Departments of 1 Pharmaceutical Sciences and 2 Pharmacy, College of Pharmacy, and Department of 4Pediatrics, College of Medicine, University of Tennessee Health Science Center, Memphis, TN 38163; 3 Children's Foundation Research Center at Le Bonheur Children's Medical Center, Memphis, TN 38103, USA
Received 19 November 2004; returned 12 January 2005; revised 21 February 2005; accepted 25 February 2005
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Abstract |
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Methods: C. albicans SC5314 was exposed to either medium alone or ciclopirox olamine at a concentration equivalent to the IC50 (0.24 mg/L) for 3 h. RNA was isolated and gene expression profiles were compared using DNA microarrays. Differential expression of select genes was confirmed by real-time reverse transcription (RT)PCR. Mutants disrupted for CDR2 and both CDR1 and CDR2, as well as a clinical isolate overexpressing CDR1 and CDR2, were examined for changes in susceptibility to ciclopirox olamine.
Results: A total of 49 genes were found to be responsive to ciclopirox olamine, including 36 up-regulated genes and 13 down-regulated genes. These included genes involved in small molecule transport (HGT11, HXT5, ENA22, PHO84, CDR4), iron uptake (FRE30, FET34, FTR1, FTR2, SIT1) and cell stress (SOD1, SOD22, CDR1, DDR48). Mutants disrupted for CDR2 and both CDR1 and CDR2, as well as a clinical isolate overexpressing CDR1 and CDR2, showed no change in susceptibility to ciclopirox olamine compared with the respective parent.
Conclusions: Consistent with the hypothesis that ciclopirox olamine acts as an iron chelator, it induced changes in expression of many genes involved in iron uptake. Despite induction of the multidrug efflux pump genes CDR1 and, to a lesser extent, CDR2 by ciclopirox olamine, these genes do not affect susceptibility to this agent.
Keywords: microarrays , gene regulation , C. albicans , antifungal activity , efflux pumps
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Introduction |
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Recently, there has been renewed interest in the hydroxypyridone anti-infective agent ciclopirox olamine.10,11 While this agent exhibits a broad spectrum of activity against a number of medically important fungi and has shown utility as a topical treatment for mucocutaneous fungal infections, its mechanism of action is poorly understood. C. albicans appears to take up ciclopirox olamine via an energy-dependent mechanism.12 The drug has been reported to inhibit RNA, DNA and protein synthesis, and to inhibit uptake of precursors of macromolecular synthesis.13,14 It has been reported to act as a chelator of free iron and thus its mechanism of action is by aiding the host defence mechanism of iron starvation against C. albicans.15 Cicloprox olamine belongs to the hydroxamic acid class of enzyme inhibitors, in which the hydroximate functionality chelates strongly to an enzyme-bound transition metal cofactor (Zn, Fe, Mn, Ni), inactivating the target enzyme. These inhibitors have found a broad range of applications as potential therapeutics for metalloproteases involved in angiogenesis and arthritis, as antineoplastic agents against histone deacetylase and as antibacterial therapeutics as inhibitors of peptide deformylase.1618 Thus, it is possible that there is a preferred enzymatic target for this inhibitor.
Niewerth et al.11 recently examined the expression of 47 C. albicans genes in response to ciclopirox olamine by reverse transcription (RT)PCR. These included genes involved in pathogenicity, drug resistance and iron transport. Additional differentially expressed genes were identified using suppression-subtractive hybridization. These investigators found the drug resistance genes CDR1 and CDR2 to be up-regulated in response to ciclopirox olamine, as well as the iron permease genes FTR1 and FTH1, the copper permease gene CCC2, the iron reductase gene CFL1 and the siderophore transporter gene SIT1. They also found that addition of iron(III) chloride reversed the effect of ciclopirox olamine against C. albicans, supporting the hypothesis that the drug acts as an iron chelator. Furthermore, ciclopirox olamine increased the susceptibility to H2O2-induced oxidative stress in C. albicans, suggesting the loss of activity of iron-dependent antioxidant enzymes.
The sequencing of the C. albicans genome has greatly facilitated the use of functional genomic approaches to study the interaction of antifungal agents with this pathogenic fungus.19 We have used DNA microarray analysis to examine azole20,21 and polyene22 resistance in this organism, as well as the response to the systemically used antifungal agents amphotericin B, 5-FC, ketoconazole and caspofungin, in both Saccharomyces cerevisiae and C. albicans.23,24 In the present study we used genome-wide expression profiling to identify genes differentially expressed in response to ciclopirox olamine.
