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The DNA-binding domain of CaNdt80p is required to activate CDR1 involved in drug resistance in Candida albicans

¹ Graduate Institute of Life Sciences, National Defence Medical Center, Taipei, Taiwan, Republic of China

² Division of Clinical Research, National Health Research Institutes, 35 Keyan Road, Zhunan Town, Miaoli County 350, Taiwan, Republic of China

³ Department of Biological Science and Technology, National Chiao Tung University, Hsinchu, Taiwan, Republic of China

Correspondence
Hsiu-Jung Lo
hjlo{at}nhri.org.tw

Received 28 March 2006
Accepted 13 July 2006

Abbreviations: ß-Gal, ß-galactosidase.

	INTRODUCTION

TOP INTRODUCTION METHODS RESULTS AND DISCUSSION REFERENCES

 
Candida albicans is the most frequently isolated fungal pathogen in humans, and has caused morbidity in seriously debilitated and immunocompromised hosts (Edwards, 1990). The prevalence of nosocomial candidaemia increased 27-fold from 1981 to 1993 at a hospital in Taiwan (Chen et al., 1997; Hung et al., 1996). In the USA, yeast infections also rank as the fourth-most-common cause of nosocomial bloodstream infection (Beck-Sague & Jarvis, 1993; Pfaller et al., 1998). Coinciding with the increased usage of antifungal drugs, incidences of drug resistance have also multiplied (Pfaller et al., 2000; Vanden Bossche et al., 1994; Yang & Lo, 2001).

Overexpression of efflux pumps, such as ATP-binding cassette (ABC) transporters, has been shown to be the major mechanism for drug resistance in clinical isolates (Karababa et al., 2004; Marger & Saier, 1993; Michaelis & Berkower, 1995). Mutations on CDR1 in C. albicans result in an increased susceptibility to azole drugs (Sanglard et al., 1996), which is consistent with the observation that overexpression of CDR1 contributes to the drug resistance in clinical isolates (Lopez-Ribot et al., 1998). Recently, two transcription factors, CaNdt80p and Tac1p, have been identified as positive regulators of CDR1, and mutations of either gene result in an increased drug susceptibility (Chen et al., 2004; Coste et al., 2004).

CaNdt80p is a C. albicans homologue of the Saccharomyces cerevisiae ScNdt80p (Chen et al., 2004). Nevertheless, CaNdt80p is reported to be involved in drug resistance in C. albicans (Chen et al., 2004), while ScNdt80p is a meiosis-specific transcription factor in S. cerevisiae (Abi-Said et al., 1997; Chu et al., 1998; Chu & Herskowitz, 1998). CaNdt80p is 672 amino acids (aa) in length and ScNdt80p 592 aa (Fig. 1a). The sequence from amino acids 221 to 592 of CaNdt80p and that from amino acids 1 to 330 of ScNdt80p share 35 % identity and 53 % similarity (Fig. 1b). Interestingly, there is no similarity between the N terminal of CaNdt80p and the C terminal of ScNdt80p (Fig. 1a) (Chen et al., 2004). The amino acid residues from 1 to 330 of ScNdt80p have been shown to be important for the DNA-binding activity. The residues responsible for the DNA-binding capability of ScNdt80p have been reported. Among them, the 177Arg residue is the most important one for ScNdt80p to bind DNA. When this 177Arg residue is mutated, the DNA-binding capability decreases 27-fold (Montano et al., 2002). Whether the putative DNA-binding domain of CaNdt80p also plays an important role in its function(s) has not been reported.

View larger version (46K): [in this window] [in a new window]   Fig. 1. Comparison of CaNdt80p in C. albicans and ScNdt80p in S. cerevisiae. (a) Overall comparison between CaNdt80p and ScNdt80p. For CaNdt80p, the promoter (pro) is in the solid black box, the activation domain (AD) is in the black diagonally hatched box, and the DNA-binding domain (BD) is in the cross-hatched box. For ScNdt80p, the promoter is in the horizontally lined box, the DNA-binding domain (BD) is in the open box, and the activation domain (AD) is in the grey diagonally hatched box. (b) Protein sequences of the DNA-binding domains of CaNdt80p and ScNdt80p. Similar residues of the two sequences are shaded. The mutated residue in CaNdt80p is indicated by an arrow, and the numbers represent the positions of amino acid residues in CaNdt80p and ScNdt80p.

