Constitutive overexpression of the MDR1 (multidrug resistance) gene, which encodes a multidrug efflux pump of the major facilitator superfamily, is a frequent cause of resistance to fluconazole and other toxic compounds in clinical Candida albicans strains, but the mechanism of MDR1 upregulation has not been resolved. By genome-wide gene expression analysis we have identified a zinc cluster transcription factor, designated as MRR1 (multidrug resistance regulator), that was coordinately upregulated with MDR1 in drug-resistant, clinical C. albicans isolates. Inactivation of MRR1 in two such drug-resistant isolates abolished both MDR1 expression and multidrug resistance. Sequence analysis of the MRR1 alleles of two matched drug-sensitive and drug-resistant C. albicans isolate pairs showed that the resistant isolates had become homozygous for MRR1 alleles that contained single nucleotide substitutions, resulting in a P683S exchange in one isolate and a G997V substitution in the other isolate. Introduction of these mutated alleles into a drug-susceptible C. albicans strain resulted in constitutive MDR1 overexpression and multidrug resistance. By comparing the transcriptional profiles of drug-resistant C. albicans isolates and mrr1Δ mutants derived from them and of C. albicans strains carrying wild-type and mutated MRR1 alleles, we defined the target genes that are controlled by Mrr1p. Many of the Mrr1p target genes encode oxidoreductases, whose upregulation in fluconazole-resistant isolates may help to prevent cell damage resulting from the generation of toxic molecules in the presence of fluconazole and thereby contribute to drug resistance. The identification of MRR1 as the central regulator of the MDR1 efflux pump and the elucidation of the mutations that have occurred in fluconazole-resistant, clinical C. albicans isolates and result in constitutive activity of this trancription factor provide detailed insights into the molecular basis of multidrug resistance in this important human fungal pathogen.
The yeast Candida albicans is usually a harmless commensal in many healthy people where it resides on mucosal surfaces of the gastrointestinal and urogenital tract, but it can also cause superficial as well as life-threatening systemic infections, especially in immunocompromised patients [1]. Infections by C. albicans are commonly treated with the antimycotic agent fluconazole that inhibits the biosynthesis of ergosterol, the major sterol in the fungal cell membrane. However, C. albicans can develop resistance to fluconazole, especially during long-term treatment of oropharyngeal candidiasis, which frequently affects HIV-infected persons and AIDS patients [2]. Molecular fingerprinting of serial C. albicans isolates from recurrent episodes of oropharyngeal candidiasis has shown that fluconazole resistance usually develops in previously susceptible strains, and such serial isolates from the same patient, so-called matched isolates, have proved an excellent tool to study the molecular basis of drug resistance [3–10]. Fluconazole resistance can be caused by different mechanisms, including alterations in the sterol biosynthetic pathway, increased expression of the ERG11 gene that encodes the target enzyme of fluconazole, sterol 14α-demethylase (Erg11p), mutations in the ERG11 gene that result in reduced affinity of Erg11p to fluconazole, and overexpression of genes encoding membrane transport proteins, which transport fluconazole out of the cell. In clinical C. albicans strains, several of these mechanisms are often combined to result in a stepwise development of clinically relevant fluconazole resistance (for a review, see [11]). A major mechanism of drug resistance in C. albicans is the constitutive upregulation of genes encoding efflux pumps that actively transport fluconazole and many other, structurally unrelated toxic compounds out of the cell. Two types of efflux pumps have been identified in C. albicans [12–15]. The CDR1 and CDR2 genes encode ATP-binding cassette (ABC) transporters, whereas MDR1 encodes a multidrug efflux pump of the major facilitator superfamily. In drug-susceptible C. albicans strains, MDR1 is expressed at low or non-detectable levels in standard laboratory media, but its expression can be induced when the cells are grown in the presence of certain toxic compounds, like benomyl, hydrogen peroxide, or diamide [16–20]. In contrast, many fluconazole-resistant, clinical C. albicans isolates constitutively overexpress MDR1 [3,4, 7–10]. Inactivation of MDR1 in such MDR1 overexpressing C. albicans isolates increased their susceptibility to fluconazole, confirming that MDR1 overexpression contributed to fluconazole resistance [21]. The increased resistance of such isolates to other metabolic inhibitors, like cerulenin, brefeldin A, and diamide was completely abolished after MDR1 deletion, indicating that the resistance of the strains to these toxic compounds was mainly or exclusively mediated by Mdr1p [22,23]. Comparison of the MDR1 promoter sequences in matched pairs of fluconazole-susceptible