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Materials and methods |
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Ciclopirox olamine was obtained from Sigma (St Louis, MO, USA). Fluconazole was obtained from ICN Biomedicals Inc. (Aurora, OH, USA). C. albicans strain SC5314, obtained from ATCC (Manassas, VA, USA), was used for microarray experiments. Strains CAF2-1 (ura3::imm434/URA3), DSY653 (
cdr2::hisG-URA3-hisG/
cdr2::hisG), and DSY654 (
cdr1::hisG/
cdr1::hisG
cdr2::hisG-URA3-hisG/
cdr2::hisG) were obtained from D. Sanglard. Clinical isolates Gu2 and Gu5 were obtained from J. Morschhäuser. Synthetic dextrose medium, containing 0.67% (w/v) yeast nitrogen base without amino acids and 2% (w/v) dextrose, was used to grow strain SC5314 for microarray experiments. The medium was buffered with 0.165 M MOPS (Sigma), and the pH was adjusted to 7.0 with NaOH. YPD broth (1% yeast extract, 2% peptone, 1% dextrose) was used for MIC assays.
IC50 determination
To ensure consistent results, IC50s were determined under the same environmental conditions used in subsequent microarray analysis, including: incubation times, culture volume (100 mL), flask size (250 mL Corning 430281 roller bottle), temperature (30°C) and shaking speed (225 rpm). All measurements of turbidity were taken at 600 nm on a Beckman DU530 spectrophotometer. Cultures with an A600 exceeding 1.0 were diluted 1:10 in water for measurement. Synthetic dextrose medium (100 mL) was inoculated from a fresh, saturated culture of C. albicans to an initial A600 of 0.005 and grown at 30°C until the A600 reached 1.0 (12 h). The culture was then diluted to an A600 of 0.1 and allowed to recover at 30°C/225 rpm until an A600 of 0.2 was reached (
2 h). Ciclopirox olamine was then added according to the desired concentration from a 1 g/L drug stock in dimethyl sulphoxide (DMSO) and an equal amount of DMSO was added to the control culture. DMSO concentrations were < 0.05%, and did not affect the growth curves of control cultures. The cultures were grown for 6 h and the A600 was measured at both 3 and 6 h. IC50 s were calculated at 6 h, in reference to the control culture. Three rounds of experiments were conducted, first with a broad range of six two-fold serial dilutions based on the MIC. The last two experiments used a narrow range of three drug concentrations (0.1, 0.2 and 0.4 mg/L). The average IC50 was obtained from these three experiments (0.24 mg/L).
Cell culture and drug exposure for microarray experiments
C. albicans was grown as described in the IC50 section above. For a single microarray experiment, a total of six 100 mL cultures were prepared, three with ciclopirox olamine added at a concentration equivalent to the average IC50 (0.24 mg/L) and three control cultures, treated with 0.024% DMSO. The A600 was measured 3 h after drug addition. Two control cultures and two drug-treated cultures were harvested by centrifugation at 3600 g, at 4°C for 5 min. The medium was completely removed by aspiration and the cell pellets were flash frozen on dry ice and stored at 80°C until RNA preparation. The remaining control and drug-treated cultures were allowed to grow for a further 3 h, at which point the A600 was measured and the percentage growth calculated.
RNA preparation
RNA was isolated using the hot phenol method.25 Frozen cells were resuspended in 12 mL of AE buffer (50 mM sodium acetate pH 5.2, 10 mM EDTA) at room temperature after which 800 µL of 25% SDS and 12 mL of acid phenol (Fisher Scientific, Houston, TX, USA) was added. The cell lysate was then incubated 10 min at 65°C with vortexing each minute, cooled on ice for 5 min, and subjected to centrifugation for 15 min at 11 952 g. Supernatants were transferred to new tubes containing 15 mL of chloroform, mixed and subjected to centrifugation at 200 g for 10 min. RNA was precipitated from the resulting aqueous layer by transferring that portion to new tubes containing one volume of isopropanol and 0.1 volume of 2 M sodium acetate pH 5.0, mixing well, and subjecting the mixture to centrifugation at 17 211 g for 35 min at 4°C. The supernatants were removed, the pellet was resuspended in 10 mL of 70% ethanol, and the RNA collected by centrifugation at 17 211 g for 20 min at 4°C. Supernatants were again removed, and RNA was resuspended in DEPC-treated water. Absorbance was measured at 260 and 280 nm, and integrity of RNA was visualized by subjecting 25 µL of the sample to electrophoresis through a 1% agarose/MOPS gel.
