JAC Advance Access originally published online on May 30, 2006
Journal of Antimicrobial Chemotherapy 2006 58(2):434-438; doi:10.1093/jac/dkl221
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Proteomic analysis of experimentally induced azole resistance in Candida glabrata
1 Department of Pharmacy, College of Pharmacy, University of Tennessee Health Science Center Memphis, TN 38163, USA 2 Department of Pharmaceutical Sciences, College of Pharmacy, University of Tennessee Health Science Center Memphis, TN 38163, USA 3 Department of Pediatrics, College of Medicine, University of Tennessee Health Science Center Memphis, TN 38163, USA 4 Children's Foundation Research Center at Le Bonheur Children's Medical Center Memphis, TN 38103, USA 5 Department of Microbiology and Immunology, Drexel University College of Medicine Philadelphia, PA 19129, USA 6 Department of Molecular Sciences, College of Medicine, University of Tennessee Health Science Center Memphis, TN 38163, USA
*Correspondence address. Children's Foundation Research Center of Memphis, Le Bonheur Children's Medical Center, 50 North Dunlap Street, Room 304 West Patient Tower, Memphis, TN 38103, USA. Tel: +1-901-572-5387; Fax: +1-901-448-1741; E-mail: drogers{at}utmem.edu
Received 22 February 2006; returned 5 April 2006; revised 3 May 2006; accepted 5 May 2006
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Abstract |
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Objectives: The aim of the present study was to identify changes in the proteome of a laboratory-derived azole-resistant strain of Candida glabrata compared with its susceptible parent strain in an effort to identify proteins that are differentially expressed in association with azole resistance.
Methods: Soluble and membrane protein fractions were isolated from mutant strain F15 (fluconazole MIC > 128 mg/L) and parent strain 66032 (fluconazole MIC = 16 mg/L) grown to mid-log phase. Soluble proteins were resolved by both two-dimensional (2D) and one-dimensional (1D) polyacrylamide gel electrophoresis (GE) whereas membrane proteins were resolved by 1D GE. Spots or bands representing differentially expressed proteins were identified by matrix-assisted desorption ionization-time of flight mass spectroscopy (MALDI-TOF MS) and peptide mass fingerprinting.
Results: A total of 22 proteins were found to be more abundantly represented, and 3 proteins were found to be less abundantly represented, in strain F15 compared with strain 66032. These included up-regulation of the ATP-binding cassette transporter Cdr1p, the ergosterol biosynthesis enzyme Erg11p, proteins involved in glycolysis and glycerol metabolism, and proteins involved in the response to oxidative stress and cadmium exposure.
Conclusions: In addition to transcriptional regulation of Cdr1p, this study identified the differential expression of several proteins that may contribute to azole resistance and suggests the possibility for a post-transcriptional mechanism for increased expression of Erg11p.
Keywords: lanosterol demethylase , efflux pumps , antifungals
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Introduction |
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Candida glabrata has emerged as a major cause of mucosal and invasive fungal infection in the United States, second only to Candida albicans.1 Fluconazole and other azoles have proven effective for the management of Candida infections; however, C. glabrata exhibits intrinsically low susceptibilities to the azole antifungals.2 Furthermore, acquired azole resistance has recently been reported in oral isolates of C. glabrata from AIDS patients, haematopoietic stem cell transplant recipients, and patients receiving radiation for head and neck cancer.3,4
The azole antifungal agents exert their activity by inhibiting the biosynthesis of ergosterol, the major membrane sterol of fungi. Specifically the azoles inhibit the activity of the cytochrome P450 enzyme, lanosterol demethylase, by binding to the haem in its active site. While the molecular basis for the intrinsically low susceptibility of C. glabrata remains unclear, several mechanisms of acquired resistance to the azole antifungal agents have been described in C. glabrata. These include up-regulation of genes encoding ATP-binding cassette (ABC) transporters encoded by CDR1 and PDH1 (also known as CDR2).5 Overexpression of these efflux pumps is presumed to prevent accumulation of sufficient effective concentrations of the azole antifungal agent in the fungal cell. Overexpression of the gene encoding the target of the azole antifungal agents, ERG11, has also been associated with acquired azole resistance.6 This presumably results in increased production of lanosterol demethylase, which exceeds the capacity of the azole antifungal agent to inhibit this enzyme.
In the present study we examined changes in the C. glabrata proteome of a laboratory-derived matched set of strains representing the acquisition of azole antifungal resistance. Our findings confirm changes in expression at the protein level for the azole resistance-associated gene CDR1 in this set. Furthermore, several proteins, including the target of the azole antifungal agents Erg11p, are newly identified as being differentially expressed in association with azole resistance in these strains and may contribute to this process.