and MDR1 overexpressing, fluconazole-resistant C. albicans isolates from the same patient demonstrated that the constitutive MDR1 upregulation in the resistant isolates was not caused by promoter mutations but by alterations in trans-regulatory factor (s) [24]. Several groups have identified sequences in the MDR1 promoter region that mediate its upregulation in drug-sensitive strains in response to inducing chemicals and its constitutive activation in drug-resistant strains [19,20,25,26]. However, in contrast to the ABC transporters CDR1 and CDR2, whose expression has recently been shown to be controlled by the transcription factor Tac1p, which is mutated in CDR1/CDR2 overexpressing C. albicans strains [27,28], the regulatory factors controlling MDR1 expression and the mutations that are responsible for its constitutive overexpression in drug-resistant clinical isolates have not yet been identified. In this study, we compared the alterations in gene expression occurring in three different MDR1 overexpressing, clinical C. albicans isolates on a genome-wide scale to identify genes that are commonly upregulated with MDR1. This approach led to the identification of a central regulator of MDR1 expression and to the elucidation of the molecular basis of MDR1 overexpression and multidrug resistance in clinical C. albicans isolates.
To identify genes that are coordinately upregulated with MDR1, we compared the transcriptional profiles of three matched pairs of fluconazole-susceptible and fluconazole-resistant clinical C. albicans isolates. F1 and G1 are the first, drug-susceptible isolates of two well-characterized series of clinical C. albicans isolates and do not detectably express MDR1, while F5 and G5 are the last isolates in each series that overexpress MDR1 and have become multidrug-resistant [8,21,23,24]. An additional isolate pair from another patient, isolates 5833 (no MDR1 expression) and 6692 (MDR1 overexpression), was obtained from Martine Raymond and has been described recently [29]. As can be seen in Figure 1, a common set of 21 genes was consistently upregulated in all three MDR1 overexpressing isolates, including all the genes encoding proteins that had previously been identified as upregulated in isolates F5 and G5 by proteome analysis [23]. In addition, seven genes were downregulated in all resistant isolates. The complete data set for all differentially expressed genes in the pairwise comparisons can be found in Table S1. Interestingly, one gene, orf19. 7372 (IPF1266), which was moderately upregulated in the MDR1 overexpressing strains, encodes a predicted zinc cluster transcription factor that has been given the preliminary name ZCF36 in the Candida Genome Database (http: //www. candidagenome. org/). As transcription factors often regulate their own expression in addition to that of their target genes, we hypothesized that this transcription factor might be involved in MDR1 expression and thereby control multidrug resistance. The results shown below demonstrate that this was indeed the case and we have therefore named orf19. 7372 as MRR1, for multidrug resistance regulator. To investigate if MRR1 affects drug resistance in C. albicans, we deleted the gene in the C. albicans model strain SC5314 as well as in the drug-resistant clinical isolates F5 and G5 using the SAT1-flipping strategy ([30], Figure 2A and 2C, lanes 1–3, and unpublished data). From each parental strain, two independent homozygous mrr1Δ mutants were constructed (see Table S2) and tested for their susceptibilities to various metabolic inhibitors to which MDR1 overexpression confers resistance [22,23,31]. Inactivation of MRR1 in the drug-susceptible strain SC5314, which does not detectably express MDR1 under standard growth conditions, did not affect its susceptibility to the tested compounds, except for diamide, which is known to induce MDR1 expression (see also below). In contrast, the mrr1Δ mutants of the MDR1 overexpressing isolates F5 and G5 completely lost their resistance to cerulenin, brefeldin A, and diamide and became as susceptible to these inhibitors as mdr1Δ mutants derived from these strains or the matched, drug-susceptible isolates F2 and G2, which were the last isolates in each series that did not detectably express MDR1 [8] (Figure 3). The heterozygous mrr1 mutants exhibited intermediate resistance. Resistance of isolates F5 and G5 to these compounds was mediated mostly or exclusively by MDR1 overexpression, since resistance was lost after deletion of MDR1 [22,23]. In contrast, MDR1 overexpression contributes only partially to fluconazole resistance of these isolates, which is caused by a combination of different mechanisms [8,21]. Interestingly, deletion of MRR1 in isolates F5 and G5 had an even stronger effect than inactivation of MDR1, suggesting that MRR1 is required for various mechanisms of fluconazole resistance. The increased fluconazole resistance of isolate F5 as compared with isolate F2 was completely lost in the mrr1Δ mutants derived from this strain, while the mrr1Δ mutants of