Departments of 1 Pharmacy and 3 Pharmaceutical Sciences, College of Pharmacy; 4 Department of Pediatrics, College of Medicine, University of Tennessee Health Science Center, Memphis, TN 38163; 2 Department of Pharmacy Practice, School of Pharmacy,University of Mississippi, Jackson, MS 39216, USA
Received 11 December 2002; returned 25 January 2003; revised 14 February 2003; accepted 18 February 2003
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Abstract |
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Methods: S. cerevisiae strain ATCC 9763 was passaged in increasing concentrations of itraconazole. Itraconazole and fluconazole MICs for the initial isolate (9763S) were 2 and 16 mg/L and for the final isolate (9763I) were 16 and 64 mg/L, respectively. Duplicate sets of total RNA from 9763S and 9763I were isolated and hybridized to Affymetrix S98 yeast arrays. To validate results, six differentially expressed genes were further examined by RTPCR.
Results: Of the nearly 6400 open reading frames represented on the array, a total of 116 genes (1.8%) were found to be differentially expressed. Cell wall maintenance genes TIR4 and CCW12, sterol metabolism gene UPC2, small molecule transport genes AUS1 and YHK8, and stress response gene CUP1-1 were expressed at a level at least 2.5-fold higher than the expression level found in 9763S. Eleven energy generation genes, ionic homeostasis genes FRE1, FRE2 and FRE4, and sterol metabolism genes ERG8 and ERG13 were expressed at least 2.5-fold lower than the expression level found in 9763S.
Conclusions: Several genes found to be differentially expressed in this study have been shown previously to be differentially expressed in the fungal response to azole treatment. In addition, the potential role of AUS1 and/or YHK8 as mediators of drug efflux is intriguing and warrants further study.
Keywords: fungi, microarray, resistance mechanisms
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Introduction |
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A common theme in azole resistance is over-expression of genes encoding efflux pumps or the azole target lanosterol demethylase, encoded by the ERG11 gene.5,813 Several groups have reported either the up-regulation of ERG11 or point mutations in this gene in azole-resistant clinical C. albicans isolates.12 Efflux pump genes that have been implicated in azole resistance are the ATP-binding cassette (ABC) transport genes CDR1 and CDR2, as well as the major facilitator gene CaMDR1 (BMR1).14 Up-regulation of efflux pump genes has been demonstrated in a number of azole-resistant C. albicans isolates and is thought to result in extrusion of azoles.
Because the Saccharomyces cerevisiae genome has been sequenced and many of the ORFs are well-characterized, it is well suited for studying changes in gene expression in response to antifungal compounds. Previous studies have discovered the pleiotropic drug resistance (PDR) gene family, including the PDR1 and PDR3 transcription factors that regulate drug efflux pumps such as the ABC transporter PDR5.1518 Subsequently, C. albicans homologues of PDR5 were identified as CDR1 and CDR2.19
Recent studies have taken advantage of microarray technology to examine the expression of thousands of genes simultaneously in an organism. Bammert & Fostel20 compared gene expression profiles of wild-type and ergosterol-deficient S. cerevisiae strains treated with azoles, finding cell wall function, mitochondrial respiration and oxidative stress genes affected by azole treatment. Comparing expression profiles of wild-type and PDR mutant strains of S. cerevisiae, DeRisi and colleagues21 found that several ABC and major facilitator transport, stress response and cell wall function genes were activated in the mutant strains.
Most recently, we have examined S. cerevisiae incubated with ketoconazole,22 and found that ergosterol biosynthesis and small molecule transport genes were primarily up-regulated. De Backer and colleagues23 studied the response of C. albicans to itraconazole where 296 genes were responsive, encompassing many different cellular roles. In a study examining azole resistance in experimentally induced and clinical C. albicans isolates, several cell stress, multidrug transport and cell wall maintenance genes were affected.24 Likewise, in a study using azole-susceptible and -resistant clinical C. albicans isolates, we found several of the same genes differentially expressed in azole resistance.25
Of all the studies highlighted above, only three examined resistant isolates using microarray techniques.2325 The paucity of information on the majority of C. albicans ORFs suggests that similar examination of S. cerevisiae may yield more information on the specific roles of responsive genes to give greater insight into genes involved in azole resistance.
The current study involves analysis of differential gene expression by microarray analysis in a strain of S. cerevisiae in which reduced susceptibility to fluconazole and itraconazole was experimentally induced. Cell wall maintenance, lipid, fatty-acid and sterol metabolism, and small molecule transport genes were among those differentially expressed. This study identifies genes associated with altered azole susceptibility in S. cerevisiae whose homologues in fungal pathogens could be exploited as drug targets.
