels of chitin synthesis (for a review, see reference 27). From these studies it could be inferred that ergosterol biosynthesis inhibitors should affect chitin synthesis. For example, an azole-induced increase in the chitin:total carbohydrate ratio was found in C. albicans grown for 24 h in the presence of itraconazole; the highest ratio was found at an itraconazole concentration of 49.6 nM (27). On the basis of these results, chitin synthases were expected to be upregulated when ergosterol levels drop. However, treatment with itraconazole did not significantly change expression of most known chitin synthases (fold changes were +2.3 for CHSI,
1 for CHS2, +1.7
for CHS3, +1.4 for CHS regulatory factor, +1.4
for SKTS, and 0 [not-change] for CHS5). The
increased chitin content may be at the origin of the enhanced
expression level (+3.3-fold) of the chitinase-encoding gene,
CHT3. A number of other genes involved in cell wall
maintenance were found to be significantly and reproducibly upregulated
(Table 1, cell wall maintenance).
Screening for high-copy-number suppressors of ketoconazole sensitivity
in S. cerevisiae (13) has shown that
overexpression of ERG11, cytochrome c oxidase,
ribosomal protein L27, or some specific genes of unknown function was
sufficient to overcome growth inhibition by ketoconazole. Upon
treatment of C. albicans with itraconazole, we found that an
overwhelming number of ribosomal proteins, including L27, were
moderately upregulated (+2.5- to +5.2-fold change; see Table 1, protein
synthesis). Cytochrome c oxidase was only mildly induced
(1.3-fold). The previously described effects on cytochrome c
oxidase activity were thought to be linked to inhibition of ergosterol
biosynthesis (27). It is not surprising that enzymes of
fungal mitochondrial membranes are affected: in contrast to animal
cells, large amounts of sterols (ergosterol) are found in the inner
membranes of fungal mitochondria (3).
Ergosterol plays an important role in lipid and fatty acid
biosynthesis. This is reflected in the responsiveness of 12 genes involved in lipid or fatty acid metabolism (Table 1, lipid, fatty acid
metabolism). One of the upregulated genes, AUR1, an inositol phosphoryl ceramide synthase, encodes an enzyme involved in fungal sphingolipid biosynthesis. Bammert and Fostel (1) have
suggested that there is an interaction between ergosterol and
sphingolipid biosynthetic pathways in yeast.
In C. albicans grown for 6 h in the presence of 0.1 µM miconazole, a shift from monounsaturated (oleic acid) to
diunsaturated (linoleic acid) fatty acids was observed, suggesting a
stimulation of the delta-9-desaturase, a product of the OLE1
gene (27). Compared with the contact time needed to
inhibit ergosterol synthesis, a longer time was needed to interfere
with fatty acid desaturation. The effects on delta-9-desaturase depend
on the concentration and contact time: in C. albicans grown
for 16 h in the presence of 10 nM miconazole (27) or
100 nM itraconazole (30), the shift from oleic acid (18:1)
to linoleic acid (18:2) was replaced by a shift from oleic to palmitic
acid (16:0), suggesting an interference with fatty acid elongation
and/or the fatty acid desaturase. We found that the level of
OLE1 expression was reduced 1.3-fold. Thus, changes in
delta-9-desaturase activity may again result from changes in membrane
fluidity as a result of azole-induced changes in the nature of the
membrane sterols.
FAA2 was upregulated 2.5-fold. The product of the
FAA2 gene, the acyl coenzyme A synthase (fatty acid
thiokinase [long-chain] acyl-activating enzyme), acts on fatty acids
from C-6 to C-20 and is involved in chain elongation.
It has previously been demonstrated that the level of induction of cell
stress (heat shock) genes is determined by the ratio of the saturated
fatty acid content to the unsaturated fatty acid content of fungal
membranes (18). Therefore, the observed changes in the
level of induction of cell stress genes (Table 1, cell stress) might
originate from azole-induced alterations of the ratio of the saturated
fatty acid content to the unsaturated fatty acid content.