isolate G5, which also contains a mutation in the ERG11 gene that results in reduced affinity of fluconazole for its target enzyme, were still more resistant than the matched isolate G2, which does not contain this mutation [8]. The results presented above suggested that MRR1 affects drug resistance by controlling expression of the MDR1 efflux pump. Therefore, we compared MDR1 promoter activity in the mrr1Δ mutants and their wild-type parental strains. For this purpose, a PMDR1-GFP (green fluorescent protein) reporter fusion from plasmid pMPG2S (see Materials and Methods) was integrated into the genome of these strains, and GFP expression was quantified by flow cytometry. As can be seen in Figure 4A, the strong MDR1 promoter activity in isolates F5 and G5 was completely abolished after deletion of MRR1, demonstrating that MRR1 is required for the constitutive activation of the MDR1 promoter in these clinical isolates. The heterozygous MRR1/mrr1Δ mutants exhibited reduced MDR1 promoter activity, showing that both MRR1 alleles contributed to MDR1 overexpression in the drug-resistant isolates. The loss of MDR1 expression in the mrr1Δ mutants was also independently confirmed by quantitative real-time reverse transcription (RT) -PCR (see Figure S1A). As mentioned above, strain SC5314 does not detectably express MDR1, and no significant MDR1 promoter activity was seen in the corresponding reporter strains (Figure 4B). However, MDR1 expression can be induced in drug-susceptible strains when the cells are exposed to certain chemicals [16–19]. We used two such compounds, benomyl and H2O2, to investigate whether MRR1 is also required for inducible MDR1 expression. These chemicals were chosen because it has recently been reported that different regions in the MDR1 promoter mediate its upregulation by benomyl and H2O2 [20]. While the deletion of one MRR1 allele had no effect on MDR1 promoter activity under these conditions, activation of the MDR1 promoter by either of the two compounds was almost completely abolished in the homozygous mrr1Δ mutants (Figure 4B). Therefore, MRR1 is required for both the constitutive MDR1 overexpression in drug-resistant, clinical C. albicans isolates and the inducible MDR1 expression in a drug-susceptible strain. Since MRR1 was upregulated in the MDR1 overexpressing clinical C. albicans isolates, we investigated whether artificial overexpression of MRR1 would result in activation of the MDR1 promoter. For this purpose, we placed the MRR1 coding region from strain SC5314 under the control of the strong ADH1 promoter in plasmid pZCF36E1 (see Materials and Methods). The PADH1-MRR1 fusion was integrated into strain CAG48B, a derivative of the fluconazole-sensitive laboratory strain CAI4, which expresses the GFP reporter gene from the endogenous MDR1 promoter (see Table S2). The resulting strain did not detectably express the GFP gene, similar to a control strain that carried an otherwise identical construct without the MRR1 gene (unpublished data), suggesting that overexpression of MRR1 was not sufficient to activate the MDR1 promoter and that the drug-resistant isolates F5 and G5 might carry gain-of-function mutations in MRR1. Therefore, we cloned and sequenced the MRR1 alleles of isolates F2, F5, G2, and G5 (see Table S3). Isolate F2 contained two polymorphic MRR1 alleles. The coding region of allele 1 (MRR1F2–1) was identical to the MRR1 sequence of strain SC5314, while allele 2 (MRR1F2–2) differed from it at 26 positions, with three of the polymorphisms resulting in amino acid exchanges. Only one allele (MRR1F5) was obtained from the matched resistant isolate F5 and this allele corresponded to allele 1 of isolate F2 except for a single C-T mutation at position 2047, which resulted in a proline-serine substitution at position 683 of Mrr1p. Direct sequencing of the PCR products confirmed the loss of heterozygosity in isolate F5 and the absence of the mutation in the MRR1 alleles of isolate F2. A similar situation was found for the isolate pair G2/G5. Isolate G2 contained two polymorphic MRR1 alleles (MRR1G2–1 and MRR1G2–2), which differed from one another at 28 positions, with two of the polymorphisms resulting in amino acid differences. G5 contained only one MRR1 allele (MRR1G5) that was identical with allele 2 of isolate G2 except for a single G-T mutation at position 2990, which resulted in a glycine–valine substitution at position 997 of Mrr1p. Therefore, both resistant isolates F5 and G5 had become homozygous for a mutated MRR1 allele, suggesting that these mutations might have caused the constitutive MDR1 overexpression and the resulting multidrug resistance. To directly test whether the P683S and G997V mutations in Mrr1p are responsible for MDR1 overexpression and drug resistance, we introduced the mutated MRR1 alleles from isolates F5 and G5 as well as the corresponding wild-type alleles from isolates F2 and G2 into the mrr1Δ mutants of strain SC5314. All four MRR1 alleles were integrated into one of the inactivated mrr1Δ alleles to ensure expression from the endogenous