	METHODS

TOP INTRODUCTION METHODS RESULTS AND DISCUSSION REFERENCES

 
Strains and media. S. cerevisiae strains, C. albicans strains, plasmids and primers used in this study are listed in Tables 1, 2, 3 and 4, respectively. Yeast peptone dextrose (YPD; 1 % yeast extract, 2 % peptone, 2 % glucose), and synthetic dextrose (SD; 0.67 % yeast nitrogen base without amino acids, 2 % glucose) were prepared as described in Sherman et al. (2002).

View larger version (31K): [in this window] [in a new window]   Fig. 2. Different chimeras having different effects on the expression of the CDR1p–lacZ reporter in S. cerevisiae. For CaNdt80p, the promoter (pro) is in the solid black box, the activation domain (AD) is in the black hatched box, and the DNA-binding domain (BD) is in the cross-hatched box. For ScNdt80p, the promoter is in the horizontally lined box, the DNA-binding domain (BD) is in the open box, and the activation domain (AD) is in the grey hatched box. The ß-Gal activity of the strain (SLO2) containing vector alone (pRS426) was defined as 1, and the ß-Gal activity of cells containing different chimeras was normalized accordingly. The mutated residue is marked by an asterisk.

Yeast extract (10 µl) was added to a 12x75 mm disposable glass tube containing 990 µl cold Z buffer (60 mM Na₂HPO₄, 40 mM NaH₂PO₄, 10 mM KCl, 1 mM MgSO₄) with ß-mercaptoethanol (2.7 ml l^–1). A total of 200 µl ONPG (Sigma, N1127; 4 mg ml^–1 in Z buffer) as enzyme substrate was added to start the reaction at t=0. The reaction was stopped by adding 500 µl 1 M Na₂CO₃. Bio-Rad Protein Assay Dye (Catalogue no. 500-0006) was used to determine the protein concentration of the cell extract. Based on the Bradford dye-binding procedure, the protein concentration of the supernatant was determined by comparison with a standard curve of A₅₉₅ versus protein concentration, obtained by the serial twofold dilution of 500 µg ml^–1 BSA down to 62.5 µg ml^–1. ß-Gal activity units were calculated (moles ONPG cleaved per minute per milligram protein) by the following equation:

where time is in minutes, volume of extract in millilitres, and protein concentration in milligrams per millilitre.

RT-PCR. We used two-step RT-PCR to detect the level of mRNA in different strains. Total RNA was isolated by the acid hot phenol method (Schmitt et al., 1990), followed by RQ1 RNase-free DNase (Promega, M610A) treatment to digest any contaminating DNA. First, 1 µg total RNA from each strain was used as template with oligo-dT as primer to synthesize the first-strand cDNA with StrataScript Reverse Transcriptase (Strategene, 600085). The product of each reaction was diluted in sterile double-distilled H₂O, then used as the template to carry out the PCR amplification. Each reaction contained 50 µl PCR mixture including 0.25 µM each primer, 0.2 mM dNTPs, 1x buffer (10 mM Tris/HCl, pH 9.0, 1.5 mM MgCl₂, 50 mM KCl) and 1 U Taq polymerase (Amersham, 27-0799-06). The reaction conditions were as follows: 95 °C for 30 s, 57 °C for 30 s and 72 °C for 80 s for 35 cycles. The primers HJL237 and HJL238 were used to detect the CaNDT80 mRNA in different C. albicans strains, and the primers HJL800 and HJL801 were used for CaNDT80 in different chimeras in S. cerevisiae strains. The primers HJL802 and HJL803 were used for the detection of ScNDT80. The primers HJL293 and HJL294, the sequences of which were deduced from the intron and exon of the C. albicans CaEFB1 gene, were used to distinguish the PCR products resulting from either the genomic DNA (928 bp DNA fragment) or the spliced RNA (564 bp DNA fragment) (Schaller et al., 2003). For S. cerevisiae, the primers HJL804 and HJL805 were used for the detection of ScEFB1.