Ergosterol concentrations are highest in the plasma membrane and in
secretory vesicles (3). Altered expression of genes whose
products are involved in vesicular transport is therefore to be
expected (Table 1, transport).
From our analysis it is clear that further studies should be carried
out with 4-aminobutyrate-2-oxoglutarate aminotransferase. In S. cerevisiae, this aminotransferase is the product of
UGA1. In itraconazole-treated C. albicans the
homologous gene is downregulated about 25.6-fold (Table 1, amino acid
metabolism), and its precise role in how itraconazole exerts its effect
is unknown.
Effect on drug transporter proteins. Failure to accumulate azole antifungals has been identified as a major cause of resistance posttreatment in several fungal isolates and species that are less sensitive to azole antifungal agents and other ergosterol biosynthesis inhibitors (16, 23, 35). Failure to accumulate antifungal agents can be the consequence of impaired drug influx or enhanced drug efflux. There is now a consensus that efflux pumps are a common cause of decreased intracellular content of ergosterol biosynthesis inhibitors (for a review, see reference 16). Two types of efflux pumps are known: membrane transport proteins belonging either to the major facilitator superfamily (MFS) or to the ATP-binding cassette (ABC) superfamily. Proteins of the MFS are energized by the proton motive force, and the ABC-type transporters utilize ATP as their source of energy. In C. albicans only the pumps encoded by CaMDR1, Flu1, CDR1, and CDR2 were shown to be involved in the mechanisms of azole efflux (16). Our observations do suggest that expression of some small-molecule transporters changes (among them members of the MFS and ABC families; Table 1); but we did not observe upregulation of CaMDR1, CDR1, or CDR2 after 24 h of treatment of C. albicans with itraconazole. One C. albicans gene with homology to S. cerevisiae YJR015w (whose protein has an unknown function) was upregulated fourfold upon itraconazole treatment. This gene does show significant similarity to known multidrug resistance proteins.
Effect on proteins of unknown function. Over 140 proteins of unknown function were responsive to treatment with itraconazole under the test conditions used. These data might provide the first hints of a possible function of those proteins in azole drug resistance and sensitivity. One ORF (Genbank accession no. AJ304854) encoding a hypothetical 25.3-kDa protein whose gene is in the TIM23-ARE2 intergenic region and which showed similarity to the S. cerevisiae YNR018w ORF was upregulated over 37-fold (the highest level of upregulation after that for ERG6). Two genes not showing homology to any gene in the sequence databases (i.e., putatively C. albicans-specific genes; Genbank accession no. AJ298306 and AJ298308, respectively) were upregulated 24- and 17-fold, respectively; another ORF (Genbank accession no. AJ298307) with 58% identity to C. albicans ERG25 was upregulated fourfold. These genes might thus be strongly linked to either azole or ergosterol function, and study of their putative roles in ergosterol biosynthesis is ongoing. Upregulation of another ORF (Genbank accession no. AJ298309) with 80% identity to C. maltosa CYP52 (N-alkane-inducible cytochrome P450) was found to be increased 2.8-fold. To the best of our knowledge, the CYP52 multigene family has so far been described only for Candida maltosa and Candida tropicalis, not for C. albicans.
Conclusion. Thousands of genes are being discovered for the first time by sequencing the genomes of various organisms, reminding us that many processes remain to be explored at the molecular level. DNA microarrays provide a natural vehicle for this exploration. The potential of DNA microarrays to elucidate mechanisms of action is supported by our findings showing that the majority of genes in the ergosterol biosynthetic pathway are responsive to itraconazole, a drug known to target this pathway. Inhibition of sterol biosynthesis results in increased levels of transcription of most of the genes in the pathway, presumably as a result of reduced feedback inhibition. In addition, we identified genes of unknown function which are strongly upregulated upon azole treatment of Candida, thus providing first hints of their putative function in this pathogen. As the C. albicans genome sequence is gradually becoming available in both private and public databases, genome-wide transcript profiling will soon be a versatile tool that can be used to provide an understanding of the modes of action of novel antifungal compounds.