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C. glabrata strains and growth conditions
C. glabrata strain 66032 (fluconazole MIC = 16 mg/L) and laboratory-derived azole-resistant mutant F15 (fluconazole MIC > 128 mg/L) were used in the study. For each of three independent experiments, an aliquot of glycerol stock from each isolate was diluted in YPD broth (1% yeast extract, 2% peptone, 1% dextrose) and grown overnight at 30°C in an environmental shaking incubator. Cultures were diluted to an optical density at 600 nm of 0.2 in 0.5 L of fresh YPD and grown as before to logarithmic phase (4.5 h) to an equivalent optical density.
Protein isolation
For isolation of soluble proteins, cells were washed in deionized distilled H2O and resuspended in 50 mM Tris–HCl with protease inhibitors. Cells were then disrupted using glass beads and a bead beater and placed on ice. After an initial centrifugation at 11 250 g at 4°C for 45 min, supernatants were collected and subjected to the same centrifugation conditions for 30 min. This supernatant was subjected to ultracentrifugation for 30 min at 440 000 g. The resulting supernatant contained the soluble protein fraction. Proteins from the resuspended pellet contained the less soluble protein fraction. Membrane proteins were isolated as described by Niimi et al.7 The protein content of the desalted and concentrated samples was measured using the Pierce-modified Bradford method.
One-dimensional (1D) and two-dimensional (2D) gel electrophoresis (GE) and imaging
For 2D-PAGE, samples were loaded onto a pH 3–10, NL, immobilized gradient 18 cm Immobiline Drystrip (Amersham Pharmacia Biotech, Uppsala, Sweden) and focused with an IPGphor isoelectric focusing (IEF) Unit (Amersham Pharmacia Biotech, San Francisco, CA, USA). The IEF strips were then equilibrated and loaded onto a 12% acrylamide gel. The proteins were electrophoresed (200 V, 7 h) with a Protein-Plus Dodeca Cell (Bio-Rad Laboratories, Inc, Hercules, CA, USA). For membrane proteins and proteins from the less soluble protein fraction, protein samples were separated by electrophoresis in 7.5% SDS–polyacrylamide gels. The 1D- and 2D-PAGE-separated proteins were stained with Coomassie Blue. Gels were washed and scanned (300 dpi resolution) with an Epson Expression 800 scanner (Epson, Singapore) and Photoshop (version 6.2.1, Adobe Systems Incorporated, San Jose, CA, USA) software. For 2D-PAGE, gel images were analysed and spots detected with PDQuest (version 7.1) 2D gel analysis software (Bio-Rad Laboratories). Spots were considered to represent differentially expressed proteins if they were up- or down-regulated 1.5-fold in three independent experiments. For 1D gels, bands were visually examined for reproducible differential expression. Differentially expressed proteins were selected for identification.
Protein identification
Protein spots or bands were excised from stained gels. Proteins were digested in the gel with sequencing-grade trypsin (Promega, Madison, WI, USA). The resulting peptides were extracted, and samples were subjected to analysis by mass spectrometry. Mass spectra were recorded on a Bruker Ultraflex MALDI-ToFToF reflecting time-of-flight mass spectrometer (Bruker Daltonics, Bremen, Germany). PROWL software (formerly Proteometrics, Inc.) was used to search a custom C. glabrata proteome database constructed from the The Génolevures Consortium database (Build 2, http://cbi.labri.fr/Genolevures/about.php). A Z-score of 1.65 ranks the search result in the 95th percentile of non-random matches of the mass dataset to the specific open reading frame.
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In order to identify differences in protein expression between the azole-resistant strain F15 and its parent strain 66032, we first examined differences in protein profiles between cellular protein extracts made from cultures of the two strains. The soluble protein fraction of these preparations was subjected to 2D GE, whereas the fraction representing less soluble proteins was subjected to 1D GE. The 2D GE (Figure 1) shows that in the soluble protein fraction the expression of 13 proteins was greater in strain F15 compared with 66032, whereas the expression of 3 proteins was greater in strain 66032 compared with F15. The 1D GE (Figure 2) shows that in the less soluble protein fraction the expression of six protein bands was greater in strain F15 compared with 66032. We also examined differences by 1D GE between the protein profiles of plasma membranes of these two isolates. Figure 2 shows that the expression of three protein bands was greater in F15 compared with 66032.
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Discussion |
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Both 1D and 2D GE have previously been used successfully to study drug resistance and drug activity in C. glabrata. Marichal et al.6 found at least 101 proteins to be differentially expressed between a matched azole-susceptible isolate and azole-resistant isolate with chromosomal duplication and increased ERG11 expression. This study, however, preceded the use of high throughput protein identification techniques and the sequencing of the C. glabrata genome, precluding the identification of these proteins. More recently Niimi et al.7 demonstrated the up-regulation of Cdr1p and Erg11p in C. glabrata in response to fluconazole exposure using 1D GE analysis of plasma membrane proteins.