Quantitative analysis of mRNA level by real-time PCR. The cells were harvested at OD₆₀₀ 0.7–0.9 after being grown in 20 ml SD liquid medium at 30 °C. Total RNA was isolated by the acid hot phenol method (Schmitt et al., 1990), followed by RQ1 RNase-free DNase (Promega, M610A) treatment to digest any contaminating DNA. A real-time PCR was performed in a Rotor-Gene 3000 instrument (Corbett Research) with a TITANIUM Taq PCR kit (BD Clontech, 639210) and SYBR Green I Nucleic Acid stain (Cambrex, 50513) to determine the level of mRNA. The sample set-up was carried out automatically by a CAS-1200 instrument (Corbett Research). The real-time PCR was performed according to the instructions of the manufacturer, and the expression of CaTEF3 in each strain was used as the loading control. The relative quantification was based on two standard curves for comparisons, and the results were given as a ratio (Kofron et al., 1999). The level of CaNDT80 mRNA isolated from the cells containing the wild-type CaNDT80 gene was assigned the value 1, and the relative level of mRNA isolated from different strains was normalized accordingly.

Site-directed mutagenesis. The Arg432 residue of CaNdt80p was altered by site-directed mutagenesis using the QuikChange Site-Directed Mutagenesis kit (Stratagene, 200519). The primers HJL562 and HJL567 were used to mutate the CaNdt80p 432Arg to Ala on LOB45 and LOB49, to construct LOB119 and LOB122, respectively. Then, LOB119 was used to transform the S. cerevisiae strain SLO1 (Chen et al., 2004) containing the CDR1p–lacZ reporter, to generate SLO85. LOB122 was linearized at the SpeI site located in the promoter of CaNDT80 before being used to transform the Candt80/Candt80 homozygous mutant YLO132 (Chen et al., 2004) to generate the YLO263 (Candt80/Candt80 : : Candt80^R432A) strain. All constructs were assessed by PCR and sequencing.

Antifungal susceptibility tests. The Etest (Pfaller et al., 2003; Tapia et al., 2003) was used to determine susceptibility to antifungal agents for C. albicans. Homogenized isolated colonies grown on SD medium overnight were transferred in 0.85 % NaCl to achieve a density of 5x10⁶ cells ml^–1. A sterile swab was dipped into inoculum suspension and then used to swab the entire agar surface of an SD plate evenly. Fluconazole Etest strips (AB BIODISK) were applied to the plate when the excess moisture had been absorbed completely. The agar dilution method was also used to determine the susceptibility to antifungal agents of C. albicans. First, cells were diluted to OD₆₀₀ 2 (2x10⁷ cells ml^–1) and were spotted at 0.5 µl per spot with a replica device (Oxoid) onto plates containing different drugs, along with 10-fold serial dilutions. Fluconazole and miconazole, in 0.1 % DMSO, were prepared to final concentrations of 5 and 0.3 µg ml^–1, respectively. Cells were grown on SD media containing 0.1 % DMSO in the absence or presence of drugs for 2 days at 30 °C.