However, as not all cellular processes are controlled at the level of gene expression (transcription), protein profiling will certainly prove to be a helpful complementary approach. In addition, one must bear in mind that genes which are expressed at a very low level might be missed by this approach, but as the technology improves, this might be remedied in the near future.| |
ACKNOWLEDGMENTS |
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We thank Ing. Ronald de Hoogt (Janssen Research Foundation) for technical support and Jackson Wan (R. W. Johnson Pharmaceutical Research Institute) for helpful advice.
This work was supported by IWT grant 960192 from the "Vlaams Instituut voor de Bevordering van het Wetenschappelijk-Technologisch Onderzoek in de Industrie" (Brussels, Belgium).
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FOOTNOTES |
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* Corresponding author. Present address: Department of Immunology (2517D), R. W. Johnson Pharmaceutical Research Institute, 3210 Merryfield Row, San Diego, CA 92121. Phone: (858) 784-3110. Fax: (858) 450-2081. E-mail: mdebacke{at}prius.jnj.com.
Present address: Arcturus, San Diego, CA 92121.
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REFERENCES |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
|
|---|
| 1. |
Bammert, G. F., and J. M. Fostel.
2000.
Genome-wide expression patterns in Saccharomyces cerevisiae: comparison of drug treatments and genetic alterations affecting biosynthesis of ergosterol.
Antimicrob. Agents Chemother.
44:1255-1263 |
| 2. | Brown, P. O., and D. Botstein. 1999. Exploring the new world of the genome with DNA microarrays. Nat. Genet. 21:33-7[CrossRef][Medline]. |
| 3. | Daum, G., N. D. Lees, M. Bard, and R. Dickson. 1998. Biochemistry, cell biology and molecular biology of lipids of Saccharomyces cerevisiae. Yeast 14:1471-1510[CrossRef][Medline]. |
| 4. |
Dimster-Denk, D.,
J. Rine,
J. Philips,
S. Scherer,
P. Cundiff,
K. DeBord,
D. Gilliland,
S. Hickman,
A. Jarvis,
L. Tong, and M. Ashby.
1999.
Comprehensive evaluation of isoprenoid biosynthesis regulation in Saccharomyces cerevisiae utilizing the Genome Reporter Matrix.
J. Lipid Res.
40:850-860 |
| 5. | Dixon, G., D. Scanlon, S. Cooper, and P. Broad. 1997. A reporter gene assay for fungal sterol biosynthesis inhibitors. J. Steroid Biochem. Mol. Biol. 62:2-3, 165-167. |
| 6. |
Eisen, M. B.,
P. T. Spellman,
P. O. Brown, and D. Botstein.
1998.
Cluster analysis and display of genome-wide expression patterns.
Proc. Natl. Acad. Sci. USA
95:14863-14868 |
| 7. | Fonzi, W. A., and M. Y. Irwin. 1993. Isogenic strain construction and gene mapping in Candida albicans. Genetics 134:717-728[Abstract]. |
| 8. |
Harousseau, J. L.,
A. W. Dekker,
A. Stamatoullas-Bastard,
A. Fassas,
W. Linkesch,
J. Gouveia,
R. De Bock,
M. Rovira,
W. F. Seifert,
H. Joosen,
M. Peeters, and K. De Beule.
2000.
Itraconazole oral solution for primary prophylaxis of fungal infections in patients with hematological malignancy and profound neutropenia: a randomized, double-blind, double-placebo, multicenter trial comparing itraconazole and amphotericin B.
Antimicrob. Agents Chemother.
44:1887-1893 |
| 9. |
Henry, K. W.,
J. T. Nickels, and T. D. Edlind.
2000.
Upregulation of ERG genes in Candida species by azoles and other sterol biosynthesis inhibitors.
Antimicrob. Agents Chemother.
44:2693-2700 |
| 10. |
Kelly, S. L.,
M. A. Quail,
J. Rowe, and D. E. Kelly.
1992.