MRR1 promoter (see Figure 2B and 2C, lanes 4–7). In each case, two independent correct transformants were used for further analysis. Figure 5A shows that, as noted above, deletion of MRR1 in strain SC5314 did not affect its susceptibility to cerulenin, brefeldin A, and fluconazole, but the mutants displayed increased sensitivity to diamide, a compound that induces MDR1 expression and is an Mdr1p substrate. Insertion of the MRR1–1 allele from isolate F2 (which is identical to MRR1 of strain SC5314, so this also represented a reinsertion of the original allele) or the MRR1–2 allele from isolate G2 complemented the hypersusceptibility of the mutants to diamide, but did not increase resistance to cerulenin, brefeldin A, and fluconazole. In contrast, insertion of the mutated alleles from isolates F5 and G5 resulted in enhanced resistance to all compounds. To obtain direct evidence that the mutated MRR1 alleles confer drug resistance by activating the MDR1 promoter and, thus, mediate overexpression of the Mdr1p efflux pump, we integrated the various MRR1 alleles into a derivative of the laboratory strain CAI4 that expresses the GFP reporter gene from the endogenous MDR1 promoter. For this purpose, we first inactivated the MRR1 wild-type alleles in the reporter strain CAG48B, as described above for the other strains, and then reinserted one of the four different MRR1 alleles. Two independent transformants were kept for each MRR1 allele. Figure 5B shows that, while the MRR1F2–1 and MRR1G2–2 alleles from the susceptible isolates had no effect, expression of the corresponding mutated MRR1F5 and MRR1G5 alleles resulted in strong MDR1 promoter activity. MDR1 upregulation by the mutated MRR1 alleles was also independently confirmed by comparing MDR1 mRNA levels in strains carrying wild-type or mutated MRR1 alleles by quantitative real-time RT-PCR (see Figure S1B). These results demonstrated that the P683S and G997V mutations in Mrr1p caused constitutive MDR1 overexpression and multidrug resistance. A mutation in the transcription factor TAC1 has recently been shown to cause constitutive upregulation of the ABC-transporters CDR1 and CDR2 as well as drug resistance in certain C. albicans strains, but only after the strains had become homozygous for the mutated allele [28]. Since the resistant isolates F5 and G5 also had become homozygous for mutated MRR1 alleles, we tested whether these alleles could mediate MDR1 overexpression and drug resistance in the presence of a non-mutated, wild-type allele. Therefore, to produce strains that contained both a wild-type and a mutated allele, the MRR1 alleles from isolates F5 and G5 were inserted into the inactivated mrr1 allele in the heterozygous MRR1/mrr1Δ mutants derived from strain SC5314 (see Figure 2B and 2C, lanes 8 and 9). The mutated alleles conferred drug resistance also in the presence of a wild-type MRR1 allele, although we observed a slightly reduced resistance as compared with the strains containing only a mutated MRR1 allele, especially for the MRR1F5 allele (see Figure 5A). To directly compare MDR1 promoter activity in strains carrying only a mutated MRR1 allele or both a mutated and a wild-type MRR1 allele, the mutated MRR1 alleles were also integrated into the inactivated mrr1 allele of the heterozygous MRR1/mrr1Δ mutant with the PMDR1-GFP reporter fusion. Figure 5B shows that both mutated MRR1 alleles were able to activate the MDR1 promoter in the presence of a wild-type MRR1 allele, but the degree of activation was lower than in strains containing only a mutated allele. Again, this effect was more pronounced for the MRR1F5 allele. Taken together, these results demonstrate that the MRR1F5 and MRR1G5 alleles can act in a semi-dominant fashion and mediate MDR1 overexpression and multidrug resistance in the presence of a nonmutated MRR1 allele, but the presence of a wild-type MRR1 allele reduces the activity of the MRR1 alleles containing gain-of-function mutations. That deletion of MRR1 from the drug-resistant C. albicans isolates F5 and G5 affected fluconazole resistance more strongly than deletion of MDR1 suggests that Mrr1p controls the expression of additional genes that contribute to the increased fluconazole resistance of these isolates. Therefore, to identify the set of genes controlled by the transcription factor Mrr1p, we compared the gene expression profiles of isogenic strains expressing either a wild-type MRR1 allele (MRR1F2–1 or MRR1G2–2) or its constitutively active, mutated counterpart (MRR1F5 and MRR1G5, respectively). In addition, we compared the gene expression profiles of the clinical isolates F5 and G5, which carry the gain-of-function MRR1 alleles, with those of their mrr1Δ derivatives. As shown in Figure 6A and 6B, 20 and 27 genes were consistently upregulated in the transformants expressing the MRR1F5 and MRR1G5 alleles, respectively, and 19 of the 28 total genes were commonly upregulated by both gain-of-function alleles. As expected, CDR1 and CDR2 were not among the Mrr1p target genes. Strikingly, the 11 most strongly upregulated