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1 Institute of Microbiology, University of Lausanne and University Hospital Center, CH-1011 Lausanne, Switzerland
2 School of Biosciences, University of Exeter, Exeter EX4 4QD, UK
Correspondence
Dominique Sanglard
Dominique.Sanglard{at}chuv.ch
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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In C. albicans, two ABC transporters, Cdr1p and Cdr2p (for Candida drug resistance) and one MFS transporter, Mdr1p (multidrug resistance) were shown to be upregulated in several azole-resistant isolates (Sanglard et al., 1995
). Each of the genes encoding these proteins can be upregulated in distinct clinical azole-resistant strains. The transcription of the gene encoding Mdr1p, MDR1, is almost absent in azole-susceptible isolates, but is measurable in specific azole-resistant cells. The gene encoding Cdr1p, CDR1, shows basal transcriptional activity in azole-susceptible isolates, but higher transcription levels in azole-resistant isolates. CDR1 is usually co-ordinately upregulated in such isolates with CDR2, a gene with no detectable transcriptional activity in azole-susceptible isolates. Interestingly, these transporters can also be induced transiently by treating cells with other drugs such as oestradiol and fluphenazine for CDR1 and CDR2, and benomyl or H2O2 for MDR1 (De Micheli et al., 2002; Gupta et al., 1998; Karababa et al., 2004), mimicking their expression in azole-resistant cells. Recently we demonstrated the crucial role of Tac1p (transcriptionnal activator of CDR genes), a Zn(2)-Cys(6) transcription factor, in the regulation of CDR1 and CDR2 in clinical azole-resistant isolates and during other drug treatments (Coste et al., 2004, 2007). We showed that Tac1p acts through the previously described drug-responsive element (DRE) located in the promoters of CDR1 and CDR2 (Coste et al., 2004; De Micheli et al., 2002). The presence of gain-of-function mutations in TAC1 is one of the mechanisms permitting development of antifungal drug resistance (Coste et al., 2006).
In S. cerevisiae, multidrug transporter genes are regulated by a network of transcription factors with leucine zipper dimerization domains (YAP1 and YAP2) or with Zn(2)-Cys(6) DNA-binding motifs (PDR1, PDR3). Alteration of the transcription factor PDR1 is associated with increased transcription of the ABC transporters PDR5, SNQ2 and YOR1 (Balzi et al., 1994; Carvajal et al., 1997). The Pdr1p and Pdr3p binding site has been delimited to a consensus [5'-TCCG(C/T)GGA-3'] known as a PDRE (pleiotropic drug resistance responsive element) (Katzmann et al., 1996) and is also present in the promoters of additional ABC-transporter genes, for example PDR10 and PDR15 (Decottignies et al., 1998; Wolfger et al., 1997). It is possible that similar transcription factor networks operate by transactivating multidrug transporters genes in C. albicans isolates resistant to azole antifungals. Such transcription factors may be involved in the regulation of MDR1 or the basal level of CDR1 transcription, neither of which is dependent on Tac1p (Coste et al., 2004).
In this paper we describe the isolation of such factors by functional complementation of a C. albicans library in a S. cerevisiae PDR1/PDR3 mutant. Using such an approach, Talibi & Raymond (1999) and Yang et al. (2001) previously isolated two distinct transcription factors (FCR1 and FCR3) that were able to restore PDR5 regulation. Using a similar approach we have isolated a further set of three genes that complement PDR1/PDR3 mutants and confer resistance to azoles in S. cerevisiae. Although these genes could restore PDR5 transcription in S. cerevisiae, their involvement in regulating CDR1, CDR2 and MDR1 expression in C. albicans could not be established, thus leading to a wider examination of their roles.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. These strains were grown either in complete medium YEPD, containing 1 % Bacto peptone (Difco), 0.5 % yeast extract (Difco) and 2 % glucose (Fluka), or in minimal medium YNB (Yeast Nitrogen Base, Difco) and the required carbon source (Fluka). Strain DSY1654 was obtained from DSY669 by inactivation of the PDR5 ORF using the kanMX6 module as outlined by Longtine et al. (1998). For solid media, 2 % agar (Difco) was added to the media. Oleate-containing medium was composed of 0.12 % sodium oleate and 0.2 % Tween 40. Other carbon sources (sodium acetate, ethanol, glycerol and sodium lactate) were adjusted to 1 % (w/v) and acetic acid to 0.05 % (v/v). Escherichia coli DH5
(Hanahan, 1985) was used as a host for plasmid constructions and propagation. It was grown in LB (Luria–Bertani broth) or on LB plates, supplemented with ampicillin (0.1 mg ml–1) when required.