Microarray hybridization
The C. albicans microarrays used in this study were manufactured by Eurogentec SA (Ivoz-Ramet, Belgium) in collaboration with the European Galar Fungail Consortium (www.pasteur.fr/recherche/unites/Galar_Fungail/). Two independent sets of RNA from control and drug-treated cells (biological replicates) were used in these studies to prepare two independent cDNA probe sets. Ten micrograms of total RNA sample were added to a mixture of 1 pmol of T20VN and oligo(dT) (1821mer) primer mix (C. albicans-specific primer mix Plus) (Eurogentec); 0.5 mM each dATP, dGTP, TTP; 20.5 µM dCTP; 37.5 µM Cy3- or Cy5-dCTP (NEN Life Sciences, Boston, MA, USA); and 10 mM dithiothreitol in a buffer containing 50 mM TrisHCl (pH 8.3), 75 mM KCl and 3 mM MgCl2. The reaction mixture was denatured at 65°C for 5 min, incubated at 42°C for 5 min, after which 1 µL of Rnasin (Promega, Madison, WI, USA) and 200 U of Superscript II reverse transcriptase (LifeTechnologies/Invitrogen, Carlsbad, CA, USA) were added to the mixture. The reaction proceeded at 42°C for 1 h after which an additional 200 U of Superscript II reverse transcriptase was added, and the reaction mixture was incubated at 42°C for an additional hour. To stop the reaction, EDTA (pH 8.0) and sodium hydroxide were added to a final concentration of 5 mM and 0.4 M, respectively, and the mixture was incubated at 65°C for 20 min. Finally, acetic acid was added to achieve a final concentration of 0.37 M.
The labelled cDNA probes were purified using Qia-Quick columns (Qiagen, Valencia, CA, USA) following the manufacturer's instructions. The cDNA probes were then fluorescently labelled. One set of cDNA probes was labelled using Cy5 for those representing RNA from drug-treated cells and Cy3 for those representing RNA from control cells. The second set was labelled using Cy3 for those representing RNA from drug-treated cells and Cy5 for those representing RNA from control cells. Five microlitres each of the Cy3- and Cy5-labelled probes were mixed with 50 µg of heat-denatured salmon sperm DNA, incubated at 95°C for 2 min and snap-cooled on ice. The mixture was added to 40 µL of hybridization buffer (DIG easy hyb; Roche, Basel, Switzerland) and applied to the array slides under glass coverslips. Hybridization was performed at 37°C overnight in a humidified chamber (Corning Life Sciences, Acton, MA, USA). To wash the slides, the coverslip was removed, and the slide was incubated at room temperature in 0.2x SSC + 0.1% SDS for 5 min with agitation, rinsed at room temperature with 0.2x SSC for 5 min with agitation, and spin-dried at 500 rpm for 5 min. Slides were scanned using a ChipReader microarray scanner (Virtek Vision Intl).
Data analysis
GenePix 1.0 software (Axon Instruments, Inc.) was used for image analysis and data visualization. The local background values were calculated from the area surrounding each spot and subtracted from the total spot signal values. These adjusted values were used to determine differential gene expression (Cy3/Cy5 ratio) for each spot. A normalization factor was applied to account for systematic differences in the probe labels by using the differential gene expression ratio to balance the Cy5 signals. In the present study, only spots with a mean balanced differential expression ratio 2 or
0.5 for both spots representing a given cDNA on the array in two independent experiments were considered to be differentially expressed.