genes were the same in strains expressing the MRR1F5 or the MRR1G5 allele. All of these (plus three additional commonly upregulated genes) were also significantly overexpressed in the drug-resistant clinical isolates F5 and G5 as compared with the matched susceptible isolates F1 and G1, respectively, and downregulated again after deletion of MRR1 from the resistant isolates F5 and G5. These 14 genes, therefore, represent a core set of genes whose expression is controlled by Mrr1p, and some of them might contribute to fluconazole resistance in the clinical isolates (see Discussion). Twenty-eight genes (including MRR1) were consistently downregulated after deletion of MRR1 in the resistant isolates F5 and G5 (see Table 1) A complete list of all genes that were found to be differentially regulated in the mrr1Δ mutants as compared with their wild-type parental strains is provided in Table S4. Fourteen of the 28 genes were also upregulated by the MRR1F5 and MRR1G5 alleles in the SC5314 background and in the resistant isolates F5 and G5 as compared with the matched susceptible isolates F1 and G1, respectively. The 14 genes were mainly those most strongly affected by the MRR1 deletion. The other genes were either not consistently upregulated above the threshold level in the SC5314 transformants expressing only one mutated MRR1 allele (e. g. , MRR1 itself) or possibly had a strain-specific dependence on MRR1. Altogether, the results of the transcriptional profiling experiments provided a comprehensive list of genes that are regulated, directly or indirectly, by Mrr1p in various C. albicans strain backgrounds. The identification of these genes, in turn, provided clues about how gain-of-function mutations in MRR1 contribute to fluconazole resistance of clinical C. albicans strains, besides causing overexpression of the MDR1 efflux pump (see Discussion).
Since the initial report by Sanglard et al. more than ten years ago [3], many studies have shown that the major mechanism of resistance to the widely used antifungal agent fluconazole in clinical C. albicans isolates is the constitutive overexpression of efflux pumps, which actively transport this drug and other toxic substances out of the cell, thereby conferring multidrug resistance. It is well established that mutations in trans-regulatory factors are responsible for the upregulation of genes encoding efflux pumps in drug-resistant C. albicans isolates [24,32]; however, until recently the identity of these regulators has remained elusive. Two main factors, namely 1) the discovery of a cis-regulatory element in the promoters of CDR1 and CDR2 with features that are typical of binding sites of zinc cluster transcription factors, and 2) the observation that homozygosity at the mating type locus was linked with the development of azole resistance in certain clinical strains, led Coste et al. to exploit the available C. albicans genome sequence information to systematically search for candidate transcription factors that might regulate the expression of these efflux pumps. This strategy resulted in the identification of the zinc cluster transcription factor TAC1, which is located near the mating type locus, as the major regulator of CDR1 and CDR2 [27]. In contrast, no obvious criteria for a similar in silico search for a regulator of MDR1, the other efflux pump that mediates drug resistance in many clinical C. albicans isolates, were evident. Promoter deletion analyses performed by several research groups have identified three different activating regions in the MDR1 promoter, two of which contain binding sites for the transcription factors Cap1p and Mcm1p [19,20,25,26]. However, each of these regions could be individually deleted from the full-length MDR1 promoter without abrogating its constitutive activation in MDR1 overexpressing C. albicans isolates, which suggested that a transcription factor other than Cap1p or Mcm1p causes the upregulation of MDR1 in drug-resistant strains. Assuming that a common mechanism might be responsible for the constitutive MDR1 upregulation in such strains, we compared the alterations in gene expression that occurred in three different MDR1 overexpressing, drug-resistant C. albicans isolates. This approach led to the identification of MRR1, a zinc cluster transcription factor that was moderately upregulated in all three resistant isolates as compared with matched, drug-susceptible isolates, suggesting that this transcription factor might contribute to MDR1 overexpression and/or drug resistance. A role of MRR1 in fluconazole resistance would not have been easily inferred from genetic analysis of commonly used C. albicans laboratory strains, because deletion of MRR1 from the model strain SC5314 did not result in hypersusceptibility of the mutants. However, the development of methods to inactivate genes in clinical C. albicans strains [21,30] allowed us to demonstrate the essential role of MRR1 in drug resistance of two different MDR1 overexpressing C. albicans isolates. Similar findings were previously obtained with the efflux