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1 Institute of Microbiology, University Hospital Lausanne, Rue du Bugnon 48, CH-1011 Lausanne, Switzerland
2 Comenius University, Faculty of Natural Sciences, Department of Microbiology and Virology, 842 15 Bratislava, Slovak Republic
Correspondence
Dominique Sanglard
Dominique.Sanglard{at}chuv.ch
| ABSTRACT |
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-galactosidase reporter system. In an azole-susceptible strain, transcription of this reporter is transiently upregulated in response to either benomyl or H2O2, whereas its expression is constitutively high in an azole-resistant strain (FR2). Two cis-acting regulatory elements within the MDR1 promoter were identified that are necessary and sufficient to confer the same transcriptional responses on a heterologous promoter (CDR2). One, a benomyl response element (BRE), is situated at position 296 to 260 with respect to the ATG start codon. It is required for benomyl-dependent MDR1 upregulation and is also necessary for constitutive high expression of MDR1. A second element, termed H2O2 response element (HRE), is situated at position 561 to 520. The HRE is required for H2O2-dependent MDR1 upregulation, but dispensable for constitutive high expression. Two potential binding sites (TTAG/CTAA) for the bZip transcription factor Cap1p (Candida AP-1 protein) lie within the HRE. Moreover, inactivation of CAP1 abolished the transient response to H2O2. Cap1p, which has been previously implicated in cellular responses to oxidative stress, may thus play a trans-acting and positive regulatory role in the H2O2-dependent transcription of MDR1. A minimal BRE (290 to 273) that is sufficient to detect in vitro sequence-specific binding of protein complexes in crude extracts prepared from C. albicans was also defined. Interestingly, the sequence includes a perfect match to the consensus binding sequence of Mcm1p, raising the possibility that MDR1 may be a direct target of this MADS box transcriptional activator. In conclusion, while the identity of the trans-acting factors that bind to the BRE and HRE remains to be confirmed, the tools developed during this characterization of the cis-acting elements of the MDR1 promoter should now serve to elucidate the nature of the components that modulate its activity.
| INTRODUCTION |
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-lanosterol demethylase, can be elevated, (ii) the affinity of Erg11p for azole antifungal agents can be decreased, (iii) the ergosterol biosynthetic pathway can be altered by inactivation of the sterol
5,6-desaturase and (iv) cells can fail to accumulate these agents due to the upregulation of multidrug transporters belonging to the ABC transporters and the major facilitator (MF) families. Some azole-resistant strains upregulate the ABC transporters CDR1 (Candida drug resistance) and CDR2, while others upregulate MF MDR1 (multidrug resistance 1) (Sanglard & Odds, 2002
Overexpression and gene inactivation studies have shown that MDR1 confers specific resistance to fluconazole, benomyl, cycloheximide, benzotriazole, methotrexate, 4-nitroquinoline-N-oxide (4-NQO) and sulfomethuron methyl (Ben-Yaacov et al., 1994
; Fling et al., 1991
; Goldway et al., 1995
; Wirsching et al., 2000b
). Disruption of MDR1 causes an increased susceptibility to methotrexate, cycloheximide and 4-NQO (Goldway et al., 1995
; Sanglard et al., 1996
). However, its inactivation does not increase susceptibility to benomyl, suggesting that other mechanisms are involved in this resistance (Goldway et al., 1995
). MDR1 is usually not expressed at detectable levels in fluconazole-susceptible C. albicans isolates but its constitutive upregulation in some fluconazole-resistant isolates reveals a correlation between azole resistance and overexpression of MDR1 (Franz et al., 1998
; Sanglard et al., 1995
; White, 1997
; White et al., 1997
).
Little is known about the transcriptional regulation of MDR1 in C. albicans. However, a dramatic transient upregulation of MDR1 can be observed in azole-susceptible strains after treatment with toxic hydrophobic agents (benomyl, methotrexate, 4-NQO, o-phenanthroline), oxidizing agents [diamide, H2O2, tert-butyl hydrogen peroxide (T-BHP) and diethylmaleate] and the alkylating agent methyl methanesulfonate (Gupta et al., 1998
; Harry et al., 2005
). Clinical isolates with increased MDR1 expression have not undergone amplification of the MDR1 gene (Wirsching et al., 2000a
). Sequence analysis of MDR1 promoters from clinical isolates compared to wild-type promoters did not reveal cis-acting mutations (Harry et al., 2005
; Wirsching et al., 2000a
). Thus MDR1 upregulation in clinical strains is most likely due to transcriptional activation resulting from mutations of factors acting in trans (Wirsching et al., 2000a
). Deletion analysis of the MDR1 promoter, performed with a luciferase reporter gene, suggested that regulatory functions could be assigned to two separate regions: an element required for induction by benomyl was assigned to the region between 399 and 299 upstream of the start codon and another region located between 601 and 500 was responsible for MDR1 induction by the oxidative agent T-BHP.
In Saccharomyces cerevisiae, drug resistance is also mediated by membrane transport proteins belonging to the ABC transporter and major facilitator superfamilies and the different mechanisms controlling the expression of these multidrug resistant genes are relatively well understood (Sa-Correia & Tenreiro, 2002
; Taglicht & Michaelis, 1998
). The homologue of MDR1, FLR1, confers resistance to multiple drugs such as fluconazole, 4-NQO, cycloheximide, methotrexate and benomyl (Alarco et al., 1997
; Broco et al., 1999
; Nguyen et al., 2001
; Tenreiro et al., 2001
). Induction of FLR1 expression by the oxidizing agents H2O2, diamide, T-BHP and diethylmaleate, as well by the alkylating agent methyl methanesulfonate (Broco et al., 1999
; Jelinsky & Samson, 1999
; Nguyen et al., 2001
), is mediated by Yap1p (also called Pdr4p or Snq3p), a basic leucine zipper (bZip) transcription factor (Nguyen et al., 2001
; Tenreiro et al., 2001
). The direct targets of Yap1p, principally genes involved in oxidative stress, have a common promoter element, the palindromic YRE (Yap1p response element: TTAC/GTAA) (Fernandes et al., 1997
; Kuge & Jones, 1994
; Toone & Jones, 1999
).