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). For C. albicans transformations, cells from 0.2 ml stationary-phase cultures were resuspended in 0.1 ml of a solution containing 200 mM lithium acetate (pH 7.5), 40 % (w/v) PEG 8000, 15 mg DTT ml–1, 250 µg denatured salmon sperm DNA ml–1. Transforming DNA (1–5 µg) was added to the yeast suspension, which was then incubated for 60 min at 43.5 °C. Transformation mixtures were plated directly on to selective plates.
Measurement of β-galactosidase activities.
To measure β-galactosidase activities in S. cerevisiae strain PB4, 100 µl crude protein extract was incubated for 30 min at 30 °C with 400 µl Z buffer (60 mM Na2HPO4, 40 mM NaH2PO4, 10 mM KCl, 1 mM MgSO4, 50 mM β-mercaptoethanol). Next, 150 µl ONPG (Roche) previously diluted to a final concentration of 1 mg ml–1 in filter-sterilized 0.1 M KH2PO4 (pH 7.0) was added to the mixture, which was further incubated for 30 min at 30 °C. The reaction was stopped by adding 400 µl 1 M Na2CO3. Reactions were then mixed by inversion and centrifuged for 1 min at 13 000 r.p.m. A420 was measured using a spectrophotometer (Spectrometer Lambda 18, Perkin Elmer). Plasmids containing the PDRE-CYC1 and PDREmut-CYC1 chimeric constructions (pDK52 and pDK53, respectively) were a generous gift from S. Moye-Rowley (University of Iowa, Iowa City, USA) and were introduced into S. cerevisiae PB4. These plasmids were introduced simultaneously with pDS139, pDS173 and pS174 (containing CTA4, ASG1 and CTF1, respectively) or the parent vector (YEp24) alone. The PDR5-lacZ (pDS450) fusion was produced by inserting the PDR5 promoter (–1246 bp) with 89 bp of coding sequence as a SmaI–HindIII fragment into YEp367 and was a kind gift of A. Delahodde and C. Jacq (Ecole Normale Supérieure, Paris, France). The β-galactosidase activities were calculated in Miller units as recommended by Ausubel et al. (1987).
Plasmids pDS1161 and pDS1181, which are derived from Yip353 and contain a CDR2(-DRE) promoter element, were described previously (Coste et al., 2004). As compared to pDS1161, pDS1181 contains TAC1 from C. albicans.
Drug susceptibility testing.
Yeast cultures were grown overnight in YEPD and diluted to a density of 1.5x107 cells ml–1 and 10-fold dilutions were performed. Cell suspensions in 4 µl aliquots were spotted onto YEPD plates containing drugs (see Fig. 3 for compound concentrations used). Plates were incubated for 48 h at 30 °C for S. cerevisiae and at 35 °C for C. albicans.
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; Hiller et al., 2006a) by chemoluminescence with an ECL kit according to the recommendations of the manufacturer (Amersham Biosciences).
Construction of C. albicans mutant strains.
Restriction maps for each gene investigated in this study and the positions of deletions are given in Fig. 1(A). For the disruption of CTA4, a 2.2 kb BamHI–EcoRV fragment containing part of the CTA4 ORF was first cloned into pBluescript KS+ to yield pDS516. A 3.7 kb SphI–BglII fragment with the URA3-blaster cassette from pMB7 was cloned into pDS516 previously digested with the same enzymes, thus removing an internal 0.5 kb from CTA4, to yield pDS522. The linear fragment obtained after digestion of pDS522 with ApaI and SacI was used to transform C. albicans.
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), respectively. The PCR fragments were cloned in the XhoI–XbaI sites of pBluescript KS+ to yield pDS656 and pDS640, respectively. Deletions within cloned regions in these plasmids were carried out by PCR with deletion primer pairs ASG1-Bgl/ASG1-Pst (deletion in ASG1 from –62 to +873) and CTF1-Bgl/CTF1-Pst (deletion in CTF1 from +1735 to +2740). The 3.7 kb PstI–BglII fragment with the URA3-blaster cassette from pMB7 was cloned into the PCR fragment previously digested with PstI and BglII to obtain disruption cassettes pDS654 and pDS644. For transformation in C. albicans, linear fragments were obtained by digestion of deletion constructs with ApaI and SacI.
