Departments of 1 Pharmacy and 2 Pharmaceutical Sciences, College of Pharmacy; 3 Department of Pediatrics, College of Medicine, University of Tennessee Health Science Center, Memphis, TN; 4 Children's Foundation Research Center, Le Bonheur Children's Medical Center, Memphis, TN; 5 Department of Clinical Sciences and Administration, University of Houston College of Pharmacy, Houston, TX; 6 Department of Biology, Indiana University Purdue University Indianapolis, Indianapolis, IN; 7 Department of Drug Disposition, Eli Lilly & Co., Lilly Corporate Center, Indianapolis, IN, USA
Received 13 January 2004; returned 8 March 2004; revised 6 May 2004; accepted 25 May 2004
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Abstract |
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Methods: C. albicans SC5314 was passed in increasing concentrations of amphotericin B to generate isolate SC5314-AR. Susceptibility testing by Etest revealed SC5314-AR to be highly resistant to both amphotericin B and fluconazole. The gene expression profile of SC5314-AR was compared with that of SC5314 using DNA microarray analysis. Sterol composition was determined for both strains.
Results: Upon examination of MICs of antifungal compounds, it was found that SC5314-AR was resistant to both amphotericin B and fluconazole. By microarray analysis a total of 134 genes were found to be differentially expressed, that is up-regulated or down-regulated by at least 50%, in SC5314-AR. In addition to the cell stress genes DDR48 and RTA2, the ergosterol biosynthesis genes ERG5, ERG6 and ERG25 were up-regulated. Several histone genes, protein synthesis genes and energy generation genes were down-regulated. Sterol analysis revealed the prevalence of sterol intermediates eburicol and lanosterol in SC5314-AR, whereas ergosterol was the predominant sterol in SC5314.
Conclusion: Along with changes in expression of these ergosterol biosynthesis genes was the accumulation of sterol intermediates in the resistant strain, which would account for the decreased affinity of amphotericin B for membrane sterols and a decreased requirement for lanosterol demethylase activity in membrane sterol production. Furthermore, other genes are implicated as having a potential role in the polyene and azole antifungal resistant phenotype.
Keywords: microarrays , polyenes , azoles , antifungal resistance , C. albicans
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Introduction |
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Amphotericin B, a fungicidal agent, has activity against a number of pathogenic fungi.3 Its mode of action is thought to involve binding to ergosterol in the fungal cell membrane, which results in the formation of pores and ultimately leakage of cellular components. Although C. albicans, Candida krusei and Candida glabrata infrequently exhibit resistance to amphotericin B, species such as Candida lusitaniae and Candida guilliermondii are intrinsically resistant to the drug.4,5 Acquired resistance to amphotericin B has been reported in C. albicans, often in conjunction with resistance to the azole antifungal agents.6
Fluconazole and other azoles are fungistatic and inhibit the cytochrome P450 enzyme lanosterol demethylase (encoded by the ERG11 gene), a key enzyme in ergosterol biosynthesis.7 Additionally, C-22 sterol desaturase, another cytochrome P450 enzyme encoded by the ERG5 gene, also appears to be a target of azole antifungal compounds.8 There are several well-documented examples in the literature of acquisition of azole resistance in clinical isolates of C. albicans.913 Moreover, many non-albicans species of Candida are either intrinsically resistant to fluconazole or acquire a resistant phenotype at a greater frequency.14,15
Several studies have focused on identifying mechanisms of resistance to amphotericin B and fluconazole in C. albicans.9,11,12,1620 To date, resistance to fluconazole has been shown to involve two general mechanisms: increased expression of drug efflux pumps and alterations in genes that encode enzymes in the ergosterol biosynthesis pathway. Drug efflux pumps demonstrated to be overexpressed in some resistant isolates are ATP-binding cassette transporters encoded by CDR1 and CDR2 and a major facilitator encoded by MDR1 (also known as BMR1) in C. albicans.9,11,1,12, A commonly altered ergosterol biosynthesis enzyme is the azole target Erg11p. This experiences amino acid substitutions that result in lowered affinity of azoles for the enzyme or is overexpressed at levels that overwhelm the drug's ability to inhibit growth.16,21
Another mechanism by which antifungal resistance can occur is through changes in other components of the ergosterol biosynthesis pathway that result in the accumulation of sterol intermediates.22 In clinical isolates of C. albicans, combined resistance to both amphotericin B and azoles has been associated with accumulation of ergosta-7,22-dienol. This is consistent with reduced activity of C-5 desaturase, encoded by ERG3.6,23,24 Such changes in ERG3 are also thought to reduce the conversion of episterol to potentially toxic metabolites that accumulate in the cell membrane during azole exposure.22