In C. albicans, Cap1p, a bZip transcription factor structurally and functionally related to S. cerevisiae Yap1p, has been implicated in multidrug resistance and oxidative stress resistance (Alarco & Raymond, 1999
; Wang et al., 2006
; Zhang et al., 2000
). It was also shown to confer fluconazole resistance in S. cerevisiae by transcriptionally activating FLR1 (Alarco et al., 1997
), suggesting it has the same sequence specificity as Yap1p. Conversely, the C. albicans MDR1 promoter harbours the putative Cap1p-binding site (TTAGTAA at 532). Together these data indicate that mechanisms regulating MDR1 and its S. cerevisiae orthologue may be grossly similar. However, while in S. cerevisiae inactivation of Yap1p abolished FLR1 transcription (Tenreiro et al., 2001
), Alarco & Raymond (1999)
showed that disruption of CAP1 in the azole-resistant strain FR2 did not suppress but rather increased the level of MDR1 expression. In C. albicans Cap1p may act as both a repressor and an activator of MDR1 transcription, since experiments performed by Harry et al. (2005)
demonstrating the importance of the YRE in the T-BHP response suggest that Cap1p is an activator of MDR1 expression in response to oxidative stress.
In this study, we used a reporter system to explore the transcriptional regulation of MDR1 in azole-resistant and azole-susceptible strains. The system has identified two different cis-acting regulatory elements and has permitted us to locate them at high resolution. The first, located between 296 and 260, was responsible for dramatic MDR1 upregulation in an azole-susceptible strain exposed to benomyl as well as for high constitutive expression in an azole-resistant strain. The second, which harbours two putative binding sites for Cap1p, is located between 561 and 520. Like the trans-acting Cap1p, this cis-acting element was not required for high constitutive expression of MDR1 in an azole-resistant strain. However, the response of MDR1 to H2O2 was dependent upon the presence of both Cap1p and its binding site within the MDR1 promoter. The benomyl-dependent response was also partially dependent upon the presence of Cap1p, raising the possibility that interactions may occur between complexes bound at the two regulatory elements.
| METHODS |
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(Hanahan, 1985
was grown in LuriaBertani (LB) broth or LB agar plates supplemented with ampicillin (0.1 mg ml1) when required.
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Construction of promoterlacZ fusions.
To generate MDR1lacZ and CDR2lacZ fusions used in drug-induction assays, the 999 bp MDR1 and 903 bp CDR2 promoters were first amplified by PCR using the Taq DNA polymerase (Roche), with primers MDR1-5'-999-KPN and MDR1-3'-PST or primers CDR2-KPN and CDR2-PST (Table 3
). The generated fragments were cloned into pAU36 (Uhl & Johnson, 2001
) previously digested with KpnI and PstI. The resulting plasmids (pBR1 and pDS295, respectively) were digested with either NruI for the MDR1lacZ fusion or SnaBI for the CDR2lacZ fusion prior to transformation into C. albicans, resulting in their integration at the MDR1 and CDR2 loci, respectively.
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DNA amplification by PCR.
DNA fragments used in cloning steps or as probes were generated by PCR with the Taq DNA polymerase (Roche) following the instructions of the manufacturer. The reaction mixture (final volume 100 µl) was composed of 10 µl of 10x Taq DNA polymerase buffer (Roche), 10 µl of MgCl2 (Roche), 1 µl of each 100 µM forward and reverse primer, 4 µl of 10 mM deoxynucleoside triphosphates (Roche), 10 U of Taq DNA polymerase and 1 µl of 0.51 µg µl1 genomic DNA. PCR was performed under the following conditions: one cycle for 4 min at 94 °C, 1 min at 5460 °C and 2 min at 72 °C; 30 cycles for 30 at 94 °C, 1 min at 5460 °C and 2 min at 72 °C followed by one terminal cycle for 10 min at 72 °C. After analysis of 1/10 vol. of PCR products by electrophoresis on 1 % agarose gel, DNA fragments were purified with the QIAquick PCR purification kit (QIAGEN) and resuspended in 50 µl sterile water.
Southern blotting.
Southern blots were carried out as described previously (Sanglard et al., 1999
). DNA probes used in this study were generated by PCR using the following pairs of primers: for the verification of genomic insertion of pMM43-derived plasmids at the LEU2 genomic locus, CALEU2-3 (5'-CACCACCACTAAAACCCCAACAAATCA-3') and CALEU2-5 (5'-ATGTCTGTTAAAACCCAAACCATTACT-3'); for insertion of pAU36-derived plasmids at the MDR1 locus, MDR1-5'-501-KPN (5'-GCGCAAAGGTACCCAATTTTCATTTTAGGAAATTTACCGAGTTT-3') and LacZ-rev (5'-ATGTCTGTTAAAACCCAAACCATTACT-3'). Radioactive DNA probes were generated by random priming using the Megaprime DNA labelling kit (Amersham). Radioactive signals were revealed by exposure to Kodak BioMax MR film (Amersham Biosciences).
Electrophoretic mobility shift assays (EMSAs).
Protein extracts from C. albicans strain CAF2-1 were prepared from 2 l cultures in YEPD grown to mid exponential phase until the density reached 1.5x107 cells ml1. An equal quantity of cells was exposed to benomyl (35 µg ml1) for 30 min or left untreated as a control. The benomyl-treated or control cells were centrifuged for 20 min at 5500 r.p.m. and each cell pellet was washed with 10 ml extraction buffer [400 mM (NH4)2SO4, 10 mM MgCl2, 1 mM EDTA, 10 % (v/v), glycerol, 7 mM mercaptoethanol, 200 mM Tris/HCl pH 8]. Washed cells were centrifuged for 10 min at 5500 r.p.m. and the pellet obtained was resuspended in 10 ml extraction buffer supplemented with a protease inhibitor mix (Roche). Glass beads (10 g) were added to the cell suspension and the resulting mixture was disrupted for 5 min at 4 °C using a Macro bead-beater (Biospec Products). The homogenized solution was kept on ice for 30 min before centrifugation at 4 °C for 30 min at 13 000 r.p.m. The supernatant was transferred to a new tube and the cell pellet was reextracted with 5 ml fresh extraction buffer. The two supernatants were combined and the resulting solution was centrifuged for 1 h at 13 000 r.p.m. at 4 °C. The supernatant was recovered and ammonium sulfate was added to a final concentration of 0.3 g ml1. After dissolution of ammonium sulfate, the protein extract was centrifuged for 10 min at 13 000 r.p.m. and 4 °C. Precipitated proteins were resuspended in 250 µl protein buffer (20 mM HEPES, 5 mM EDTA, 7 mM mercaptoethanol, 1 mM PMSF and 20 % glycerol) supplemented with protease inhibitors (Roche). Protein concentrations of the crude extracts were determined using the Bradford method.