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). Ura– colonies for each heterozygous mutant were next transformed with the appropriate linearized disruption cassettes. The verification of homozygous mutants was performed by PCR and Southern blot analysis (see Fig. 1
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Complementation of homozygous mutants was achieved by expression of CTA4, ASG1 or CTF1 under the control of the ADH1 promoter. ORFs of CTA4, ASG1 and CTF1 were amplified by PCR with primer pairs CTA4-5/CTA4-3, ASG1-5/ASG1-3 and CTF1-5/CTF1-3, respectively (Table 2). PCR fragments were next cloned into the expression plasmid pYPB1-ADH1pL (Leberer et al., 1996) digested by XhoI and BglII to yield plasmids pDS1538, pDS1536 and pDS1537, respectively. These plasmids were transformed into homozygous mutants into which the URA3 marker had been regenerated as mentioned above (see Table 1).
Southern blots.
DNA probes used in this study were generated by digestion of pDS139, pDS173 and pDS174 as described in Fig. 1(A). 32P-DNA labelled probes were generated by random priming (Feinberg & Vogelstein, 1984). Southern blots were performed as described previously (Sanglard et al., 1995). Radioactive signals were revealed by exposure to Kodak BioMax MR films (Amersham Biosciences).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Screening of a C. albicans genomic library in a pdr1
pdr3 S. cerevisiae mutant
In order to isolate such factors, we used a strategy reliant upon the complementation of the fluconazole hypersusceptibility of a S. cerevisiae mutant (DSY669), in which PDR1 and PDR3 were inactivated, with a C. albicans genomic library constructed in the vector YEp24 (Sanglard et al., 1997). After screening of approximately 105 independent transformants on selective medium containing 5 µg fluconazole ml–1, 34 fluconazole-resistant clones were obtained. After rescue from S. cerevisiae, the different plasmids were assayed for their ability to transactivate PDR5 expression using a lacZ reporter system. The plasmids conferring lacZ-positive expression were analysed by restriction analysis, revealing plasmids with three different restriction patterns as shown in Fig. 1(A). lacZ-negative plasmids were not further analysed. Each lacZ-positive plasmid with a unique restriction pattern was transformed back into DSY669 and transformants tested in plates containing fluconazole to confirm drug resistance phenotypes.
Characteristics of the cloned genes
Plasmids pDS139, pDS173 and pDS174 conferred resistance to multiple compounds when compared to the S. cerevisiae strain containing only the parent plasmid YEp24. As shown in Fig. 2, pDS139, pDS173 and pDS174 conferred resistance not only to fluconazole but also to itraconazole, rhodamine 6G and cycloheximide. The presence of pDS173 and pDS174 in DSY669 conferred enhanced resistance to cycloheximide as compared to pDS139. Nucleotide sequences of the insert flanking regions were determined and compared with the C. albicans genome data in order to obtain the entire sequence of the inserts. In each of the inserts distinct ORFs with Zn(2)-Cys(6) binuclear cluster domains could be located. The minimal complementing sizes for pDS139, pDS173 and pDS174 contained three ORFs which, according to the Candida Genome Database (CGD), corresponded to orf19.7374 (CTA4), orf19.166 (ASG1) and orf19.1499 (CTF1), respectively.
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). The best hit of CTA4 with the S. cerevisiae genome is OAF1, an oleate-activated transcription factor, which acts alone and as a heterodimer with Pip2p and activates genes involved in β-oxidation of fatty acids and peroxisome organization and biogenesis (Karpichev & Small, 1998). Interestingly, CTA4 is situated in an 11 kb genomic region of chromosome 3 containing two other genes with Zn(2)-Cys(6) motifs. Such gene ‘clusters’ have already been identified for similar transcription factors, including Tac1p, which is flanked by two other genes with the same DNA-binding motifs (Coste et al., 2004, 2006, 2007). The closest homologue of orf19.166 in S. cerevisiae is YIL130w (ASG1), with no clear function yet. It was named ASG1 in the Candida Genome Database due to this similarity. CTF1 has strong hits with GAL4 in S. cerevisiae and with transcription factors regulating cutinase activity in several other fungal species.