In the present study, we generated a strain of C. albicans that is resistant to both amphotericin B and fluconazole by serially passaging C. albicans strain SC5314 in increasing concentrations of amphotericin B until resistance was achieved. Gene expression in the resulting resistant strain SC5314-AR and its parent strain SC5314 was compared for over 6000 genes by microarray analysis. This analysis identified the differential expression of genes involved in ergosterol biosynthesis, cell stress and resistance to other inhibitors of the ergosterol biosynthesis pathway. Analysis of sterol content in the strains confirmed the hypothesis that resistance to fluconazole and amphotericin B was the result of accumulation of sterol intermediates consistent with inactivation of lanosterol demethylase, and potentially due, in part, to the increased expression of several ergosterol biosynthesis genes.27
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Materials and methods |
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Cultures of C. albicans SC5314 were passed in increasing concentrations of amphotericin B. Specifically, the culture of SC5314 was initiated by adding 100 µL of glycerol stock to 15 mL of 0.25 mg/L amphotericin B in YPD broth (1% yeast extract, 2% peptone, 2% dextrose). Cultures were grown at 30°C in an environmental shaking incubator, and cells were passed when cultures were turbid (13 days between passages). With each passage, the concentration of amphotericin B in YPD broth was doubled until the concentration used for the final passage was 128 mg/L. Aliquots of the parent strain and the final strain SC5314-AR were stored at 70°C as glycerol stocks.
Determination of stability of the antifungal resistant phenotype
Stability of the resistant phenotype was tested by growing the SC5314-AR strain in 10 mL of YPD broth (without drug) for a total of 60 doublings. Once every 24 h, an aliquot of the previous day's saturated culture was added to 10 mL of fresh YPD broth to an OD600 of 0.2, the culture was grown to an OD600 of 0.60.8, an aliquot was taken to prepare a glycerol stock and the remainder of the culture was allowed to grow to saturation until the next day. The MICs of amphotericin B and fluconazole were then determined for each of the glycerol stocks collected.
Susceptibility testing of C. albicans isolates
MICs were determined with fluconazole [(FLU) 0.016256 mg/L] and amphotericin B [(AMB) 0.00232 mg/L] Etest strips (AB Biodisk North America Inc., Piscatway, NJ, USA), with solidified (1.5%) 100 mm RPMI + MOPS agar plates serving as the medium. A standardized cell suspension (0.5 McFarland) in sterile 0.85% NaCl was prepared by transferring three to four colonies from a 24 h culture onto potato dextrose agar (Remel, Lenexa, KS, USA). Plates were then inoculated by pouring 5 mL of the standardized cell suspension onto the agar. After allowing the suspension to achieve a uniform distribution, moisture was aspirated with a vacuum pipette and the plates allowed to dry at ambient temperature for 15 min. Antifungal strips were then placed onto the agar. Plates were then inoculated at 37°C and MICs recorded at 24 h. The susceptibility endpoint for fluconazole was read at the intersection of the first discernable growth-inhibition ellipse, whereas that of amphotericin B was read at the intersection of the scale with the first completely clear ellipse.
RNA isolation
For each of two independent sets of cultures, an aliquot of glycerol stock from each isolate was diluted in YPD broth and grown overnight at 30°C in an environmental shaker. Cultures were diluted to an OD600 of 0.10.2 in 200 mL of fresh YPD (1% yeast extract, 2% peptone, 2% dextrose) and grown at 30°C for 3 h for subsequent RNA isolation. RNA was isolated using the hot phenol method.25 Briefly, cells were collected by centrifugation and snap-frozen in liquid nitrogen. Frozen cells were then resuspended in 12 mL of AE buffer (50 mM sodium acetate pH 5.2, 10 mM EDTA) at room temperature, after which 800 µL 25% SDS and 12 mL of acid phenol (pH 4.55.5; Fisher Scientific) were added. The cell lysate was then incubated for 10 min at 65°C with vortexing each min, snap-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 isopropanol and 0.1 volume 2 M sodium acetate pH 5, mixing well, and subjecting the mixture to centrifugation at 17 211 g for 35 min at 4°C. The supernatants were removed, the pellet resuspended in 10 mL of 70% ethanol and RNA collected by centrifugation at 17 211 g for 20 min at 4°C. Supernatants were again removed, and RNA was resuspended in 0.51 mL of diethylpyrocarbonate (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% agaroseMOPS gel.