Double-stranded oligonucleotide probes were prepared by annealing 5' end labelled oligonucleotides with their complementary strands. The sequences of the probes are described in Figs 7 and 8![]()
. Single-strand oligonucleotides (5 pmol) were incubated at 37 °C for 45 min with 10 U T4 polynucleotide kinase (Roche) and 40 µCi (1.48 MBq) [
-32P]ATP (3000 Ci mmol1, 10 µCi µl1) in a final volume of 10 µl. The excess of [
-32P] ATP was eliminated using the Nucleotide Removal kit (Qiagen). The last elution step was performed with 100 µl of sterile water. After addition of the unlabelled oligonucleotides, the annealing of the complementary strands was performed by progressive temperature reduction after a 3 min treatment at 95 °C.
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Measurement of
-galactosidase reporter activities.
Cells were grown overnight in 3 ml YEPD at 30 °C under constant agitation. Cultures were then diluted in 5 ml fresh medium to obtain a cell density of approximately 5x106 cells ml1 and were regrown under agitation until the density reached 1.5x107 cells ml1. At this point, 1 ml aliquots from cultures that were not exposed to drugs (azole-resistant strains FR2 or MMY412) were centrifuged at 4 °C for 5 min at 5500 r.p.m. Exposure of other strains (azole-susceptible strains DSY449 and MMY411) to different drugs was carried out for defined time periods at 30 °C with constant agitation. After drug exposure, cell density was measured and cultures were centrifuged at 4 °C and 5500 r.p.m. for 10 min. The cell pellets were washed with 1 vol. Z buffer (60 mM Na2HPO4, 40 mM NaH2PO4, 10 mM KCl, 1 mM MgSO4) and centrifuged at 4 °C for a further 10 min. The washed cell pellets were resuspended in 150 µl Z buffer supplemented with 38.6 mM mercaptoethanol. After addition of 50 µl chloroform and 20 µl 0.1 % SDS, cells were disrupted by vortexing. To each aliquot was added 700 µl ONPG (1 mg ml1; Sigma) in Z buffer supplemented with mercaptoethanol and preheated at 30 °C. The mixtures were incubated at 37 °C until they developed a yellow colour (20 min to 3 h). The enzyme was inactivated by the addition of 500 µl 1 M Na2CO3. The mixtures were centrifuged at 13 000 r.p.m. for 10 min and the OD420 was read. The
-galactosidase activities were calculated using the following equation:
-galactosidase activity (Miller units)=(1000xOD420)/(txvxOD600), where t is the time of reaction expressed in min and v is the volume of culture used in the assay expressed in ml.
Measurement of Renilla luciferase reporter activities.
Cells were grown overnight in 3 ml YEPD at 30 °C under constant shaking, then diluted in 5 ml fresh medium to obtain a cell density of approximately 1.5x107 cells ml1. Benomyl (35 µg ml1) was added to the cell suspensions and cultures were incubated with constant shaking at 30 °C for a further 30 min. After drug exposure, cells were centrifuged for 10 min at 5500 r.p.m. The pellet was washed twice with 1 ml cold sterile water followed by 1 ml cold RLUC buffer (0.5 M NaCl, 0.1 M K2HPO4, 1 mM EDTA, 0.6 mM sodium azide). The final pellet was resuspended in 200 µl cold RLUC buffer supplemented with protease inhibitors (Roche) and an equal volume of chilled acid-washed glass beads was added. The cells were disrupted at 4 °C for 30 s using a Minibead beater (Biospec Products) and then centrifuged at 4 °C for 10 min at 13 000 r.p.m. The supernatants containing soluble proteins were stored at 20 °C or used directly for measurement of luciferase activities.
The relative luciferase activities of the different clones were measured using the Dual-Luciferase kit (Promega). Five microlitres of the crude protein extracts was added to 25 µl LAR II buffer according to the recommendations of the manufacturer and firefly luciferase activities were measured using a Lumat LB 9507 luminometer (E. G. and G. Berthold). Twenty-five microlitres of freshly prepared STOP and GLO buffer was then added to the reactions and Renilla luciferase activities were measured. Specific luciferase activities, expressed in Renilla luciferase units [RLU; i.e. U (µg protein)1] were calculated after determination of the protein concentration of cell extracts by the Bradford method.
Statistical analysis.
For the analysis of promoter deletions, a mathematical model was fitted using non-linear least squares. The choice of the model was based on the observation that, starting from a maximal response (H1 in the equation), the response decreases to a minimum level (H0) over a very short interval of length. The centre of this interval (A) represents the critical length for loss of response, while the width of this interval indicates the abruptness of the change (the slope B). The final equation was y=H0+(H1H0)/{1+exp[(Ax)/B]}, where y is the response and x the promoter length. For the mutant strains (cap1
/
), the same equation was used with parameters written in lower case (h0, h1, a, b). The fit of these equations contained eight parameters. The differences between the parameters determined for the wild type strain and the corresponding mutant were next tested using the generalized F test.
Comparisons between two sets of values (e.g. promoter bearing transversion or deletion versus wild type promoter) were performed using the nonparametric MannWhitney test.
| RESULTS |
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/
) at the MDR1 locus as described in Methods. We chose to use this strain because the absence of CDR1 may reduce drug efflux and thereby enhance the sensitivity of drug-dependent MDR1 activation.
Upregulation of MDR1 can occur in C. albicans azole-resistant strains or in azole-susceptible strains exposed to diverse drugs including benomyl and H2O2 (Gupta et al., 1998
; Harry et al., 2005
). Therefore we tested the response of the reporter to a panel of compounds known as inducers of MDR1. In order to test the MDR1 response specificity, we also tested the response to oestradiol and fluphenazine, compounds known to induce CDR2 (de Micheli et al., 2002
). For this purpose, a CDR2lacZ reporter system was integrated in DSY449 in the same fashion as for the MDR1lacZ construct. The relative increase of MDR1lacZ and CDR2lacZ reporter activities after treatment of C. albicans with these compounds is shown in Table 6
. Upon exposure to benomyl, H2O2 or diamide, MDR1lacZ reporter activities increased more than 10-fold as compared to untreated conditions, while no increase was observed for the CDR2lacZ promoter fusion. In contrast, oestradiol and fluphenazine treatment specifically increased CDR2lacZ reporter activities (by approximately 100-fold). Thus, the response of the two reporter genes to five different agents phenocopies the effects on the endogenous MDR1 and CDR2 genes. Exposure of C. albicans to methotrexate, sulfomethuron methyl and phenanthroline had no influence on transcription of either reporter. Based on these results, responses to the hydrophobic compound benomyl and to the oxidizing agent H2O2 were selected as readouts for the functional dissection of the MDR1 promoter. As the chemical properties of these drugs are different, we reasoned that they might reveal different modes of activation of the MDR1 promoter.