Sterol 14-demethylase: target of the azole antifungal agents, p. 155-187.
In
P. B. Fernandes (ed.), New approaches for antifungal drugs. Birkhauser, Boston, Mass.
|
| 11. |
Kontoyiannis, D. P.,
N. Sagar, and K. D. Hirschi.
1999.
Overexpression of Ergl 1p by the regulatable GAL1 promoter confers fluconazole resistance in Saccharomyces cerevisiae.
Antimicrob. Agents Chemother.
43:2798-2800 |
| 12. |
Lamb, D. C.,
S. Maspahy,
D. E. Kelly,
N. J. Manning,
A. Geber,
J. E. Bennett, and S. L. Kelly.
1999.
Purification, reconstitution, and inhibition of cytochrome P-450 sterol, 22-desaturase from the pathogenic fungus Candida glabrata.
Antimicrob. Agents Chemother.
43:1725-1728 |
| 13. | Launhardt, H., A. Hinnen, and T. Munder. 1998. Drug-induced phenotypes provide a tool for the functional analysis of yeast genes. Yeast 14:935-942[CrossRef][Medline]. |
| 14. |
Marbois, B. N., and C. F. Clarke.
1996.
The COQ7 gene encodes a protein in Saccharomyces cerevisiae necessary for ubiquinone biosynthesis.
J. Biol. Chem.
271:2995-3004 |
| 15. | Mariceau, C., J. Joets, D. Pousset, M. Guilloton, and F. Karst. 1996. FEN2: a gene implicated in the catabolite repression-mediated regulation of ergosterol biosynthesis in yeast. Yeast 12:531-539[CrossRef][Medline]. |
| 16. | Marichal, P. 1999. Mechanisms of resistance to azole antifungal compounds. Curr. Opin. Anti-infective Investig. Drugs 1:318-333. |
| 17. |
Marichal, P.,
J. Gorrens,
L. Laurijssens,
K. Vermuyten,
C. Van Hove,
L. Le Jeune,
P. Verhasselt,
D. Sanglard,
M. Borgers,
F. V. S. Ramaekers,
F. Odds, and H. Vanden Bossche.
1999.
Accumulation of 3-ketosteroids induced by itraconazole in azole resistant clinical Candida albicans isolates.
Antimicrob. Agents Chemother.
43:2663-2670 |
| 18. | Matthews, R. C., B. Maresca, J. P. Burnie, A. Cardona, L. Carrutu, S. Conti, G. S. Deepe, A. M. Florez, S. Franceschelli, E. Garcia, L. S. Gargano, G. S. Kobayashi, J. G. McEwen, B. L. Ortiz, A. M. Oviedo, L. Polonelli, J. Ponton, and A. Restrepo. 1998. Stress proteins in fungal diseases. Med. Mycol. 36(Suppl. 1):45-51. |
| 19. | M'baya, B., M. Fegueur, M. Servouse, and F. Karst. 1989. Regulation of squalene synthase and squalene epoxidase activities in Saccharomyces cerevisiae. Lipids 24:1020-1023[Medline]. |
| 20. | Odds, F. C. 1996. Antifungal therapy, p. 35-48. In C. C. Kibbler, D. W. R. Mackenzie, and F. C. Odds (ed.), Principles and practice of clinical mycology. John Wiley & Sons, Chichester, United Kingdom. |
| 21. | Parks, L. W. 1978. Metabolism of sterols in yeast. Crit. Rev. Microbiol. 6:301-341. |
| 22. |
Rosamond, J., and A. Allsop.
2000.
Harnessing the power of the genome in the search for new antibiotics.