Microarray design and preparation
The C. albicans microarray was manufactured by Eurogentec SA in collaboration with the European Galar Fungail Consortium (www.pasteur.fr/recherche/unites/Galar_Fungail/). Primers for each of the 6039 putative ORFs in the C. albicans genome were designed to amplify a specific region of each ORF. Amplicons were an average length of 300 bp and were spotted in duplicate, along with 27 control genes, using a ChipWriter Pro robotic array printer.
Probe preparation and microarray hybridization
Ten mg of total RNA sample was added to a mixture of T20VN and Oligo(dT) primer mix; dNTPs including Cy3- or Cy5-dCTP; and DTT in a buffer containing Tris-HCl, KCl and MgCl2. The reaction mixture was denatured at 65°C for 5 min and incubated at 42°C for 5 min, after which Rnasin and Superscript II reverse transcriptase (RT) were added to the mixture. The reaction proceeded at 42°C for 1 h, after which additional Superscript II RT was added, and the reaction mixture incubated at 42°C for an additional hour. To stop the reaction, EDTA and NaOH were added, the mixture was incubated at 65°C for 20 min and acetic acid was added. Five microlitres each of the Cy3- and Cy5-labelled probes were mixed with heat-denatured salmon sperm DNA, incubated at 95°C for 2 min and snap-cooled. The mixture was added to hybridization buffer and applied to the array slides under glass coverslips. Hybridization was performed at 37°C overnight in a humidified chamber. To wash the slides, the coverslip was removed and the slide incubated at room temperature in 0.2 x SSC (20 x SSC stock consists of 3 M sodium chloride, 0.3 M sodium citrate) + 0.1% SDS for 5 min, rinsed at room temperature with 0.2 x SSC for 5 min and spin-dried for 5 min. Slides were scanned using a ChipReader microarray scanner.
Data analysis
GenePix 1.0 software was used for image analysis. The local background values were calculated from the area surrounding each feature and subtracted from the total feature signal values. These adjusted values were used to determine differential gene expression for each feature. 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. Only features with a mean balanced differential expression ratio 1.5 or
1.5 (increased or decreased by 50%) for both features 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 BLASTn searches using the Stanford database (http://www-sequence.stanford.edu/group/candida), GenBank, and the CandidaDB database (http://www.pasteur.fr/Galar_Fungail/CandidaDB/).
cDNA synthesis and RT-PCR
cDNA was synthesized using the protocol and reagents from Invitrogen Corp. (Carlsbad, CA, USA). Briefly, 2 µg of total RNA from samples not used in microarray hybridization was mixed with 2 µL random hexamers (50 ng/mL) in 10 µL of 1 mM dNTP (equimolar solution of dATP, dCTP, dGTP and TTP) solution. The mixture was incubated at 65°C for 5 min then snap-cooled on ice. A reaction mixture was added to the denatured RNA to give the following final concentrations: RT buffer [20 mM Tris-HCl (pH 8.4), 50 mM KCl], 5 mM MgCl2, 0.01 M dithiothreitol (DTT), and 2 U RNaseOUT Recombinant Ribonuclease Inhibitor. After a 2 min incubation at 25°C, 50 U Superscript II RT was added and the reaction incubated at 25°C for an additional 10 min. The reaction was then allowed to proceed at 42°C for 50 min followed by termination at 70°C for 15 min and snap-cooling on ice. Finally, 2 U RNase H was added and the reaction incubated for 20 min at 37°C.
PCR was performed 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 of each forward and reverse primer, 2.5 U Taq polymerase (Sigma, St. Louis, MO, USA) 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 1 min, gene-specific annealing temperature for 1 min and 72°C for 2 min; and one repetition of 72°C for 5 min. The amount of cDNA used per reaction was based upon the volume of each cDNA sample required to normalize the intensity of 18S rRNA PCR products. Equivalent volumes of PCR product were applied to a 3% agarose gel and separated by gel electrophoresis in 1 x TAE (50 x TAE stock consists of 2 M Tris-acetate, 0.05 M EDTA). Primer sequences used in PCR are listed in Table 1.
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Sterols were isolated as previously described and analysed by gas chromatography.26 Sterol analysis was performed using a DB-5 capillary column (15 m x 0.25 mm x 0.25 µm; J&W Scientific, Folsom, CA, USA) and an HP5890 Series II gas chromatograph equipped with Hewlett-Packard CHEMISTATION software. The gas chromatograph was programmed from 195280°C (1 min at 195°C, then an increase at 20°C/min until 240°C and from 240280°C at 2°C/min). The linear velocity was 30 cm/s using nitrogen as the carrier gas. All injections were run in the spitless mode.