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A single transformant containing each deletion construct was chosen for testing of reporter activity in the absence and presence of benomyl. As shown in Fig. 2
(a), the reporter under control of the proximal 1459 bp of the MDR1 promoter exhibited a 26.1±4.4-fold increase of activity upon exposure to benomyl. Progressive deletions (99 to 141 bp) from 1459 to 302 (with respect to the ATG start codon) did not significantly alter the relative increase of reporter activity due to benomyl exposure. However, deletion of the region between 302 and 201 resulted in a 10-fold decrease in benomyl-dependent reporter activity, indicating that the minimal domain required for dramatic benomyl-dependent upregulation must lie within this region. This element was named BRE (for benomyl response element). To further delimit the BRE, small deletions of the region between nucleotides 302 and 222 were generated (Fig. 2b
). Progressive 4 to 7 bp (302 to 279) and 18 to 20 bp deletions (279 to 222) revealed that benomyl-dependent MDR1 transcription was significantly lowered by removal of sequence downstream of 296. The benomyl-dependent increase in reporter activity was 22.9±3.1-fold for the construct containing the MDR1 promoter region terminating at 296. It decreased to 4.7±0.7- and 6.4±1.1-fold for constructs with promoters terminating at 290 and 286, respectively. A further decline of relative reporter activity to 2.1±0.5-fold at 279 was also observed. These data indicate that the 5' extremity of the BRE lies between 296 and 279. A residual twofold increase in benomyl-dependent transcription appears to be independent of the BRE and suggests that the basal MDR1 promoter can still respond to the drug.
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-galactosidase activities measured in this strain (Fig. 4
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The partial retention of reporter activity in constructs lacking parts of the BRE may suggest that this regulatory region is composed of several discrete functional domains but we cannot rule out the involvement of other compensatory mechanisms. Moreover, while 5' deletions only delimit the 5' extremity of regulatory elements (i.e. 296, see Fig. 2b
), the results presented above aid the localization of central elements of the BRE. Deletions D2 (295 to 287), D3 (286 to 278) and D4 (277 to 269) all led to a decrease of benomyl-dependent MDR1 response and of reporter activity in strain FR2, thus indicating that they all contain nucleotides important for the functionality of the BRE. Since these three deleted regions are sequential in the MDR1 promoter, it can be concluded that at least all the nucleotides of the central deleted region D3 (286 to 278) are part of the BRE.
Cap1p, a bZip transcription factor, acts as a trans-acting factor of MDR1 regulation
The presence of two Cap1p-like binding sites within the cis-acting HRE (TTAG/CTAA; at 549 and 532) suggested that Cap1p, a bZip transcription factor involved in oxidative stress, could play a role in regulating H2O2-dependent MDR1 expression by binding to these sequences. To further explore this hypothesis, promoter deletion analysis was undertaken in MMY411 (cap1
/
), a strain derived from DSY449 that lacks CAP1. For all promoter fragments tested, the reporter activities obtained in the absence or presence of H2O2 treatment were not significantly different in this strain. The relative levels of reporter activity after H2O2 exposure were between 0.8±0.1- and 1.7±0.6-fold higher than that recorded when the strain was not treated with H2O2 (Fig. 3a
, black columns). Thus, H2O2-dependent expression is essentially completely lost in the cap1
/
strain irrespective of whether the HRE is present. Therefore Cap1p is likely to be the main positive regulator involved in the response of the MDR1 promoter to H2O2. We also examined the benomyl-dependent response of reporters under control of MDR1 promoter fragments in the cap1
/
strain. Inactivation of CAP1 did not prevent the different reporter constructs from responding to benomyl. Reporter constructs carrying at least 302 nucleotides of the proximal MDR1 promoter showed a benomyl-dependent increase in activity (13.0±5.1-fold, black columns Fig. 2a
). However, since the level of the responses was generally twofold lower than that recorded in the corresponding wild-type strain (25.9±5.3-fold, white columns Fig. 2a
), we cannot exclude the possibility that Cap1p plays a minor role in mediating the transient response to benomyl. Finally, we observed no significant differences between the specific reporter activities measured in the azole-resistant strain FR2 (58.2±5.5 U) and the corresponding cap1
/
mutant MMY412 (51.3±7.5 U, Fig. 4a
), indicating that Cap1p is not necessary for constitutive high expression of MDR1 in FR2.
Importance of the cis-acting HRE for MDR1 regulation
As the response of MDR1 to benomyl was partially reduced in the strain lacking CAP1, the deletion of a region (552 to 521) within the HRE (delimited between 561 and 520) was performed in the full-length MDR1 promoter and reporter activities were measured under benomyl exposure in DSY449. As shown in Fig. 6
(a), deletion of the region between 552 and 521 within the full-length MDR1 promoter did not alter the response to benomyl: the increase in reporter activity after benomyl exposure (25.7±5.0-fold) was not significantly different from that in the control (30.1±3.1-fold). The simultaneous deletion of the BRE and HRE resulted in a similar effect to the deletion of the BRE alone (2.1±0.5-fold versus 1.3±0.2-fold increase in reporter activity). Consistent with the results shown in Fig. 2(a)
, these results indicate that the upregulation of MDR1 under benomyl exposure is not dependent on the HRE. The HRE was also not involved in high constitutive expression of MDR1 in strain FR2. In this strain, reporter activities were identical in the presence (66.4±5.2 U) or absence (73.6±3.7 U) of the HRE (Fig. 6b
). Moreover, reporter activities after the simultaneous deletion of BRE and HRE (23.6±2.6 U) were also similar to those observed in the absence of the BRE (16.3±0.7 U) (Fig. 6b
).
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In summary, our results show that the HRE is not essential for MDR1 upregulation by benomyl or in an azole-resistant isolate but is required for H2O2-dependent regulation of this gene. In the presence of H2O2, Cap1p is likely to act in trans on the MDR1 promoter through the HRE. While we cannot exclude an indirect role for Cap1p in the benomyl response, it does not act through either of the cis-acting elements that we have identified in MDR1.