Science
287:1973-1976 |
| 23. | Sanglard, D., F. Ischer, M. Monod, and J. Bille. 1996. Susceptibilities of Candida albicans multidrug transporter mutants to various antifungal agents and other metabolic inhibitors. Antimicrobiol. Agents Chemother. 40:2300-2305[Abstract]. |
| 24. | Smith, S. J., J. H. Crowley, and L. W. Parks. 1996. Transcriptional regulation by ergosterol in the yeast Saccharomyces cerevisiae. Mol. Cell. Biol. 16:5427-5432[Abstract]. |
| 25. | Truan, G., J.-C. Epinat, C. Rougeulle, C. Cullin, and D. Pompon. 1994. Cloning and characterization of a yeast cytochrome b5-encoding gene which suppresses ketoconazole hypresensitivity in a NADPH-P450 reductase-deficient strain. Gene 149:123-127[CrossRef][Medline]. |
| 26. |
Turi, T. G., and J. C. Loper.
1992.
Multiple regulatory elements control expression of the gene encoding S. cerevisiae cytochrome P450, Ianosterol 14 alpha-demethylase (ERG11).
J. Biol. Chem.
267:2046-2056 |
| 27. | Vanden Bossche, H. 1985. Biochemical targets for antifungal azole derivatives hypothesis on the mode of action. Curr. Top. Med. Mycol. 1:313-351[Medline]. |
| 28. | Vanden Bossche, H. 1990. Importance and role of sterols in fungal membranes, p. 135-157. In P. J. Kuhn, A. P. J. Trinci, M. J. Jung, M. W. Goosey, and L. G. Copping (ed.), Biochemistry of cell walls and membranes in fungi. Springer-Verlag, Berlin, Germany. |
| 29. | Vanden Bossche, H. 1995. Chemotherapy of human fungal infections, p. 431-484. In H. Lyr (ed.), Modern selective fungicides. Gustav Fischer Verlag, Jena, Germany. |
| 30. | Vanden Bossche, H., J. Heeres, L. J. J. Backx, P. Marichal, and G. Willemsens. 1993. Discovery, chemistry, mode of action, and selectivity of itraconazole, p. 263-283. In J. W. Rippon, and R. A. Fromtling (ed.), Cutaneius antifungal agents. Marcel Dekker, Inc, New York, N.Y. |
| 31. | Vanden Bossche, H., and L. Koymans. 1998. Cytochromes P450 in fungi. Mycoses 41(Suppl. 1):32-38. |
| 32. | Vanden Bossche, H., G. Willemsens, and P. Marichal. 1987. Anti-Candida drugs: the biochemical basis for their activity. Crit. Rev. Microbiol. 15:57-72[Medline]. |
| 33. | Vanden Bossche, H., G. Willemsens, W. Cools, P. Marichal, and W. Lauwers. 1983. Hypothesis on the molecular basis of the antifungal activation of N-substituted imidazoles and triazoles. Biochem. Soc. Trans. 11:665-667[Medline]. |
| 34. |
Vanden Bossche, H.,
P. Marichal,
L. Le Jeune,
M. C. Coene,
J. Gorrens, and W. Cools.
1993.
Effects of itraconazole on cytochrome P-450-dependent sterol 14-demethylation and reduction of 3-ketosteroids in Cryptococcus neoformans.
Antimicrob. Agents Chemother.
37:2101-2105 |
| 35. | Vanden Bossche, H., F. Dromer, I. Improvisi, M. Lozano Chiu, J. H. Rex, and D. Sanglard. 1998. Antifungal drug resistance in pathogenic fungi. Med. Mycol. 36(Suppl. 1):119-128. |
| 36. | Wheat, J., P. Marichal, H. Vanden Bossche, A. Le Monte, and P. Connolly. 1997. Hypothesis on the mechanism of resistance to fluconazole in Histoplasma capsulatum. Antimicrob. Agents Chemother. 41:410-414[Abstract]. |
| 37. |
White, T. C.
1997.
Increased mRNA levels of ERG16, CDR, and MDR1 correlate with increases on azole resistance in Candida albicans isolates from a patient infected with human immunodeficiency virus.
Antimicrob. Agents Chemother.
42:1488-1494 |
Antimicrobial Agents and Chemotherapy, June 2001, p. 1660-1670, Vol. 45, No. 6
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.6.1660-1670.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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