DNA sequences were annotated on the basis of results of BLASTn searches using GenBank (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi), the Stanford sequencing database (Stanford University, Palo Alto, CA, USA; http://www-sequence.stanford.edu/group/candida)19 and the CandidaDB database (http://www.pasteur.fr/Galar_Fungail/CandidaDB/).26
Quantitative real time RTPCR
An aliquot of the RNA preparations from untreated and treated samples, used in the microarray experiments, was saved for quantitative real time RTPCR follow-up studies. First strand cDNAs were synthesized from 2 µg of total RNA in a 21 µL reaction volume using the SuperScript First-Strand Synthesis System for RTPCR (Invitrogen) as per the manufacturer's instructions. Quantitative real time PCRs were performed in triplicate using the 7000 Sequence Detection System (Applied Biosystems, Foster City, CA, USA). Independent PCRs were performed using the same cDNA for both the gene of interest and 18S rRNA, using the SYBR® Green PCR Master Mix (Applied Biosystems). Gene-specific primers were designed for the gene of interest and 18S rRNA using Primer Express® software (Applied Biosystems) and the Oligo Analysis & Plotting Tool (Qiagen). Primer sequences are listed in Table 1. The PCR conditions consisted of AmpliTaq Gold activation at 95°C for 10 min, followed by 40 cycles of denaturation at 95°C for 15 s and annealing/extension at 6°C for 1 min. A dissociation curve was generated at the end of each PCR cycle to verify that a single product was amplified using software provided with the 7000 Sequence Detection System. The change in fluorescence of SYBR Green I dye in every cycle was monitored by the system software, and the threshold cycle (CT) above background for each reaction was calculated. The CT value of 18S rRNA was subtracted from that of the gene of interest to obtain a CT value. The
CT value of an arbitrary calibrator (e.g. untreated sample) was subtracted from the
CT value of each sample to obtain a
CT value. The gene expression level relative to the calibrator was expressed as 2
CT.
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The MIC of each clinical isolate or mutant and parent strain was determined by the microbroth dilution method in 96-well plate format. Briefly, ciclopirox olamine was prepared in DMSO at a concentration of 10 mg/mL, and two-fold serial dilutions were prepared in 100 µL of YPD medium from 25 mg/L to 0.025 mg/L in a 96-well plate. For each C. albicans mutant, a 10 mL culture was grown to an OD600 of 0.40.6 in YPD medium. The culture was then diluted to an OD600 of 0.01 in YPD medium and 100 µL was added to each well, for a total of 200 µL/well at an OD600 of 0.005. Plates were incubated at 30°C for 36 h and the MIC determined by visual inspection. To determine the percentage growth for fluconazole-treated cells each well was mixed, 100 µL was removed and diluted 1:10 in H2O, and the absorbance read at 600 nm on a spectrophotometer. Percentage growth was then calculated. Assays were performed in triplicate.
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Results and discussion |
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In the present study we employed a similar experimental design with regard to choice of drug concentration, exposure time and medium selection to a previous study we conducted in S. cerevisiae, for the purpose of examining the effects of antifungal agents on global gene expression.23 Owing to the expense of these arrays, we were limited to a single concentration and single time-point. An exposure time of 3 h (two doubling times) was chosen as this should allow a long enough exposure to elicit drug-specific effects, but would be short enough for the resulting gene expression profiles to not be over-represented with secondary transcriptional changes. The IC50 was chosen for this study to provide a concentration sufficient enough to elicit drug-specific responses without producing secondary effects that might be more indicative of a general stress response. IC50s were determined under the exact conditions in which the RNA would be isolated for subsequent analysis. For our experiment we included two biological replicates representing independent cell cultures, drug treatments and RNA sample isolations. These biological replicates also incorporated the use of dye swapping, i.e. the control sample for one experiment was labelled with Cy3 and the experimental sample labelled with Cy5, whereas in the other experiment the labelling of samples with these dyes was reversed. We found a total of 49 genes to be responsive to ciclopirox olamine (Table 2). Of these, 36 were up-regulated and 13 were down-regulated. The complete dataset for both gene expression profiling experiments can be found at http://www.dcp.utmem.edu/rogerslab/data.html.
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To validate the differential expression of genes identified by microarray analysis we performed real-time RTPCR for 12 genes of interest. These genes were chosen for their involvement in iron uptake, oxidative stress response and drug resistance. Indeed there was good correlation between real time RTPCR and microarray data (Figure 1).