The BRE and HRE are functional in a heterologous promoter
To further assess the cis-acting role of the BRE in response to benomyl, the element was placed in a minimal 198 bp CDR2 promoter lacking its own regulatory element (DRE). The CDR2 promoter is not responsive to benomyl and is therefore convenient for addressing the involvement of the BRE in this process. A BRECDR2 chimeric promoter was constructed and used with a Renilla luciferase reporter system. Because the sequences immediately flanking the BRE may modulate its function, a 50 bp MDR1 promoter sequence was used which spanned nucleotides 309 to 260. The reporter system was inserted at the LEU2 genomic locus of strains DSY449 and FR2. As shown in Fig. 7
(a), the presence of the BRE conferred benomyl responsiveness to the minimal CDR2 promoter. Up to 12.0±2.1-fold increase in reporter activity could be measured in benomyl-treated cells as compared to the control, untreated cells. Activity was reduced however as compared to the native 592 bp MDR1 promoter (72.8±5.6-fold). When two copies of the BRE were fused to the CDR2 promoter, the increase in reporter activity (96.8±46.4-fold) was comparable to that of the MDR1 promoter. To assess the role of the HRE in the benomyl-dependent response, a 41 bp MDR1 promoter sequence spanning nucleotides 556 to 516, and containing the HRE with Cap1p-binding sites, was placed upstream of the BRECDR2 promoter. The increase in reporter activity induced by benomyl (17.6±1.6-fold) was not significantly different from that observed with the BRECDR2 promoter (12±2.1-fold). When the HRE alone was fused to the CDR2 promoter, almost no increase in reporter activity could be detected (1.6±0.5-fold), indicating that the HRE is not involved in the benomyl response of MDR1. In FR2, the BRECDR2 chimeric promoter mediated high reporter activity. As shown in Fig. 7(b)
, the specific luciferase activity obtained with the BRECDR2 fusion (1.9±0.45x106 RLU) was only 2.5-fold lower than that obtained with the MDR1 promoter (4.9±1.0x106 RLU). Insertion of two copies of the BRE upstream of the minimal CDR2 promoter gave reporter activities (5.8±1.6x106 RLU) similar to those measured with the MDR1 promoter. Since the specific reporter activities measured with the HREBRECDR2 and the BRECDR2 fusions (1.7±0.3x106 and 1.9±0.5x106 RLU, respectively) were similar and only background reporter activity was detected with the HRE alone (<104 RLU), the HRE was not considered to be involved in the high levels of expression of MDR1 in FR2. The data presented in Fig. 7
also contribute to a more precise localization of the functional BRE. Placing the 5'extremity at position 296 as previously determined, the 3' extremity can be proposed to be at position 260, since this corresponds to the position identified in the testing of BRE functionality.
We investigated whether the HRE could confer an H2O2-dependent response to the minimal CDR2 promoter in strain DSY449 (Fig. 7c
). However, when the HRE alone was fused to the minimal CDR2 promoter, the relative increase in reporter activity (1.9±0.7-fold) was not different from the background activity increase measured with the basal CDR2 promoter (2.0±1.0-fold). The relative increase in reporter activities after H2O2 exposure obtained with the BRECDR2 promoter was similar (2.9±1.8-fold) to that with the HRECDR2 promoter. However, when the HRE was placed upstream of the BRECDR2 promoter, a 43.0±3.1-fold increase in reporter activity generated by H2O2 exposure was measured, which was 1.6-fold higher than with the MDR1 promoter (27.3±3.7-fold). Thus, consistent with our observation that deletion of either element reduced the H2O2-dependent response of the MDR1 promoter, both elements are required to convey significant H2O2-dependent upregulation to a heterologous promoter.
Trans-acting factors bind to the BRE
To assess the possible interaction between the BRE and putative transcription factors present in C. albicans protein extracts, EMSAs were performed with double-stranded probes corresponding to several regions of the BRE. In this experiment, it was possible to observe the formation of a DNAprotein complex with labelled probe 1 (296 to 259; Fig. 8a
), which contained the BRE delimited by 5' promoter deletion analysis and insertion into another promoter context (between 296 and 260). This complex was present at higher levels, as revealed by the intensity of the band, when a protein extract from benomyl-exposed cells was used in comparison to that observed with proteins from untreated cells. The proteinDNA complex was specific for the BRE, since competition with increasing amounts of unlabelled BRE resulted in the disappearance of the complex (Fig. 8a
). Furthermore, in the presence of a molar excess of an unlabelled probe, corresponding to a promoter region of the RTA4 gene, the formation of the proteinDNA complex could be still observed. Finally, using this labelled RTA4 as a probe (probe 2), formation of a proteinDNA complex with a similar migration pattern was not observed.
Labelled probes harbouring transversions identical to those used in promoter analysis (T1 to T4: probes 36) were also incubated with C. albicans protein extracts. However, as illustrated in Fig. 8(b)
, no specific band could be detected, thus corroborating the negative effects of these transversions upon reporter activities. Moreover, EMSAs carried out with labelled probes harbouring internal deletions (deletion D5: probe 7, 282 to 277 and deletion D6: probe 8, 285 to 274) showed no specific binding (Fig. 8b
). This result is consistent with the finding that deletions within this region strongly reduced reporter activities (Fig. 5b
). These results show that the promoter regulatory region previously delimited (296 to 260) corresponds to that which can bind sequence specific protein complexes in vitro.
To further delimit the BRE sequence necessary for the binding in trans of specific protein complexes, band-shift assays were performed with probe lengths reduced at both the 5' and 3' extremities of probe 1 (296 to 259) containing the BRE. Removal of 5 and 6 bp at the 5' extremity of probe 1 (respectively probes 9 and 10) and of 5 to 13 bp at its 3' extremity (probes 1517) did not abolish the formation of a DNAprotein complex (Fig. 9a
). The intensity of bands for protein complexes with probes lacking 7 bp at the 5' extremity (probe 11) or 14 bp at the 3' extremity (probe 18) was slightly reduced as compared to the full-length probe 1. Finally, removal of at least 8 bp at the 5' extremity (probes 1214) or at least 15 bp at the 3' extremity (probes 1922) resulted in the absence of protein complex formation (Fig. 9a
). The results show that the minimal protein-binding region of the BRE is likely to be from 290 to 273. As expected, a specific DNA protein complex could be revealed (Fig. 9b
) by using a labelled probe covering this region (probe 23).