pump MDR1 itself, whose disruption in a C. albicans laboratory strain did not result in fluconazole hypersusceptibility, whereas MDR1 inactivation in MDR1 overexpressing strains reduced or abolished their resistance to fluconazole and other metabolic inhibitors [14,21,33]. We found that MRR1 is not only responsible for the constitutive overexpression of MDR1 in drug-resistant isolates, but also mediates the inducible MDR1 expression in a drug-susceptible strain. The transcription factors Cap1p and Mcm1p have been implicated in the induction of MDR1 expression by H2O2 and benomyl, respectively [20]. Interestingly, deletion of MRR1 almost completely abolished the induction of the MDR1 promoter in response to both of these stimuli, indicating that Mrr1p has a more central and essential role in the control of MDR1 expression and, depending on the environmental conditions, may cooperate in different ways with these other transcription factors to regulate expression of the efflux pump. The identification of MRR1 as the central regulator of MDR1 expression also enabled us to elucidate the genetic alterations that had occurred in drug-resistant C. albicans isolates and were responsible for MDR1 overexpression and multidrug resistance. In two clinical isolates we found different single nucleotide substitutions in MRR1 that resulted in amino acid exchanges in Mrr1p. The ability of the mutated MRR1 alleles to activate the MDR1 promoter and confer drug resistance when expressed in a C. albicans laboratory strain confirmed that these were indeed gain-of-function mutations that resulted in constitutive activation of the transcription factor. The two mutations, which were found in different regions of Mrr1p, most likely relieve the transcription factor from repression by an autoinhibitory domain or by another negatively acting factor that keeps Mrr1p in its inactive state in the absence of inducing signals. In both cases the MDR1 overexpressing, drug-resistant isolate had become homozygous for the mutated MRR1 allele, which suggested that Mrr1p containing a gain-of-function mutation would not be able to activate the MDR1 promoter in the presence of wild-type Mrr1p. However, we found that both mutated MRR1 alleles were able to induce MDR1 expression and cause drug resistance in the presence of a wild-type allele, albeit at slightly reduced levels as compared with strains containing only the mutated allele. This indicates that the gain-of-function alleles acted in a semi-dominant fashion. The comparison of heterozygous and homozygous mrr1Δ mutants with their drug-resistant parental strains showed that the presence of two rather than only one mutated MRR1 allele resulted in higher MDR1 promoter activity and drug resistance. Therefore, the increased activity of the transcription factor in strains carrying two copies of a mutated MRR1 allele instead of only one, coupled with a slight negative effect of wild-type Mrr1p on the activity of the activated form, appears to provide sufficient advantage during antimycotic therapy to select for the loss of heterozygosity once a gain-of-function mutation has occurred in one of the two MRR1 alleles. Such a loss of heterozygosity readily occurs in C. albicans either by loss of one chromosome and duplication of the homologous chromosome or by mitotic recombination between the two homologous chromosomes, as has been well documented in several recent studies [28,34,35]. Interestingly, deletion of the transcription factor MRR1 from MDR1 overexpressing C. albicans isolates reduced fluconazole resistance of these strains even more than deletion of MDR1 itself, suggesting that the gain-of-function mutations in MRR1 contribute to fluconazole resistance of these strains by other mechanisms, in addition to causing overexpression of the efflux pump. We, therefore, aimed to find out which of the many alterations in gene expression seen in the drug-resistant C. albicans clinical isolates (see Figure 1 and Table S1) were caused by the MRR1 mutations. When introduced into strain SC5314, both mutated MRR1 alleles caused the upregulation of 19 genes including MDR1 (note that some genes of the IFD family may in fact be alleles of the same gene, e. g. , those that were originally designated as IFD1 and IFD5 in CandidaDB but have now been assigned the same orf19 name). Some additional genes were reproducibly upregulated by only one of the two mutated alleles (one for MRR1F5 and eight for MRR1G5), which could be explained by differential effects of the P683S and G997V mutations on Mrr1p activity at the respective target promoters. The expression of 14 of the 19 genes that were upregulated by both mutated MRR1 alleles in the SC5314 background was found to be downregulated after deletion of MRR1 in both drug-resistant clinical isolates F5 and G5. A considerable number of additional genes were affected by inactivation of MRR1 in the clinical isolates (see Table S4), but only 28 genes were downregulated in the mrr1Δ mutants of both parental strains, indicating that the other effects of MRR1 deletion