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Materials and methods |
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Cultures of S. cerevisiae isolate ATCC 9763 (ATCC, Manassas, VA, USA) were passed in increasing concentrations of itraconazole (formulated for injection; Ortho Biotech, Raritan, NJ, USA). Aliquots of the parent strain (designated 9763S) and the final strain (9763I) were stored as glycerol stocks. Cultures grown for RNA isolation were initiated by diluting an aliquot of glycerol stock in YPD broth (1% yeast extract, 2% bacto-peptone, 2% glucose; from Sigma Chemical Co.) in the absence of itraconazole and incubating overnight at 30°C in an environmental shaking incubator. Cultures were then diluted to an optical density at 600 nm (OD600) of 0.2 in fresh YPD in the absence of itraconazole and grown as before to mid-logarithmic phase (OD600 = 0.50.8). At this point, an aliquot of this culture was prepared as a glycerol stock and used for susceptibility testing while RNA was isolated from the remainder of the culture.
MIC determination
The MICs of itraconazole, fluconazole, amphotericin B, flucytosine and caspofungin were determined by broth microdilution as described by the National Committee for Clinical Laboratory Standards (NCCLS).26 The determinations of the MICs for the isolates were carried out before experimental use and are listed in Table .
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RNA was isolated from two sets of independently grown cultures of isolates with the hot phenol method, suspended in diethylpyrocarbonate (DEPC)-treated water, and stored at 70°C until use.27 Absorbances were measured at 260 and 280 nm, and integrity of RNA was visualized by subjecting a portion of the sample to electrophoresis through a 1% agaroseMOPS gel. RNA was isolated from two independent sets of cultures for use in microarray hybridizations, while a third set of RNA was isolated independently for subsequent RTPCR analysis.
Microarray hybridization
Microarray hybridizations were carried out with the Affymetrix S98 array using protocols as described by Affymetrix, Inc. (Santa Clara, CA, USA). The Affymetrix S98 Yeast Genome array contains 6400 oligonucleotide probe sets designed from sequence data of S. cerevisiae strain S288C and
600 additional probe sets representing putative ORFs identified by SAGE analysis, mitochondrial proteins, TY proteins, plasmids and a small number of ORFs for strains other than S288C. Probes for hybridization were synthesized from sets of 9763I and 9763S S. cerevisiae RNA samples generated from two independent experiments. Ten micrograms of total RNA were subjected to first- and second-strand cRNA synthesis incorporating biotin-labelled nucleotides. Biotinylated product was then fragmented and subsequently hybridized overnight with the microarray chips and the manufacturers hybridization buffer. Hybridized microarrays were washed according to manufacturers protocols and subjected to a signal enhancement protocol consisting of an initial incubation with a streptavidinphycoerythrin conjugate followed by a goat anti-streptavidin biotinylated antibody and a final staining with the streptavidinphycoerythrin conjugate. The microarrays were then scanned with the GeneArray scanner using an argon ion laser excitation source and emission detected by a photomultiplier tube through a 570 nm long pass filter.
Data analysis
Data were analysed with Affymetrix Microarray Suite 5.0 software. Genes were considered to be differentially expressed if the element was assigned at least one present call for either the 9763S or 9763I hybridization in both experiments, normalized signal ratios were 2.5 or
0.4 in both experiments, and the change in gene expression was in the same direction in both experiments. Genes were annotated on the basis of results of BLASTn searches in GenBank (http://www.ncbi.nlm.nih.gov/entrez/) and the Saccharomyces Genome Database (SGD; http://genome-www.stanford.edu/Saccharomyces/).
cDNA synthesis and RTPCR
Two micrograms of total RNA from each sample was denatured in the presence of 1 µg oligo(dT) primer stock (ResGen/Invitrogen, Carlsbad, CA, USA) at 70°C after which the mixture was chilled on ice, and a master mix containing 50 mM TrisHCl pH 8.3, 75 mM KCl, 3 mM MgCl2, dATP, dCTP, dGTP and TTP at 1.25 mM each, and 25 U Superscript II reverse transcriptase (Gibco BRL/Invitrogen, Carlsbad, CA, USA) was added to each tube. The reaction mixture was incubated for 10 min at room temperature, followed by a 60 min incubation at 37°C and a 5 min incubation at 90°C.
PCR was carried out by mixing 1 µL of the appropriate dilution of cDNA (empirically determined for each gene to give product in the linear range), 0.5 µg each forward and reverse primer, 2.5 U Taq polymerase, and 0.1% Triton X-100 in EasyStart Micro50 PCR tubes, and subjecting the reaction mixture to the following reaction conditions: one repetition of 94°C for 5 min; 32 repetitions of 94°C for 30 s, gene-specific annealing temperature for 30 s, 72°C for 1 min and one repetition of 72°C for 5 min. Equivalent volumes of PCR product were applied to a 3% agarose gel and separated by gel electrophoresis in 1 x TAE. Primers for RTPCR are listed in Table .
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Results and discussion |
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One potential limitation of the present study is that the intravenous formulation of itraconazole was used to generate 9763I. It is therefore possible that some of the changes in gene expression induced by exposure to this formulation were the result of exposure to ß-cyclodextrin. However, the use of the intravenous formulation resulted in the generation of a strain with reduced susceptibility to both itraconazole and fluconazole that expressed genes consistent with a response to azole exposure as outlined below.