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| DISCUSSION |
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Surprisingly, the HRE had no impact on high constitutive expression of MDR1, since absence of this element did not alter MDR1 expression (Fig. 6
). Moreover, when fused to the promoter CDR2, the HRE was unable to confer a high reporter activity in FR2. In the background of an FR2-derived mutant lacking CAP1, MDR1lacZ reporter activities were also unchanged as compared to the parent strain. These results highlight the existence of distinct transcriptional programmes responsible for MDR1 upregulation. While the influence of the HRE appears restricted to the H2O2 response, the deletion of CAP1 also decreased, but did not eliminate, the benomyl-dependent response of MDR1 (Fig. 2
). Thus, our work reveals that there is some degree of crosstalk between the elements that mediate distinct responses. As the benomyl response of MDR1 is dependent on the BRE, we speculate that Cap1p may cooperate with unknown factor(s) acting on the BRE.
The second cis-acting element of the MDR1 promoter, the BRE, was responsible both for the benomyl-dependent response of MDR1 and for high constitutive expression of this gene in the azole-resistant strain FR2. The delimitation of the minimal regulatory element that constitutes the BRE requires still further promoter analysis. The promoter truncations we examined have only delimited the 5' extremity of the regulatory elements (i.e., 296, see Fig. 2
); internal deletions and transversions (Fig. 5
) demonstrated that nucleotides between 286 and 278 are part of the BRE, but do not give a precise position of the 3' extremity of the regulatory element. Nevertheless, the 3' functional extremity of the BRE can be deduced from results obtained with the BRECDR2 promoter fusion. They suggest that the position of the 3' extremity must lie upstream of position 260, therefore defining the functional BRE between 296 and 260. In the light of results obtained by EMSA with oligonucleotides corresponding to the sequence of the BRE, the minimal oligonucleotide length still enabling sequence-protein binding was determined to be located between 290 and 273 (Figs. 8 and 9![]()
) and thus lies fully within the functionally defined BRE. The convergence of both methods to this specific region of the promoter strengthens its implication as a regulatory element. In vivo and in vitro footprinting analysis of the BRE can now be applied to aid precise delimitation of this regulatory element and the further characterization of the proteins that bind to it.
The functional cis-acting BRE identified in our work (296 to 260) lies close to a cis-acting benomyl-responsive region, as delimited by a separate study (Harry et al., 2005
). In that study, removal of the region between 399 and 299 led to a 20-fold diminution of the benomyl-dependent increase in reporter gene activities, whereas a weaker diminution (two- to fourfold) was observed when the region between 299 and 200 was absent from the MDR1 promoter. Thus, while both studies indicate that the BRE-containing sequences are important, we have not seen evidence of a second, more distal benomyl-responsive element. This discrepancy could be the result of several factors, including the strain background or the type of gene reporter system used to assess promoter activity. Above all, the genomic site of reporter gene insertion might contribute to such differences. In the present study, the MDR1lacZ reporter system was integrated at the MDR1 genomic locus, whereas the Renilla luciferase reporter system used in the study of Harry et al. (2005)
was inserted at the ADE2 genomic locus. A very recent study by Hiller et al. (2006)
also shows the existence of several cis-acting domains in the MDR1 promoter. This promoter analysis was performed with a GFP reporter system integrated at the ACT1 locus in an azole-susceptible strain exposed to benomyl (3 h at 50 µg ml1) and in an azole-resistant isolate expressing MDR1 at high levels. This experimental context is similar to the one used here, except that the drug exposure time was longer and the gene reporter system was different and not integrated at the MDR1 locus. The authors identified three different regulatory regions (region 1: 397 to 301; region 2: 588 to 500; region 3: 287 to 209). In their study, region 2 was implicated in the benomyl-dependent MDR1 response and regions 1 and 3 were required for constitutive high expression of MDR1. Thus while there is some consensus of agreement on the delimitation of regulatory regions, there are also significant discrepancies between the findings of Hiller et al. (2006)
and the other two studies, this work and Harry et al. (2005)
. Most notably, our results clearly implicate the HRE in the H2O2 response of MDR1, while Hiller et al. (2006)
found that the region encompassing this element (region 2) was responsible for benomyl-dependent MDR1 upregulation, a feature not confirmed in our study. The discrepancies between these different studies could have several origins, as mentioned above. The details of which regulatory elements respond to particular signals may also depend on chromatin-mediated factors and the gene's global context within the genome. Thus we believe it is of importance to have integrated the reporter constructs at the MDR1 locus. Moreover, the demonstration that we can recapitulate the functions of the HRE and BRE in the context of another promoter further validates the transcriptional cis-acting roles that we assign to them. Furthermore, we demonstrated protein binding to the BRE, thus confirming that this cis-acting element may interact with as yet unknown factor(s).
A BRE-like sequence is present in the promoter of genes co-regulated with MDR1 and contains a Mcm1p-binding site
In a previous study, we determined, by transcript profiling experiments, that MDR1 was co-regulated with other genes in an azole-resistant strain and in a susceptible strain exposed to benomyl (Karababa et al., 2004
). We therefore expected to identify a common regulatory cis-acting motif resembling the BRE in the promoters of these genes. Alignment of promoter sequences revealed that nine of the 16 commonly upregulated genes indeed contain one or two sequences that match the signature of the BRE (Fig. 10a
). Conserved sequence 1 (287 to 267) in the MDR1 promoter matches the previously delimited BRE (296 to 260) and differs by 4 nucleotides from the consensus established after promoter sequences alignment (5'-TCCMMWTTWGKAAAKTYHCCG-3'). While the presence of the BRE signature in these promoters is therefore consistent with their co-regulation, additional work will be required to confirm that the BRE is important for the regulation of these genes. A second BRE-like sequence (496 to 476) was identified in the MDR1 promoter (see Fig. 10a
); however, promoter deletion analysis revealed that this sequence is not involved in MDR1 regulation either in DSY449 exposed to benomyl or in FR2. Furthermore, EMSA with a probe corresponding to this second sequence failed to reveal protein-binding activity (data not shown).
|
In summary, the work presented here has enabled the precise delimitation of two cis-acting regulatory elements (HRE and BRE) in the MDR1 promoter. We have shown that Cap1p is likely to be one trans-acting factor for the HRE; however, confirmation of its role and the full identification of the trans-acting components with which it interacts will necessitate further investigation. Future studies will also clarify whether mutations in MCM1 can be responsible for high constitutive expression of MDR1 in a manner analogous to the recent finding that mutations in TAC1, a key trans-acting regulator of the ABC-transporter genes CDR1 and CDR2, are responsible for the high CDR1/CDR2 expression of clinical strains (Coste et al., 2006
).
| NOTE ADDED IN PROOF |
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| ACKNOWLEDGEMENTS |
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| REFERENCES |
|---|
|
|
|---|
Alarco, A. M., Balan, I., Talibi, D., Mainville, N. & Raymond, M. (1997). AP1-mediated multidrug resistance in Saccharomyces cerevisiae requires FLR1 encoding a transporter of the major facilitator superfamily. J Biol Chem 272, 1930419313.