Pdr1p and Pdr3p in S. cerevisiae are the major Zn(2)-Cys(6) transcription factors interacting with PDRE. It can be expected that the C. albicans ORFs cloned here would also contain this conserved fungal DNA-binding domain. Since these genes complemented PDR1 and PDR3 defects and most likely PDR5 expression in S. cerevisiae, a quantitative analysis of lacZ reporter activities was performed in S. cerevisiae carrying a PDR5-lacZ fusion. The analyses revealed that the presence of ASG1 produced the highest reporter activities (215±32 U), which were comparable to the values (250±21 U) obtained with PDR3. CTA4 and CTF1 conferred only weak reporter activities (4.7±1.3 and 38±5 U, respectively) (Table 3). To show that these activities were dependent on the presence of a PDRE, chimeric promoter constructs in the promoter of CYC1 containing the wild-type PDRE or a mutated PDRE (PDREmut) were co-transformed into S. cerevisiae with the plasmids containing the C. albicans genes. CYC1-PDRE-dependent lacZ reporter activities were in general lower than those measured with the PDR5-lacZ fusion, and the CTF1-dependent activity was about 10-fold lower as compared to CTA4 and ASG1 or even PDR3 (Table 3). The lower CYC1-PDRE-dependent lacZ reporter activities can be attributed to differences in the number of PDRE sequences or other regulatory elements existing between the chimeric CYC1 promoter and the PDR5 promoter. Mutations in PDRE resulted in a decrease of reporter activity as compared to the wild-type PDRE for the three transcription factors. The CTA4-, ASG1- and CTF1-dependent reporter activity decreased by approximately 4-, 11-fold and 10-fold, respectively, when the PDRE was mutated (Table 3). Although PDREs were not mutated in the PDR5 promoter in this study because of their redundancy, these results suggest that the proteins encoded by CTA4, ASG1 and CTF1 could transactivate PDR5 through PDRE.
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). No significant PDR5-lacZ reporter activity was measured upon TAC1 expression in S. cerevisiae (see Table 3), thus suggesting that only a specific subset of C. albicans Zn(2)-Cys(6) finger transcription factors are able to interact with PDR5.
Role of CTA4, ASG1 and CTF1 in C. albicans
In order to get insight into the function of the genes cloned by functional complementation in S. cerevisiae, they were disrupted in C. albicans. The resulting mutants were analysed by Southern blotting to verify each disruption. As shown in Fig. 1(B), each mutant yielded restriction patterns expected from the disruption of each gene.
The CTA4, ASG1 and CTF1 mutants were tested in drug susceptibility assays performed on YEPD plates containing fixed drug concentrations. To avoid possible interference of the positioning of URA3 in these assays, all mutants and the parent wild-type were restored to Ura+ prototrophy by integrating URA3 at the RPS10 locus (Brand et al., 2004). Fig. 3(A) shows that the disruption of CTA4, ASG1 and CTF1 had no effect on fluconazole susceptibility. Other azoles including voriconazole and itraconazole were tested but no significant changes were observed (data not shown). The expression of the ABC transporters Cdr1p and Cdr2p, which are functional homologues of Pdr5p, was verified on cell extracts for each C. albicans mutant by Western blot analysis with Cdr1p and Cdr2p antibodies. Each mutant was also exposed to 10 µg fluphenazine ml–1 for 20 min, which is a condition known to induce both CDR1 and CDR2 (Coste et al., 2007). As observed in Fig. 4, Cdr1p and Cdr2p levels could still be increased after fluphenazine addition to all mutants, thus suggesting that CTA4, ASG1 and CTF1 played no role in the drug-dependent transcriptional activation of ABC transporters important for azole resistance in C. albicans. Slight variations were also observed in basal Cdr1p levels of the constructed mutants, especially in the cta4/ and ctf1/ mutants (Fig. 4). Given that these differences have no impact on the fluconazole susceptibility of these mutants (see Fig. 3A), it is likely that the variations in basal Cdr1p levels can be attributed to altered transfer efficiencies of the Western blot on the nylon membrane.