The S. cerevisiae genome was examined with microarrays to compare differences in gene expression between 9763I and 9763S. From both independent experiments, a total of 116 genes (1.8% of all the ORFs) was reproducibly differentially expressed in 9763I, i.e. at least 2.5-fold higher or lower than the expression level found in 9763S. Of the 116 genes, there were 64 up-regulated genes (Table ) and 52 down-regulated genes (Table
). Six genes of interest from the array data were further examined by RTPCR (Figure 1).
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Three lipid, fatty-acid and sterol metabolism genes were differentially expressed, including ERG8 and ERG13, which were down-regulated in 9763I. Whereas several studies have demonstrated the increase in ERG gene expression in response to azole treatment in S. cerevisiae20,22 and C. albicans23 or in C. albicans azole resistance,25 none document down-regulation of ERG genes. However, there are clinical C. albicans isolates that contain a significant fraction of membrane sterols other than ergosterol.28 It is possible that isolate 9763I contains an increase in alternative sterols in its membrane that may contribute to the increase in MIC and allow for down-regulation of ERG8 and ERG13.
Sterol uptake genes
UPC2, encoding a transcription factor that regulates sterol biosynthesis uptake, was up-regulated in the present study. The role of UPC2 was examined recently in a microarray analysis comparing wild-type and upc2-1 mutant strains of S. cerevisiae.29 As a result of the gain-of-function mutation, UPC2 itself was up-regulated in the mutant strain as were several other genes also found to be differentially expressed in the present study: TIR4, AUS1 (YOR011W), FIT2, HEM13, HEM14 and YGR131W. When UPC2 was deleted in the upc2-1 strain, sterol uptake was significantly diminished in anaerobic conditions. Deletion mutants of eight other genes found to be over-expressed in the upc2-1 strain were constructed, but only the yor011w strain (AUS1 deletion mutant) exhibited a significant decrease in sterol uptake.29 AUS1 is an ABC transport gene similar to the CDR genes and is up-regulated under aerobic conditions in response to azole exposure.22 CDR1 can transport steroid molecules as well as elicit drug efflux;30 similarly, it may be possible that AUS1 is a mediator of drug efflux as well as sterol transport. Regardless, AUS1 is up-regulated both in response to azole treatment and as a result of reduced azole susceptibility in S. cerevisiae; therefore, the specific function of AUS1 in this context should be examined further.
Energy generation, ionic homeostasis and small molecule transport genes
Most of the energy generation gene products down-regulated in this study are mitochondrial electron transport proteins. We speculate that these genes are down-regulated in order to lessen the amount of endogenously generated reactive oxygen species (ROS) in the cell. This would be in response to generation of ROS due to the action of azoles on the cell membrane. Kobayashi and colleagues31 demonstrated the production of ROS in C. albicans by miconazole and fluconazole, and there was a strong inverse correlation between the level of ROS production and the MIC. The authors speculated that isolates may exhibit resistance mechanisms that involve scavenging ROS. In the present study, the metallothionein CUP1-1 was up-regulated over seven-fold and has been shown to be up-regulated in the ROS response.32 Three ionic homeostasis genes, FRE1, FRE2 and FRE4, and two metal ion transport genes, SIT1 and CTR3, were down-regulated in 9763I. In previous studies, we have found that iron transport genes were down-regulated in fluconazole-resistant clinical C. albicans isolates.27
Amino acid metabolism genes
Five amino acid metabolism genes were found to be differentially expressed in 9763I compared with 9763S. Found to be up-regulated among these is ScMET14, a gene whose product is induced in response to oxidative stress.33 In microarray analysis carried out previously to examine the S. cerevisiae response to cadmium, a potent cell poison that causes oxidative stress, 50 genes were up-regulated four-fold or more in response to oxidative stress. Most were stress response genes, glutathione synthesis genes and sulphur assimilation genes including ScMET14.33
Cell wall maintenance genes
CCW12 and TIR4, up-regulated in 9763I, both encode cell wall mannoproteins that, although their specific biological functions are unknown, have dramatic effects on the cell if not expressed. In a study demonstrating that the CCW12 gene product was associated with the cell wall in S. cerevisiae, disruption of the gene led to pronounced sensitivity to Calcofluor White and Congo Red as well as decreased mating efficiency and decreased level of agglutination.34 The gene TIR4 is one of nine members of the seripauperin family of cell wall mannoproteins, the DAN/TIR family. These genes are similarly regulated, and all nine are expressed only during anaerobic conditions. Additionally, when TIR1, TIR3 or TIR4 is disrupted, cells cannot grow during anaerobis.35 Since the up-regulation of TIR4 in the present study does not coincide with the up-regulation of other DAN/TIR genes or with the down-regulation of their aerobic-growth counterparts CWP1 and CWP2, it is possible that TIR4 has additional roles in response to conditions elicited by azoles.
Conclusions
The present study utilizes an experimentally induced isolate of S. cerevisiae with reduced susceptibility to fluconazole and itraconazole as a model for finding novel molecules associated with azole resistance. Previous studies have focused on PDR and ERG genes in S. cerevisiae azole resistance. Here we found genes that have previously not been associated with this phenotype. These molecules represent potential new targets to examine in relation to azole resistance.
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Acknowledgements |
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Footnotes |
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References |
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