Strain F15 has previously been shown to overexpress CDR1 and, to a lesser extent, PDH1.5 In the present study we identified two up-regulated protein bands that were in the molecular weight range of these ABC transporters; however, both bands were identified by MALDI-TOF MS as Cdr1p. We did not observe overexpression of a band representing Pdh1p in the membrane fraction of strain F15. Detection of two Cdr1p bands suggests differences in post-translational modification, such as glycosylation, phosphorylation or proteolytic processing.
We also observed the overexpression of Erg11p in azole-resistant strain F15. This critical enzyme in the ergosterol biosynthesis pathway represents the major target of the azole antifungal agents. Up-regulation of ERG11 mRNA is a documented mechanism of azole resistance in C. albicans, and up-regulation of this gene in azole-resistant C. glabrata strains has been reported.4 However, we have shown previously that expression of ERG11 mRNA is the same in strains 66032 and F15.5 This raises the possibility of post-transcriptional regulation of this enzyme.
Homologues of several proteins found to be up-regulated in strain F15 have also been shown to be induced in Saccharomyces cerevisiae in response to oxidative stress and/or cadmium exposure and appear to be under the control of the transcription factor Yap1p.8 These include Shm2p, Eno1p, Pgk1p, Tsa1p, Ahp1p and Ynl134cp. Miconazole and fluconazole have been shown to induce the production of endogenous reactive oxygen species (ROS) in Candida species in a dose-dependent fashion.9 Both azole-induced ROS and Candida cell death was attenuated by the antioxidant pyrrolidinedithiocarbamate. Furthermore, in multiple clinical isolates, azole-induced ROS production inversely correlated with the azole MIC. These data suggest a role for ROS production in the mechanism of action of the azole antifungal agents. Indeed, isolates of C. glabrata lacking the ERG11 gene have been shown to be more susceptible to oxidative effects of neutrophils and H2O2 than isolates with ERG11 intact.10 It is therefore possible that pharmacological inhibition of the ERG11 gene product, lanosterol demethylase, with an azole antifungal agent may produce a similar effect. This suggests that imparting to the fungal cell such enhanced susceptibility to oxidative damage may be part of the mechanism of action of the azole antifungals. Up-regulation of proteins involved in the Yap1p-dependent oxidative stress response in the present study is consistent with this hypothesis.
We have confirmed the up-regulation of the CDR1 product in a laboratory-derived azole-resistant C. glabrata isolate. Furthermore, we have demonstrated the up-regulation of proteins involved in ergosterol biosynthesis, glycolysis and glycerol catabolism, and the Yap1p-dependent stress response in association with azole resistance. Further study of the post-transcriptional mechanism by which Erg11p is constitutively overexpressed, as well as the direct role of these proteins in the azole resistance phenotype in clinical isolates of C. glabrata, is warranted.
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None to declare.
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Acknowledgements |
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We thank Massoumeh Z. Hooshdaran for technical assistance associated with this work.
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References |
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1 Bodey GP, Mardani M, Hanna HA, et al. (2002) The epidemiology of Candida glabrata and Candida albicans fungemia in immunocompromised patients with cancer. Am J Med 112:380–5.[CrossRef][ISI][Medline]
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Bennett JE, Izumikawa K, Marr KA. (2004) Mechanism of increased fluconazole resistance in Candida glabrata during prophylaxis. Antimicrob Agents Chemother 48:1773–7.
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Redding SW, Kirkpatrick WR, Saville S, et al. (2003) Multiple patterns of resistance to fluconazole in Candida glabrata isolates from a patient with oropharyngeal candidiasis receiving head and neck radiation. J Clin Microbiol 41:619–22.
5 Vermitsky JP, Earhart KE, Smith L, et al. (2006) PDR1 regulates multidrug resistance in Candida glabrata: gene disruption and genome-wide expression studies. Mol Microbiol in press.
6 Marichal P, Vanden Bossche H, Odds FC, et al. (1997) Molecular biological characterization of an azole-resistant Candida glabrata isolate. Antimicrob Agents Chemother 41:2229–37.[Abstract]
7 Niimi M, Nagai Y, Niimi K, et al. (2002) Identification of two proteins induced by exposure of the pathogenic fungus Candida glabrata to fluconazole. J Chromatogr B Analyt Technol Biomed Life Sci 782:245–52.[CrossRef][ISI][Medline]
8
Vido K, Spector D, Lagniel G, et al. (2001) A proteome analysis of the cadmium response in Saccharomyces cerevisiae. J Biol Chem 276:8469–74.
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Kobayashi D, Kondo K, Uehara N, et al. (2002) Endogenous reactive oxygen species is an important mediator of miconazole antifungal effect. Antimicrob Agents Chemother 46:3113–7.
10 Kan VL, Geber A, Bennett JE. (1996) Enhanced oxidative killing of azole-resistant Candida glabrata strains with ERG11 deletion. Antimicrob Agents Chemother 40:1717–9.[Abstract]
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