depended on the strain background. It is striking that of the core set of Mrr1p target genes (i. e. , the 14 genes that were upregulated by the mutated MRR1 alleles in the SC5314 background as well as upregulated in the drug-resistant isolates F5 and G5 and downregulated in the mrr1Δ mutants of these strains) many encode putative oxidoreductases (see Table 1). IPF7817 is a member of the NAD (P) H oxidoreductase family that is strongly induced during oxidative stress [36,37]. It is supposed to be involved in the regulation of intracellular redox homeostasis, as mutants in which the gene was inactivated had increased intracellular levels of reactive oxygen species (ROS) and, presumably as a compensatory mechanism, upregulated other redox-related genes [38]. IPF17186 is a member of the ThiJ/PfpI protein family [39]. Its ortholog in S. cerevisiae, also named HSP31, is induced by the transcription factor Yap1p in response to oxidative stress. Also, an hsp31Δ mutant is hypersensitive to a subset of ROS generators, suggesting that Hsp31p may protect the cell against oxidative stress [40]. GRP2 is a homolog of S. cerevisiae GRE2, which encodes a stress-induced NADH-dependent methylglyoxal reductase that is regulated by the pleiotropic drug resistance regulator Pdr1p. A gre2Δ mutant exhibited a growth defect under conditions of membrane stress and activated ERG genes as a compensatory mechanism, and it displayed an increased sensitivity to ergosterol biosynthesis inhibitors [41]. IFD1, IFD4, IFD5, IFD6, and IFD7 encode proteins of the aldo-keto reductase family and are homologs of the putative aryl-alcohol dehydrogenase YPL088w of S. cerevisiae. Interestingly, YPL088w is regulated by the transcriptional regulators Yrr1p and Yrm1p, which are involved in the control of multidrug resistance [42]. Similarly, IPF5987 encodes a member of the aldo-keto reductase family, and its ortholog in S. cerevisiae is also transcriptionally regulated by Yrr1p and Yrm1p [42]. MDR1 and other Mrr1p target genes, almost all of which have also been found to be upregulated in another MDR1 overexpressing clinical C. albicans isolate [18], are induced in the presence of chemicals that exert oxidative stress upon the cells, like hydrogen peroxide or diamide. They are also induced by the microtubule destabilizing agent benomyl [16,18–20]. Interestingly, benomyl treatment causes lipid peroxidation and glutathione depletion in rats and these effects were blocked by treatment with antioxidants, suggesting that the in vivo toxicity of benomyl may be associated with oxidative stress to cellular membranes [43]. Therefore, it seems possible that the activation of Mrr1p by all these compounds may be a response to oxidative damage of the cells, and the function of many of the target genes that are induced by activated Mrr1p could be to restore the intracellular redox balance. This may also explain how the upregulation of these genes contributes to fluconazole resistance of clinical C. albicans strains containing MRR1 gain-of-function mutations. In addition to inhibiting ergosterol biosynthesis, azoles have been shown to increase the level of endogenous reactive oxygen species in C. albicans cells, and the decrease in cell viability associated with miconazole treatment was significantly prevented by addition of an antioxidant [44]. These observations suggest that ROS plays a role in the mechanism of action of azole antifungal agents and that ROS detoxification mechanisms may contribute to azole resistance. In contrast to other compounds that cause oxidative stress, like hydrogen peroxide or diamide, fluconazole does not induce MDR1 expression [8,16]. Therefore, the constitutive upregulation of MDR1 and other Mrr1p target genes in strains containing MRR1 gain-of-function mutations provides protection of the cells against this antifungal agent. A major regulator of the oxidative stress response in C. albicans is the bZIP transcription factor Cap1p [36,45,46]. Like MDR1, several other Mrr1p target genes (IPF7817, IPF17186, and GRP2) contain a YRE element, the putative binding site for Cap1p, in their promoters. Their expression is induced by hydrogen peroxide in a Cap1p-dependent manner [18,20,36,45]. Therefore, Cap1p and Mrr1p may act together to regulate expression of these genes in response to oxidative stress. Cap1p also controls the expression of other genes that are involved in the oxidative stress response, like the thioredoxin reductase TRR1, the glutathione reductase GLR1, the glutathione S-transferase GTT1, and the superoxide dismutase SOD2 [36,45], and which were not found among the Mrr1p target genes. Unlike CAP1 inactivation [45], MRR1 deletion or MRR1 gain-of-function mutations had no effect on the susceptibility of C. albicans to H2O2 (unpublished data), which in contrast to diamide is not a substrate of the Mdr1p efflux pump [23]. Therefore, the Cap1p target genes that are not controlled by Mrr1p and that are typical oxidative stress-response genes seem to be more important for the resistance of C. albicans to hydrogen peroxide, while