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Antimicrobial Agents and Chemotherapy, July 2004, p. 2700-2703, Vol. 48, No. 7
0066-4804/04/$08.00+0     DOI: 10.1128/AAC.48.7.2700-2703.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Constitutive Activation of the PDR16 Promoter in a Candida albicans Azole-Resistant Clinical Isolate Overexpressing CDR1 and CDR2

Xavier De Deken and Martine Raymond^*

Institut de Recherches Cliniques de Montréal, Montréal, Québec, Canada H2W 1R7

Received 4 December 2003/ Returned for modification 7 January 2004/ Accepted 8 March 2004

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Candida albicans azole-resistant clinical isolates overexpressing the CDR1 and CDR2 genes (multidrug transporters) also overexpress the PDR16 gene (phosphatidylinositol transfer protein). We show here that the PDR16 promoter displays higher transcriptional activity following integration in an azole-resistant isolate than in the matched azole-susceptible one. Thus, the upregulation of PDR16 in the resistant strain results from a mutation acting in trans.

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Many Candida albicans azole-resistant clinical isolates overexpress the CDR1 and CDR2 genes encoding multidrug transporters of the ATP-binding cassette (ABC) superfamily (10). The overexpression of CDR1 and CDR2 in these strains is concomitant and stable and most likely results from activating mutations occurring in a transcription factor controlling the expression of both genes (2). We recently found that clinical isolates overexpressing CDR1 and CDR2 also overexpress the orthologue of the PDR16 gene, which encodes a phosphatidylinositol transfer protein involved in azole tolerance in the budding yeast Saccharomyces cerevisiae (7, 8; S. Saidane, S. Weber, X. De Deken, G. St-Germain, and M. Raymond, submitted for publication). We also found that PDR16 expression is coinduced with CDR1 and CDR2 upon fluphenazine (FPZ) treatment, suggesting that the three genes belong to the same transcriptional network (S. Saidane et al., submitted). These findings implied that the upregulation of PDR16 in azole-resistant strains results from a trans-acting mutation. To address this question, we have cloned and functionally characterized the PDR16 promoter in C. albicans experimental and clinical strains.

(This work was presented in part [abstr. M-397] at the 43rd Interscience Conference on Antimicrobial Agents and Chemotherapy, Chicago, Ill., 14 to 17 September 2003.)

The PDR16 gene is present in two different contigs in the SC5314 genome database (contig19-20087 and contig19-10087; Stanford Genome Technology Center, http://www-sequence.stanford.edu/group/candida/index.html), which correspond to the two alleles of PDR16. The –1.2-kb upstream sequences from the two PDR16 alleles (PDR16prom1, –1218 to –1 bp; and PDR16prom2, –1202 to –1 bp) (positions relative to the translation start site set at +1) were retrieved and analyzed by using the TRANSFAC database (11). This analysis revealed the presence of putative cis-acting regulatory elements, such as a TATA box, a CCAAT box, a yeast AP-1 binding site (YRE), and two GATA binding sites (GATA) (Fig. 1). We also identified four putative binding sites for transcription factors of the zinc cluster family, which are widely implicated in azole resistance in S. cerevisiae (1, 5, 6) (ZCB1 to -4; Fig. 1). These factors bind to two CGG triplets present in a direct, inverted, or everted orientation with variable spacing (4, 9). The CDR1 and CDR2 promoters contain a 21-bp conserved drug-responsive element (DRE) responsible for their constitutive upregulation in an azole-resistant clinical isolate that contains two CGG triplets in the direct orientation (2). No exact match for the 21-bp DRE sequence was found in the PDR16 promoter, suggesting differences between the regulation of PDR16 expression and that of CDR1 and CDR2 expression. The two PDR16 promoter alleles are highly similar (98% identity), with a few base differences scattered along the sequences and one gap of 18 nucleotides upstream of the putative TATA box (Fig. 1). Since these polymorphisms could give rise to different transcriptional activities, we cloned and characterized the two alleles of the PDR16 promoter, using the CaEGFP reporter and mycophenolic acid resistance (MPA^R) selection system (12).

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FIG. 1. Alignment of the 1.2-kb promoter regions of the two PDR16 alleles. The primers with the restriction sites used for the cloning of PDR16prom1 and PDR16prom2 are indicated by arrows. Putative TATA and CCAAT boxes along with a number of potential cis-regulatory elements are identified. YRE, yeast AP-1 response element; GATA, GATA binding site; ZCB, zinc cluster binding site; +1, translation start site.