	RESULTS AND DISCUSSION

TOP INTRODUCTION METHODS RESULTS AND DISCUSSION REFERENCES

 
ScNdt80p failed to activate CDR1p–lacZ in S. cerevisiae

CaNdt80p was annotated as the C. albicans homologue of the S. cerevisiae transcription factor ScNdt80p due to the high degree of identity between the C terminal of CaNdt80p and the N terminal of ScNdt80p, a known DNA-binding domain (Fig. 1). Interestingly, there is no similarity between the N terminal of CaNdt80p and the C terminal of ScNdt80p (Chen et al., 2004). Thus, to investigate the closeness of functionality between these two proteins, we wished to determine whether ScNdt80p, like CaNdt80p, can activate CDR1p–lacZ in S. cerevisiae. The ß-Gal activity of CDR1p–lacZ was determined by liquid assay, and the results are summarized in Fig. 2. CaNDT80 in a high-copy-number vector (pRS426) increased the expression of CDR1p–lacZ approximately fivefold in S. cerevisiae SLO5, consistent with our previous report (Chen et al., 2004). On the other hand, ScNDT80 in the same vector did not affect the expression of CDR1p–lacZ in S. cerevisiae SLO71. Since ScNDT80 is tightly regulated and expressed only during meiosis (Pak & Segall, 2002), we constructed a chimera in which ScNDT80 was under the control of the CaNDT80 promoter (LOB116) to further determine the effect of ScNdt80p in activating the expression of CDR1p–lacZ. The expression of CDR1p–lacZ was not induced by ScNdt80p in S. cerevisiae SLO79 (Fig. 2), even though the ScNDT80 gene under the control of the CaNDT80 promoter was expressed (Fig. 3a, lane 12). The fact that ScNdt80p failed to activate CDR1p–lacZ may be due to the difference in activation domain between ScNdt80p and CaNdt80p.

View larger version (23K): [in this window] [in a new window]   Fig. 3. Expression of different chimeras in S. cerevisiae and both CaNDT80 and Candt80R432A alleles in C. albicans. (a) RT-PCR was performed with primers specific for ScEFB1, ScNDT80 and CaNDT80. Total RNA was isolated from different S. cerevisiae strains. The template for lane 1 was genomic DNA from the parental S. cerevisiae strain (10560-2B). The remaining templates were cDNAs from different S. cerevisiae strains: lane 2, SLO2; lane 3, SLO5; lane 4, SLO71; lane 5, SLO79; lane 6, SLO77; lane 7, SLO75; lane 8, SLO81; lane 9, SLO85; lane 10, water as a negative control; lane 11, SLO5; and lane 12, SLO79. Lanes 2–10 were from reactions containing primers for amplifying CaNDT80, and lanes 11–12 were for ScNDT80. (b) RT-PCR was performed with primers specific for CaEFB1 and CaNDT80, separately. The total RNAs were isolated from different C. albicans strains. The template for lane 1 was genomic DNA from the wild-type C. albicans strain (SC5314), and the remaining templates were cDNAs from different strains: lane 2, Candt80/Candt80 : : CaNDT80 (YLO137); lane 3, Candt80/Candt80 (YLO133); lane 4, Candt80/Candt80 : : Candt80R432A (YLO263); lane 5, water as negative control. The sizes of the RT-PCR products are shown on the right.

CaNdt80p with a mutation of the DNA-binding domain failed to activate CDR1p–lacZ in S. cerevisiae

The DNA-binding capability of ScNdt80p decreased approximately 19-, 27- and 14-fold when the DNA-binding domain contained a mutation at residues Arg111, Arg177 and Arg254, respectively. Several homologues of ScNdt80p from higher eukaryotes, including proteins from Neurospora crassa, Dictyostelium discoideum, Caenorhabditis elegans, Drosophila melanogaster and humans, have been reported (Montano et al., 2002). Interestingly, Arg177 and Arg254 are absolutely conserved, and Arg111 is highly conserved (Arg or Lys), in all these proteins (Montano et al., 2002). Since the DNA-binding domain of CaNdt80p is important for activating CDR1p–lacZ in S. cerevisiae, CaNdt80p with mutations of the DNA-binding domain could fail to activate CDR1p–lacZ. To assess this possibility, we mutated the Arg432 amino acid of CaNdt80p, corresponding to ScNdt80p residue Arg177, known to be the most important residue for the DNA-binding activity of ScNdt80p (Fig. 1b) (Montano et al., 2002). We found that overexpression of Candt80^R432A in the SLO85 strain failed to activate CDR1p–lacZ in S. cerevisiae (Fig. 2). In addition, this is also consistent with the concept that this domain of CaNdt80p is important for the activity associated with DNA binding.