Albertson, G. D., Niimi, M., Cannon, R. D. & Jenkinson, H. F. (1996). Multiple efflux mechanisms are involved in Candida albicans fluconazole resistance. Antimicrob Agents Chemother 40, 28352841.[Abstract]
Ben-Yaacov, R., Knoller, S., Caldwell, G. A., Becker, J. M. & Koltin, Y. (1994). Candida albicans gene encoding resistance to benomyl and methotrexate is a multidrug resistance gene. Antimicrob Agents Chemother 38, 648652.
Broco, N., Tenreiro, S., Viegas, C. A. & Sa-Correia, I. (1999). FLR1 gene (ORF YBR008c) is required for benomyl and methotrexate resistance in Saccharomyces cerevisiae and its benomyl-induced expression is dependent on pdr3 transcriptional regulator. Yeast 15, 15951608.[CrossRef][Medline]
Chang, V. K., Fitch, M. J., Donato, J. J., Christensen, T. W., Merchant, A. M. & Tye, B. K. (2003). Mcm1 binds replication origins. J Biol Chem 278, 60936100.
Coleman, D. C., Bennett, D. E., Sullivan, D. J., Gallagher, P. J., Henman, M. C., Shanley, D. B. & Russell, R. J. (1993). Oral Candida in HIV infection and AIDS: new perspectives/new approaches. Crit Rev Microbiol 19, 6182.[Medline]
Coste, A., Turner, V., Ischer, F., Morschhauser, J., Forche, A., Selmecki, A., Berman, J., Bille, J. & Sanglard, D. (2006). A mutation in Tac1p, a transcription factor regulating CDR1 and CDR2, is coupled with loss of heterozygosity at chromosome 5 to mediate antifungal resistance in Candida albicans. Genetics 172, 21392156.
de Micheli, M., Bille, J., Schueller, C. & Sanglard, D. (2002). A common drug-responsive element mediates the upregulation of the Candida albicans ABC transporters CDR1 and CDR2, two genes involved in antifungal drug resistance. Mol Microbiol 43, 11971214.[CrossRef][Medline]
Fernandes, L., Rodrigues-Pousada, C. & Struhl, K. (1997). Yap, a novel family of eight bZIP proteins in Saccharomyces cerevisiae with distinct biological functions. Mol Cell Biol 17, 69826993.[Abstract]
Fling, M. E., Kopf, J., Tamarkin, A., Gorman, J. A., Smith, H. A. & Koltin, Y. (1991). Analysis of a Candida albicans gene that encodes a novel mechanism for resistance to benomyl and methotrexate. Mol Gen Genet 227, 318329.[CrossRef][Medline]
Fonzi, W. A. & Irwin, M. Y. (1993). Isogenic strain construction and gene mapping in Candida albicans. Genetics 134, 717728.[Abstract]
Franz, R., Kelly, S. L., Lamb, D. C., Kelly, D. E., Ruhnke, M. & Morschhauser, J. (1998). Multiple molecular mechanisms contribute to a stepwise development of fluconazole resistance in clinical Candida albicans strains. Antimicrob Agents Chemother 42, 30653072.
Ghannoum, M. A., Rex, J. H. & Galgiani, J. N. (1996). Susceptibility testing of fungi: current status of correlation of in vitro data with clinical outcome. J Clin Microbiol 34, 489495.[Abstract]
Goldway, M., Teff, D., Schmidt, R., Oppenheim, A. B. & Koltin, Y. (1995). Multidrug resistance in Candida albicans: disruption of the BENr gene. Antimicrob Agents Chemother 39, 422426.
Gupta, V., Kohli, A., Krishnamurthy, S., Puri, N., Aalamgeer, S. A., Panwar, S. & Prasad, R. (1998). Identification of polymorphic mutant alleles of CaMDR1, a major facilitator of Candida albicans which confers multidrug resistance, and its in vitro transcriptional activation. Curr Genet 34, 192199.[CrossRef][Medline]
Hanahan, D. (1985). Techniques for transformation of E. coli. In DNA Cloning: a Practical Approach 1: Core Techniques, pp. 109135. Edited by D. M. Glover. Oxford: IRL Press.
Harry, J. B., Oliver, B. G., Song, J. L., Silver, P. M., Little, J. T., Choiniere, J. & White, T. C. (2005). Drug-induced regulation of the MDR1 promoter in Candida albicans. Antimicrob Agents Chemother 49, 27852792.
Hiller, D., Stahl, S. & Morschhauser, J. (2006). Multiple cis-acting sequences mediate upregulation of the MDR1 efflux pump in a fluconazole-resistant clinical Candida albicans isolate. Antimicrob Agents Chemother 50, 23002308.
Jelinsky, S. A. & Samson, L. D. (1999). Global response of Saccharomyces cerevisiae to an alkylating agent. Proc Natl Acad Sci U S A 96, 14861491.
Karababa, M., Coste, A. T., Rognon, B., Bille, J. & Sanglard, D. (2004). Comparison of gene expression profiles of Candida albicans azole-resistant clinical isolates and laboratory strains exposed to drugs inducing multidrug transporters. Antimicrob Agents Chemother 48, 30643079.
Keleher, C. A., Goutte, C. & Johnson, A. D. (1988). The yeast cell-type-specific repressor alpha 2 acts cooperatively with a non-cell-type-specific protein. Cell 53, 927936.[CrossRef][Medline]
Kuge, S. & Jones, N. (1994). YAP1 dependent activation of TRX2 is essential for the response of Saccharomyces cerevisiae to oxidative stress by hydroperoxides. EMBO J 13, 655664.[Medline]
Law, D., Moore, C. B., Wardle, H. M., Ganguli, L. A., Keaney, M. G. & Denning, D. W. (1994). High prevalence of antifungal resistance in Candida spp. from patients with AIDS. J Antimicrob Chemother 34, 659668.