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A number of genes involved in small molecule transport were differentially expressed in response to ciclopirox olamine including up-regulation of the hexose transporters HGT11 and HXT5. HXT5 is homologous to ScHXT5 in S. cerevisiae, which is involved in the uptake of fructose, glucose and mannose.27 Its up-regulation in response to ciclopirox olamine may reflect an increased need for energy generation within the cell or a decrease in ATP levels due to iron starvation. Also up-regulated was ENA22, a homologue of ScENA5 that appears to encode a transporter involved in the uptake of sodium.28
Of particular interest was the differential expression of genes involved in iron uptake. This included the up-regulation of FET34, FTR1, FTR2 and SIT1. FET34 is a homologue of ScFET3 that encodes a copper-metalloenzyme, which, as mentioned above, is a ferro-oxidase critical for uptake of Fe(II).29 FTR1 and FTR2 are homologues of ScFTR1, a gene encoding a component of the high affinity iron and transport complex.30 SIT1 is homologous to ScARN1, which encodes a putative siderophore transporter also believed to be involved in iron uptake.31,32 Up-regulation of these genes suggests depletion of iron and is consistent with the hypothesis that ciclopirox olamine acts as an iron chelator. Furthermore, up-regulation of FTR1, FET34 and SIT1 are consistent with ciclopirox-induced changes in gene expression observed in the study by Niewerth et al.11
Also of interest was the down-regulation of FRE30, ZRT2 and CTR1. FRE30 is homologous to ScFRE3, which, like ScFRE2, encodes a ferric reductase induced by low iron levels.33 ZRT2 encodes a putative zinc transporter homologous to ScZRT1, which has been observed to be down-regulated in ScMAC1 gain-of-function mutants. ScMac1 acts as a transcriptional activator in copper deficient cells, but is inhibited when copper is plentiful. CTR1 encodes a high affinity transporter that is required for the uptake of copper in C. albicans.34 As copper is essential for iron uptake, down-regulation of CTR1 in response to ciclopirox olamine is surprising. It is possible that while ciclopirox depletes Fe(III) in the medium, copper concentrations are sufficient as to not require increased uptake.
Genes involved in carbohydrate metabolism and energy generation
Several genes involved in carbohydrate metabolism were up-regulated in response to cicopirox olamine. These included genes encoding enzymes of the tricarboxylic acid and glyoxylate cycles such as isocitrate dehydrogenase (NAD+) subunit 2 (IDH2), malate synthase (MLS1) and isocitrate lyase (ICL1). Also up-regulated were the ATP synthase genes ATP1, ATP2 and ATP8. Up-regulation of these genes may reflect an increased need for energy by the cell. Since many enzymes critical to cellular respiration are dependent upon iron, reduced iron availability would lead to a reduced availability of cellular energy.
Genes involved in cell stress
Exposure to ciclopirox olamine also elicited changes in the expression of cell stress genes. Among these was the up-regulation of SOD1 and down-regulation of SOD21. Both of these genes encode superoxide dismutases that are important in protecting the cell from damaging superoxide radicals. SOD1 encodes a cytoplasmic copper, zinc superoxide dismutase, which has been shown to confer protection against macrophage-produced free radicals and is a virulence factor of C. albicans.35 SOD21 encodes a mitochondrial manganese superoxide dismutase that protects the organism from intracellularly derived free radicals.36
Also up-regulated were the ATP-dependent binding cassette (ABC) transporters CDR1 and CDR4, the first of which has been implicated in azole antifungal resistance.37 Up-regulation of CDR1 and CDR2 in response to ciclopirox olamine was also reported by Niewerth et al.11 Indeed we observed a 1.9-fold up-regulation of CDR2 in the present study.
Susceptibility of mutant strains to ciclopirox olamine
Up-regulation of the genes encoding the Cdr1 and Cdr2 transporters in response to ciclopirox olamine may be part of a general stress response to noxious agents. However, as this drug has been shown to accumulate in high amounts within the cell, it is possible that up-regulation of CDR1 and CDR2 serve to extrude the drug. To test this hypothesis, we examined the susceptibility to ciclopirox in mutant strains lacking CDR2 (DSY653), and both CDR1 and CDR2 (DSY654), as compared with the parent isolate CAF2-1. We also examined the susceptibility to ciclopirox in a fluconazole-resistant clinical strain that exhibits overexpression of CDR1 and CDR2, but not MDR1 (Gu5) compared with its matched parent isolate (Gu2). As expected, strains DSY653 and DSY654 exhibited hyper-susceptibility, and strain Gu5 showed marked resistance to fluconazole (Table 3). No change in susceptibility to ciclopirox olamine was observed in any of these strains. This suggests that while CDR1 and CDR2 are up-regulated in response to ciclopirox olamine, susceptibility to this agent is not affected by these efflux pumps. Indeed in the study by Niewerth et al.,11 no change in susceptibility or tolerance to ciclopirox olamine could be observed after incubation with the drug for 6 months.
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We have examined changes in the gene expression profile of C. albicans in response to ciclopirox olamine. Among the genes affected, we identified changes in gene expression that are consistent with the hypothesis that ciclopirox olamine acts as a chelator of iron. We also observed changes in expression of genes encoding the multidrug resistance efflux pumps CDR1 and CDR2. Although these genes confer resistance to other agents such as the azole antifungals, we demonstrate that these genes appear to have no effect on the susceptibility of C. albicans to ciclopirox olamine.
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Acknowledgements |
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