Construction of the Candt80/Candt80 : : Candt80^R432A C. albicans strain

Though we showed that the DNA-binding domain of CaNdt80p is important for activating CDR1p–lacZ in S. cerevisiae, we were still not sure if the DNA-binding domain of CaNdt80p indeed plays a role in drug resistance in C. albicans. To test this hypothesis, we reintroduced the Candt80^R432A mutated allele into the locus of CaNDT80 in the Candt80/Candt80 mutant to determine if this mutated allele, like the wild-type CaNDT80 gene, could restore the drug susceptibility phenotype to the Candt80/Candt80 mutant. After assessing the constructed genome with PCR and sequencing, the expression of this mutated allele in the reintroduced Candt80/Candt80 : : Candt80^R432A strain was also determined by RT-PCR (Fig. 3b), along with the Candt80/Candt80 : : CaNDT80 strain. First, there was no genomic DNA contamination in the preparations of total RNA, since none of the 928 bp intron-containing CaEFB1 DNA fragment was amplified from cDNA samples (Fig. 3b, lanes 2–4). The wild-type CaNDT80 (Fig. 3b, lane 2) and Candt80^R432A (Fig. 3b, lane 4) alleles were expressed. We also determined the level of both the CaNDT80 and the Candt80^R432A alleles by real-time PCR, and the results indicated that both alleles were expressed at the same level.

The Candt80^R432A allele with a mutation of the DNA-binding domain failed to complement Candt80/Candt80 mutant cells

Consequently, to further elucidate the function of the DNA-binding domain of CaNdt80p in C. albicans, we analysed the drug susceptibility of different strains by the Etest assay and by the agar dilution method. The Etest results are shown in Fig. 4(a). The MIC to fluconazole of the Candt80/Candt80 cells was 0.5 mg l^–1. The MICs to fluconazole of the Candt80/Candt80 mutant cells containing a copy of the wild-type CaNDT80 gene (Candt80/Candt80 : : CaNDT80) increased to 1.5 mg l^–1. Thus, the wild-type CaNDT80 gene conferred drug resistance on the Candt80/Candt80 mutant cells. In contrast, the Candt80/Candt80 : : Candt80^R432A cells were as sensitive to the drug as the Candt80/Candt80 mutant cells. The results of the agar dilution method are shown in Fig. 4(b). All cells tested grew in the absence of drug. The Candt80/Candt80 mutant cells with the wild-type CaNDT80 allele were more susceptible to the drug than the wild-type cells. This may be due to the dosage effect described previously (Chen et al., 2004; Kohler & Fink, 1996). The Candt80/Candt80 : : CaNDT80 cells with the wild-type CaNDT80 allele were more resistant to drugs than the Candt80/Candt80 mutant cells, consistent with the Etest results and our previous report that CaNdt80p is involved in drug resistance (Chen et al., 2004). It was noteworthy that the Candt80/Candt80 : : Candt80^R432A cells with the mutated Candt80^R432A allele were as sensitive to drugs as the Candt80/Candt80 mutant cells. The results of both the Etest assay and the agar dilution method suggested that, unlike the wild-type CaNDT80 allele, Candt80^R432A failed to complement the lack of drug resistance of the Candt80/Candt80 cells.

View larger version (72K): [in this window] [in a new window]   Fig. 4. The Candt80R432A allele failed to complement Candt80/Candt80 mutant cells. Susceptibility to azole drugs was determined by both the Etest assay (a) and the agar dilution method (b). Fluconazole strips (FL, 0.016–256 µg ml–1) were used for the Etest. For agar dilution, cells were grown on media in the absence of drug, or in the presence of 5 µg fluconazole ml–1 or 0.3 µg miconazole ml–1. The results of both assays for four tested strains, CaNDT80/CaNDT80 (SC5314), Candt80/Candt80 : : CaNDT80 (YLO137), Candt80/Candt80 (YLO133), and Candt80/Candt80 : : Candt80R432A (YLO263) were photographed after 2 days growth at 30 °C.

	ACKNOWLEDGEMENTS

 
This work was in part supported by grants 94-2320-B-400-001 and 94-2320-B-009-001 from the Taiwan National Science Council, and grant CL-094-PP-05 from the Taiwan National Health Research Institutes.

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