A DNA fragment containing the CaEGFP gene, the MPA^R marker, and the 3'-CDR4 region was PCR amplified from plasmid pGFP54 (12), using primers GFPf and 3CDR4r, which introduce XhoI and SstII sites, respectively (Table 1). The resulting fragment was cloned into the pBluescriptII-KS vector (Stratagene) cut with XhoI and SstII, yielding plasmid pCaEGFP1. The 5'-CDR4 region was PCR amplified from pGFP54 by using primers 5CDR4f and 5CDR4r, introducing ApaI and XhoI sites, and was inserted between the ApaI-XhoI sites of pCaEGFP1, producing plasmid pCaEGFP2 (primer 5CDR4r also introduces a NotI site for promoter cloning) (Table 1). The two PDR16 promoter alleles were obtained from SC5314 genomic DNA by PCR amplification with primers PDR16f and PDR16r, introducing NotI and XhoI sites, and cloned between the NotI and XhoI sites of pCaEGFP2, yielding plasmids pCaEGFP3 (PDR16prom1) and pCaEGFP4 (PDR16prom2). The two cloned promoter alleles were verified by automated DNA sequencing. The promoter cassettes were released by digestion of pCaEGFP3 and pCaEGFP4 with ApaI and SstII (Fig. 2A) and transformed into SC5314 by the lithium acetate method (3). A promoterless cassette was similarly processed with pCaEGFP2. Integrants were selected based on their MPA^R phenotype (12) and analyzed by Southern blotting for integration of the cassettes at the CDR4 locus, using a probe corresponding to the 3' region of CDR4 (positions 2818 to 4901 in CDR4) (Fig. 2B). In SC5314, the probe detected two HindIII restriction fragments of 8.1 and 3.0 kb, suggesting that the CDR4 locus contains a polymorphic HindIII restriction site. Correct integration at the CDR4 locus occurred in the three clones (CA01, control; CA02, PDR16prom1; CA03, PDR16prom2), as judged from the disappearance of the 8.1-kb HindIII fragment and the appearance of a new 4.8-kb HindIII fragment hybridizing with the probe. The three clones also exhibited an additional band of 12.0 kb, suggesting that the cassettes were also integrated at another locus. Nevertheless, the integrants displayed the same integration profile and copy number, therefore allowing a quantitative comparison among the clones.

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TABLE 1. Primer combinations used to generate the pCaEGFP constructs used in this study

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FIG. 2. Constitutive and inducible activity of the PDR16 promoter. (A) Integration of the PDR16prom-CaEGFP fusions at the CDR4 locus. Restriction maps of the CDR4 locus are presented without (top) and with (bottom) insertion of the PDR16prom-CaEGFP fusion constructs. The ApaI and SstII restriction sites were used to release the integration cassette before transformation. The probe used to verify the correct integration is indicated by a thick line. (B) Southern blot analysis of HindIII-digested genomic DNA from the CaEGFP integrants. Hybridization with the labeled CDR4 probe yields two fragments of 8.1 and 3.0 kb for the wild-type strain (SC5314) and three fragments of 12.0, 4.8, and 3.0 kb for the integrants. The size of the hybridizing fragments is indicated on the left. (C) FACS analysis of the CaEGFP transformants. The cells were treated with 50 or 100 µM FPZ for 135 min at 30°C and analyzed by FACS (FACScan; Becton Dickinson). Each histogram represents a total of 104 events. The fold increase was calculated from the geometric log means of the fluorescence intensity for each curve. The values represent the means of two independent experiments performed in duplicate. CA01, promoterless construct; CA02, PDR16prom1; CA03, PDR16prom2.

Fluorescence-activated cell sorting (FACS) analysis of clones containing the PDR16prom1 (CA02) or PDR16prom2 (CA03) sequences displayed increased fluorescence intensities of 11.8 ± 0.2- and 10.9 ± 0.3-fold, respectively, when compared to control cells having integrated the empty vector (CA01) (Fig. 2C). These results showed that the PDR16 promoter possesses a basal transcriptional activity and that the two alleles are similarly active. FPZ treatment increased the fluorescence intensities of the CA02 and CA03 clones in a dose-dependent manner to a maximum of fivefold at 100 µM, indicating that (i) PDR16 induction by FPZ occurs at the transcriptional level and (ii) the –1.2-kb upstream sequences contain the cis-acting elements necessary for this induction (Fig. 2C).