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), which also belong to the MFS family, we also verified whether CTA4, ASG1 and CTF1 could regulate MDR1, which encodes an MFS transporter of C. albicans involved in resistance to fluconazole (Sanglard et al., 1995). Western blot analysis with Mdr1p antibodies was performed on cell extracts of each mutant exposed to benomyl, a drug known to induce MDR1 (Rognon et al., 2006). This analysis revealed that the deletion of CTA4, ASG1 and CTF1 had no effect on the drug-induced production of Mdr1p (Fig. 4). Taken together, the results suggest that CTA4, ASG1 and CTF1, although capable of replacing the function of transcription factors involved in the regulation of multidrug transporters in S. cerevisiae, are not fulfilling the same function in C. albicans – at least as far as CDR1, CDR2 and MDR1 upregulation is concerned.
Since it is known that transcription factors from different fungal species can share similar structural domains, but still display species-specific functions, we decided to perform additional phenotypic assays with the homozygous mutants. Mutant behaviour was examined on media containing different carbon sources (sodium acetate, ethanol, sodium citrate, sodium lactate, glycerol and sodium oleate) or different compounds that could help detect alterations in cell wall integrity pathways (SDS, Congo red) or response to different pHs (3–9). The phenotypic screening revealed reduced growth of the asg1/ mutant on media containing sodium acetate, acetic acid, ethanol and sodium citrate (Fig. 3B). This phenotype could be reversed by overexpression of ASG1 under the control of the ADHI promoter. Additional phenotypic tests on media containing glucose and sodium acetate or acetic acid revealed growth defects only in the ASG1 mutant (Fig. 3C), indicating that these metabolites might exert toxicity in C. albicans. The asg1/ mutant was still able to assimilate glycerol, sodium lactate and sodium oleate (Fig. 3D), therefore suggesting that the growth defect could not be attributed to a general defect in carbon source assimilation through the TCA or glyoxylate cycles. Our results suggest rather that ASG1 controls a specific metabolic step, and further studies are under way to elucidate the role of this gene in carbon source assimilation.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). We addressed the possibility that PDR5 could be a likely target of these C. albicans genes in S. cerevisiae and be responsible for the drug resistance phenotypes shown in Fig. 2 by introducing plasmids containing the C. albicans genes into a strain lacking PDR5, PDR1 and PDR3. The absence of PDR5 did not change (with the exception of the S. cerevisiae strain containing CTF1 on cycloheximide-containing plates) the drug resistance phenotypes dependent on the C. albicans genes (Fig. 2B). The data therefore suggest that the C. albicans genes have additional gene targets in S. cerevisiae, probably including other PDRE-containing genes. The functional complementation procedure used to identify C. albicans genes also revealed, in separate studies, FCR1 and FCR3 (fluconazole resistance) (Talibi & Raymond, 1999; Yang et al., 2001). FCR1 encodes a transcription factor with a Zn(2)-Cy(6) DNA-binding motif as is the case for CTA4, ASG1 and CTF1, while FCR3 encodes a transcription factor with a bZip leucine zipper motif. The function of FCR1 in C. albicans has been addressed by the construction of a mutant lacking both FCR1 alleles; however, the mutant was more resistant to azoles than the wild-type, making FCR1 a negative regulator of azole resistance. The role of FCR1 in the regulation of CDR genes was not addressed in the study published by Talibi & Raymond (1999). A recent study reported that FCR1 overexpression could however decrease CDR1 upregulation upon exposure to fluconazole (Shen et al., 2007). Our own investigations have not revealed FCR1 as a key regulator of CDR1 and CDR2 (data not shown). The data presented here also did not reveal a functional link between the cloned C. albicans genes and the regulation of CDR genes or MDR1 (Fig. 4). One likely explanation for these differences in species-specific gene activation is that the DNA-binding motifs of the cis-acting elements responsible for the regulation of the PDR5, CDR and MDR1 genes are different. Pdr1p and Pdr3p target a sequence motif or PDRE (5'-TCCG/aC/tGG/cA/G-3'), where variable bases in the consensus motif are shown in lower case (DeRisi et al., 2000). This motif is not equivalent to the DRE [5'-CGGA(A/T)ATCGGATATTTTTTTT-3'] common to CDR1 and CDR2 (De Micheli et al., 2002) or to the benomyl-responsive element (BRE) described recently in MDR1 (Rognon et al., 2006) and also by other studies (Harry et al., 2005; Hiller et al., 2006b; Riggle & Kumamoto, 2006). Even though CTA4, ASG1 and CTF1 (as well as FCR1) show little similarity to PDR1 and PDR3, there may still be sufficient similarity to enable the activation of PDR5 through a PDRE. These transcription factors all have in common a Zn(2)-Cys(6) DNA-binding domain, which targets sequences with direct, inverted or everted palindromic repeats containing a 5'-CGG-3' motif, with various numbers of nucleotides in the intervening spacer region (MacPherson et al., 2006). It is possible therefore that when expressed in S. cerevisiae, the C. albicans proteins could interact with PDRE because of the presence of this 5'-CGG-3' motif. The exact DNA-binding motifs of Cta4p, Asg1p, Ctf1p and Fcr1p as well as their target genes remain to be established in C. albicans. Interestingly, a recent study reported that CTA4 was critical for nitrosative stress response in C. albicans. Cta4p was shown to bind a palindrome sequence (5'-CCGTCGG-3') in the promoter of YHB1, encoding a flavohaemoglobin responsible for the conversion of nitric oxide to nitrate (Chiranand et al., 2008). This sequence closely resembles the PDRE core sequence [5'-TCCG(C/T)GGA-3'] (Devaux et al., 2001) and it is therefore not too surprising that CTA4 is able to mediate PDR5 transcription as shown in the present study.