Gas chromatography/mass spectrometry (GC/MS) analyses of sterols were performed using a Thermoquest Trace 2000 gas chromatograph interfaced to a Thermoquest Voyager mass spectrometer. The GC separations were performed on a DB-5MS fused silica column (20 m x 0.18 mm x 0.18 µm film thickness; J&W Scientific). The injector temperature was 190°C, whereas the oven temperature was programmed to remain at 100°C for 1 min followed by a temperature ramp of 6.0°C/min to a final temperature of 300 °C. The temperature was held at 300°C for 25 min. Helium was the carrier gas with a linear velocity of 50 cm/s in the spitless mode. The mass spectrometer was operated with the following settings: electron impact ionization mode at an electron energy of 70 eV, scanning from 40850 atomic mass units at 0.6 s intervals, and an ion source temperature of 150°C.
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Results |
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Microarray analysis revealed 133 genes differentially expressed by at least 50% in SC5314-AR, with 27 genes up-regulated and 106 genes down-regulated (Tables 2 and 3). Additionally, differential expression of several genes of interest found in the data set (ERG5, ERG6, ERG25, DDR48, RTA2, UBI4, and FTR1) was validated by RT-PCR and is shown in Figure 2.
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Discussion |
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In addition to ERG5, ERG6 and ERG25, the stress response gene DDR48, iron transport genes FTR1 and FET34, the hexose transporters IPF3282, HGT11 and HGT12, RBT5 and RTA2 are among those found to be up-regulated in SC5314-AR. The role of these genes in either azole or amphotericin B resistance remains unclear; however, the Saccharomyces cerevisiae homologue of RTA2, ScRTA1, has been shown to confer resistance to 7-aminocholesterol, which exerts its activity by inhibiting the ERG2 and ERG24 gene products.28 Previous studies from our laboratory examining gene expression profiles of azole-resistant clinical isolates of C. albicans found DDR48, a gene putatively involved in stress response, to be up-regulated.19,20 Similarly, RTA3, which shares the same S. cerevisiae homologue to RTA2 found in the present study, is up-regulated in azole resistance in C. albicans.19,20 Further study of these genes in the context of antifungal resistance is warranted.
Of the 106 genes down-regulated in SC5314-AR, the large majority are involved in protein synthesis (Table 3). These protein synthesis genes, the histone genes HTB1, HHT21, HHF21, HHF22 and the energy generation genes ATP7, COX9, TIM11, IPF11271 and IPF14452 are probably observed to be down-regulated due to the fact that SC5314-AR grows at a much slower rate than SC5314. Interestingly, the differences in growth rate are consistent with mutation, inactivation or deletion of ERG11 in SC5314-AR. Sanglard et al.27 documented much slower growth rates for erg11 deletion strains than for erg3 deletion and wild-type strains. Similar to our findings, they showed profound changes in sterol composition in the deletion mutants compared with the wild-type strain, as well as resistance to both fluconazole and amphotericin B in the mutant strains.
Other down-regulated genes in the present study include stress response genes GPX2 and CRD2, heat shock proteins HSP90, SSA1 and SSE1, and the polyubiquitin gene UBI4. CRD2 and GPX2-related gene GPX1 have been shown in previous studies to be differentially expressed in azole resistance; however, unlike this study, each is up-regulated.19,20 UBI4, previously found to be down-regulated in azole-resistant C. albicans isolates, is down-regulated in the present study as well.19,20
The strain SC5314-AR was passaged for 60 doublings in the absence of drug to determine whether the strain was stably resistant to amphotericin B and fluconazole. Upon measuring susceptibility of cells tested at each passage, it was determined that the strain lost its phenotype sometime after the third passage (
28 doublings). However, in shorter-term cultures in the absence of drugsuch as those from which RNA was obtained for the initial microarray experiments and follow-up RT-PCR, the MICs of the antifungals were obtained, and the sterol analysis was performedthe MICs remained stable.
This study examines changes in the gene expression profile of C. albicans in association with experimentally induced amphotericin B and fluconazole resistance. Whereas many of these changes appear to be associated with the cell growth rate, others may be more directly involved in the fluconazole- and amphotericin B-resistant phenotype of, and are consistent with the altered sterol profile obtained for, SC5314-AR. Although an experimentally induced resistant strain is used in this study, comparison of SC5314-AR to its parent strain by microarray and sterol analyses offers insight into the adaptations the organism is capable of making in order to achieve a resistant phenotype.
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Acknowledgements |
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Footnotes |
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