We have recently characterized a matched pair of azole-susceptible (isolate 5457) and -resistant (isolate 5674) clinical isolates (S. Saidane et al., submitted). Isolate 5674 exhibits cross-resistance to fluconazole, ketoconazole, and itraconazole, which correlates with the constitutive overexpression of the CDR1 and CDR2 genes. We also showed that PDR16 is overexpressed in strain 5674. To investigate the molecular mechanism responsible for PDR16 upregulation in this strain, the PDR16prom-CaEGFP fusion cassettes were integrated at the CDR4 locus in strains 5457 and 5674. As shown in Fig. 3A, strains 5457 and 5674, unlike SC5314, gave rise to a single HindIII fragment of 8.1 kb hybridizing with the CDR4 probe. All the integrants exhibited an 8.1-kb fragment and a 4.8-kb fragment, a pattern corresponding to the correct integration of the cassette only at the CDR4 locus. Using FACS analysis, we compared the transcriptional activities of the two PDR16 promoter alleles in the 5457- and 5674-derived integrants. Our results showed that 5457 clones carrying the PDR16prom1 and PDR16prom2 constructs display a slight but reproducible increase in fluorescence intensity (1.5-fold) as compared to the control cells carrying a promoterless cassette, demonstrating that the PDR16 promoter is active in strain 5457 (Fig. 3B). The lower activity of the PDR16 promoter in strain 5457 compared to that of SC5314 (Fig. 2C) could be a consequence of a lower level of PDR16 expression in 5457, as observed in Northern blot experiments (data not shown), and/or the integration of the cassette at two loci in strain SC5314, as opposed to only one locus in strain 5457. The two 5674 clones carrying the PDR16prom1 or PDR16prom2 constructs displayed increased fluorescence intensities of 3.7- and 5.3-fold, respectively, compared to those of the control cells, demonstrating a high constitutive activity of the two PDR16 promoter alleles in the 5674 strain (Fig. 3B). Since CDR4 is not overexpressed in strain 5674 (data not shown), we can conclude that the observed induction is specific for the PDR16 promoter and is not a consequence of the integration locus.

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FIG.3. High constitutive activity of the PDR16 promoter in azole-resistant strain 5674. (A) Southern blot analysis of HindIII-digested genomic DNA from the clinical isolate integrants. Hybridization with the labeled CDR4 probe yields one fragment of 8.1 kb for the wild-type strains (5457 and 5674) and one additional fragment of 4.8 kb for the integrants. The size of the hybridizing fragments is indicated on the left. (B) Activity of the PDR16 promoter alleles in the different integrants. The fold increase was calculated as for Fig. 2. The values represent the means of two independent experiments performed in duplicate. CA08 and CA09, 5457 + promoterless cassette; CA10 and CA11, 5457 + PDR16prom1; CA12 and CA13, 5457 + PDR16prom2; CA14, 5674 + promoterless cassette; CA15 and CA16, 5674 + PDR16prom1; CA17 and CA18, 5674 + PDR16prom2.

The fact that a PDR16 promoter sequence cloned from an azole-susceptible strain (SC5314) is activated in strain 5674 demonstrates that the constitutive overexpression of PDR16 in that strain results from a mutation acting in trans. Transcriptional dissection of the PDR16 promoter should enable identification of the cis-acting sequences and trans-acting factors regulating PDR16 expression. This knowledge could also yield important information regarding the regulation of CDR1 and CDR2 in azole-resistant isolates and the pleiotropic drug resistance network in C. albicans.

 
We thank Joachim Morschhäuser for providing the pGFP54 plasmid and Sandra Weber for excellent technical assistance and critical reading of the manuscript. The PDR16 sequence for C. albicans was obtained from the Stanford Genome Technology Center (SGTC) website at http://www-sequence.stanford.edu/group/candida.

Sequencing of C. albicans at the SGTC was accomplished with the support of the NIDR and the Burroughs Wellcome Fund. This work was supported by a research grant to M.R. from the Canadian Institutes of Health Research (MT-15679). X.D.D. is a Postdoctoral Fellow from the Fonds de la Recherche en Santé du Québec (FRSQ), and M.R. is an FRSQ Senior Scientist.

 
* Corresponding author. Mailing address: Institut de Recherches Cliniques de Montréal, 110 Pine Ave. West, Montréal, Québec, Canada H2W 1R7. Phone: 514-987-5770. Fax: 514-987-5764. E-mail: raymonm{at}ircm.qc.ca.

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Antimicrobial Agents and Chemotherapy, July 2004, p. 2700-2703, Vol. 48, No. 7
0066-4804/04/$08.00+0     DOI: 10.1128/AAC.48.7.2700-2703.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

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