Marr, K. A., Seidel, K., White, T. C. & Bowden, R. A. (2000). Candidemia in allogeneic blood and marrow transplant recipients: evolution of risk factors after the adoption of prophylactic fluconazole. J Infect Dis 181, 309316.[CrossRef][Medline]
Moran, G. P., Sanglard, D., Donnelly, S. M., Shanley, D. B., Sullivan, D. J. & Coleman, D. C. (1998). Identification and expression of multidrug transporters responsible for fluconazole resistance in Candida dubliniensis. Antimicrob Agents Chemother 42, 18191830.
Nguyen, D. T., Alarco, A. M. & Raymond, M. (2001). Multiple Yap1p-binding sites mediate induction of the yeast major facilitator FLR1 gene in response to drugs, oxidants, and alkylating agents. J Biol Chem 276, 11381145.
Passmore, S., Maine, G. T., Elble, R., Christ, C. & Tye, B. K. (1988). Saccharomyces cerevisiae protein involved in plasmid maintenance is necessary for mating of MAT
cells. J Mol Biol 204, 593606.[CrossRef][Medline]
Passmore, S., Elble, R. & Tye, B. K. (1989). A protein involved in minichromosome maintenance in yeast binds a transcriptional enhancer conserved in eukaryotes. Genes Dev 3, 921935.
Rex, J. H., Rinaldi, M. G. & Pfaller, M. A. (1995). Resistance of Candida species to fluconazole. Antimicrob Agents Chemother 39, 18.[Medline]
Rottmann, M., Dieter, S., Brunner, H. & Rupp, S. (2003). A screen in Saccharomyces cerevisiae identified CaMCM1, an essential gene in Candida albicans crucial for morphogenesis. Mol Microbiol 47, 943959.[CrossRef][Medline]
Sa-Correia, I. & Tenreiro, S. (2002). The multidrug resistance transporters of the major facilitator superfamily, 6 years after disclosure of Saccharomyces cerevisiae genome sequence. J Biotechnol 98, 215226.[CrossRef][Medline]
Sanglard, D. & Odds, F. C. (2002). Resistance of Candida species to antifungal agents: molecular mechanisms and clinical consequences. Lancet Infect Dis 2, 7385.[CrossRef][Medline]
Sanglard, D., Kuchler, K., Ischer, F., Pagani, J. L., Monod, M. & Bille, J. (1995). Mechanisms of resistance to azole antifungal agents in Candida albicans isolates from AIDS patients involve specific multidrug transporters. Antimicrob Agents Chemother 39, 23782386.[Abstract]
Sanglard, D., Ischer, F., Monod, M. & Bille, J. (1996). Susceptibilities of Candida albicans multidrug transporter mutants to various antifungal agents and other metabolic inhibitors. Antimicrob Agents Chemother 40, 23002305.[Abstract]
Sanglard, D., Ischer, F., Calabrese, D., Majcherczyk, P. A. & Bille, J. (1999). The ATP binding cassette transporter gene CgCDR1 from Candida glabrata is involved in the resistance of clinical isolates to azole antifungal agents. Antimicrob Agents Chemother 43, 27532765.
Spellman, P. T., Sherlock, G., Zhang, M. Q., Iyer, V. R., Anders, K., Eisen, M. B., Brown, P. O., Botstein, D. & Futcher, B. (1998). Comprehensive identification of cell cycle-regulated genes of the yeast Saccharomyces cerevisiae by microarray hybridization. Mol Biol Cell 9, 32733297.
Taglicht, D. & Michaelis, S. (1998). Saccharomyces cerevisiae ABC proteins and their relevance to human health and disease. Methods Enzymol 292, 130162.[Medline]
Tenreiro, S., Fernandes, A. R. & Sa-Correia, I. (2001). Transcriptional activation of FLR1 gene during Saccharomyces cerevisiae adaptation to growth with benomyl: role of Yap1p and Pdr3p. Biochem Biophys Res Commun 280, 216222.[CrossRef][Medline]
Toone, W. M. & Jones, N. (1999). AP-1 transcription factors in yeast. Curr Opin Genet Dev 9, 5561.[CrossRef][Medline]
Uhl, M. A. & Johnson, A. D. (2001). Development of Streptococcus thermophilus lacZ as a reporter gene for Candida albicans. Microbiology 147, 11891195.
Wang, Y., Cao, Y. Y., Jia, X. M., Cao, Y. B., Gao, P. H., Fu, X. P., Ying, K., Chen, W. S. & Jiang, Y. Y. (2006). Cap1p is involved in multiple pathways of oxidative stress response in Candida albicans. Free Radic Biol Med 40, 12011209.[CrossRef][Medline]
White, T. C. (1997). Increased mRNA levels of ERG16, CDR, and MDR1 correlate with increases in azole resistance in Candida albicans isolates from a patient infected with human immunodeficiency virus. Antimicrob Agents Chemother 41, 14821487.[Abstract]
White, T. C., Pfaller, M. A., Rinaldi, M. G., Smith, J. & Redding, S. W. (1997). Stable azole drug resistance associated with a substrain of Candida albicans from an HIV-infected patient. Oral Dis 3, S102S109.
White, T. C., Marr, K. A. & Bowden, R. A. (1998). Clinical, cellular, and molecular factors that contribute to antifungal drug resistance. Clin Microbiol Rev 11, 382402.
Wirsching, S., Michel, S., Kohler, G. & Morschhauser, J. (2000a). Activation of the multiple drug resistance gene MDR1 in fluconazole-resistant, clinical Candida albicans strains is caused by mutations in a trans-regulatory factor. J Bacteriol 182, 400404.
Wirsching, S., Michel, S. & Morschhauser, J. (2000b). Targeted gene disruption in Candida albicans wild-type strains: the role of the MDR1 gene in fluconazole resistance of clinical Candida albicans isolates. Mol Microbiol 36, 856865.[CrossRef][Medline]
Wynne, J. & Treisman, R. (1992). SRF and MCM1 have related but distinct DNA binding specificities. Nucleic Acids Res 20, 32973303.
Zhang, X., De Micheli, M., Coleman, S. T., Sanglard, D. & Moye-Rowley, W. S. (2000). Analysis of the oxidative stress regulation of the Candida albicans transcription factor, Cap1p. Mol Microbiol 36, 618629.[CrossRef][Medline]
Received 3 July 2006;
revised 17 August 2006;
accepted 5 September 2006.
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