Very recently, the transcription factor MRR1, which also possesses a Zn(2)-Cys(6) DNA-binding motif, was discovered as a major regulator of MDR1 (Morschhauser et al., 2007). Interestingly, CTA4 is most similar among C. albicans ORFs to MRR1 and is situated only 1 kb downstream of MRR1. Along with orf19.7371, these three genes constitute a cluster of transcription factors within a 10 kb region. Although we could not demonstrate any involvement of CTA4 in MDR1 regulation, the physical association of these genes is intriguing in the framework of drug resistance, since TAC1, the regulator of CDR1 and CDR2, is also found in a cluster of three transcription factor genes with the same DNA-binding motifs. These two gene clusters, which both include genes involved in a similar function (drug resistance), suggest a common evolutionary process that probably resulted from gene duplication events associated with functional specialization. Synteny of these clusters among other sequenced yeast genomes, including Candida tropicalis, Candida parapsilosis, Pichia stipitis and Debaryomyces hansenii, is conserved and suggests the conservation of important functional traits (data not shown).
Since no association between azole resistance and CTA4, ASG1 and CTF1 could be made in C. albicans, additional phenotypic growth assays were performed. Systematic phenotypic screening tests on different carbon sources had been successfully applied in S. cerevisiae transcription factor mutants to reveal their functional categorization (Akache et al., 2001). Even though C. albicans transcription factors can share similar functions in S. cerevisiae that have been attributed by functional assays or sequence comparisons, their function in C. albicans can be considerably divergent, a notion described as ‘transcriptional rewiring’ (Martchenko et al., 2007). We addressed this possibility by performing a restricted number of phenotypic tests, which revealed growth defects on acetate- and ethanol-containing media when ASG1 was inactivated.
In C. albicans, acetate or ethanol metabolism involves gluconeogenesis via the glyoxylate cycle, thus raising the possibility that ASG1 controls specific metabolic steps of this pathway. However, because the ASG1 mutant was not compromised in oleate assimilation (Fig. 3D), this possibility remains unlikely. Toxicity of sodium acetate or acetic acid was revealed only in the ASG1 mutant (Fig. 3C). This toxicity was not pH-dependent, since independent experiments performed with this mutant on media buffered from pH 3 to pH 9 did not result in growth alteration as compared to the wild-type (data not shown). The exposure of C. albicans to acetic acid (40–60 mM, corresponding to a content of 0.3 %) triggers apoptotic events (Phillips et al., 2003) that are dependent on the RAS signalling pathway (Phillips et al., 2006). We have not yet investigated the involvement of ASG1 in acetic acid-dependent apoptosis of C. albicans; however, it is possible that this transcription factor acts as a downstream target of the RAS signalling pathway. Transcript profiling experiments using the ASG1 mutant in the presence/absence of sodium acetate and acetic acid are currently under way in our laboratory to identify ASG1-dependent genes in C. albicans and thus we hope to elucidate more precisely the role of this gene in carbon source assimilation and its hypothetical relationship to the RAS pathway.
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ACKNOWLEDGEMENTS |
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Edited by: J. F. Ernst
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Received 18 December 2007;
revised 18 February 2008;
accepted 18 February 2008.
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