the Mrr1p target genes contribute to fluconazole resistance, presumably because the two compounds cause different types of damage within the cells. The precise function of most of the Mrr1p target genes is currently unknown and their potential involvement in an oxidative stress response remains speculative. Alternatively, it is possible that fluconazole treatment causes the accumulation of other toxic molecules that are eliminated by the combined action of the oxidoreductases and other gene products whose expression is regulated by Mrr1p. The identification of MRR1 as the major regulator of MDR1 expression and the elucidation of the mutations in clinical isolates that cause constitutive activity of this transcription factor represent a major step forward in our understanding of multidrug resistance development in C. albicans. Important questions that can now be addressed include how Mrr1p is normally activated in response to inducing signals, how gain-of-function mutations cause constitutive activation of the transcription factor, and how Mrr1p interacts with other putative MDR1 regulators like Cap1p and Mcm1p to control expression of its target genes.
C. albicans strains used in this study are listed in the supporting Table S2. All strains were stored as frozen stocks with 15% glycerol at −80 °C. The strains were routinely grown in YPD medium (10 g yeast extract, 20 g peptone, 20 g glucose per liter) at 30 °C. To prepare solid media, 1. 5% agar was added before autoclaving. For induction of the MDR1 promoter with benomyl or H2O2, overnight cultures of reporter strains were diluted 10−2 in three flasks with fresh YPD medium and grown for 3 h. Fifty μg/ml of benomyl or 0. 005% H2O2 was then added to one of the cultures and the cells were grown for an additional hour. The fluorescence of the cells was quantified by FACS analysis. The coding region of the MRR1 gene of C. albicans strain SC5314 was amplified by PCR with the primers ZCF36–1 and ZCF36–2 (primer sequences are given in Table S5). The PCR product was digested at the introduced SalI and BglII restriction sites and substituted for the OPT4 open reading frame (ORF) in the XhoI/BglII-digested pOPT4E1 [47] to generate pZCF36E1. The sequence of the cloned MRR1 gene was identical to that found in the genome sequence of C. albicans strain SC5314 (orf19. 7372). An MRR1 deletion construct was generated in the following way: A SacI-SacII fragment containing MRR1 upstream sequences from positions −314 to +15 with respect to the start codon was amplified with the primers ZCF36–3 and ZCF36–4, and an XhoI-ApaI fragment containing MRR1 downstream sequences from positions +3260 to +3557 was amplified with the primers ZCF36–5 and ZCF36–6. The MRR1 upstream and downstream fragments were cloned on both sides of the SAT1 flipper cassette in plasmid pSFS1 [30] to result in pZCF36M2 in which the MRR1 coding region from positions +16 to +3259 (66 bp before the stop codon) is replaced by the SAT1 flipper (see Figure 2A). DNA fragments containing the N-terminal part and upstream sequences of the MRR1 alleles of the clinical C. albicans isolates F2, F5, G2, and G5 were amplified with the primers ZCF36–3 and ZCF36seq6, digested at the introduced SacI site and at an internal EcoRI site, and cloned in pBluescript to generate pZCF36NF2A and D, pZCF36NF5A, pZCF36NG2B and D, and pZCF36G5A, respectively. DNA fragments containing the MRR1 C-terminal part and downstream sequences were amplified with the primers ZCF36–1 and ZCF36–6, digested at the internal EcoRI site and at the introduced SacI site, and cloned in pBluescript to obtain pZCF36CF2B and C, pZCF36CF5C, pZCF36CG2A and D, and pZCF36CG5A, respectively. The complete ORFs and upstream sequences of the MRR1–1 alleles of isolates F2 and G2 were also amplified with the primers ZCF36–3 and ZCF36–8 and cloned in pBluescript to yield pZCF36TF2–1 and pZCF36TG2–1, respectively. To express wild-type and mutated MRR1 alleles in mrr1Δ mutants, an EcoRI-SalI fragment from pZCF36E1 containing the C-terminal part of MRR1, the ACT1 transcription termination sequence, and part of the caSAT1 selection marker was cloned into the EcoRI/SalI-digested pZCF36NF2A to obtain pZCF36K1. An MRR1 downstream fragment was then amplified from SC5314 genomic DNA with the primers ZCF36–7 and ZCF36–6, digested at the introduced NsiI and ApaI sites, and cloned together with a BglII-PstI fragment from pZCF36E1 containing the ACT1 transcription termination sequence and the caSAT1 selection marker into the BglII/ApaI-digested pZCF36K1 to generate pZCF36K2, which contains the MRR1–1 allele of isolate F2 (which is identical to the MRR1 gene of strain SC5314). Plasmid pZCF36K3, which contains the mutated MRR1 allele from isolate F5, was obtained by substituting an EcoRI-PstI fragment from pZCF36CF5C for the corresponding fragment in pZCF36K2. For expression of the MRR1–2 allele of isolate G2, an NdeI-EcoRI fragment from pZCF36NG2B and an EcoRI-PstI fragment from pZCF36CG2D were substituted for the corresponding region in pZCF36K2, resulting in pZCF36K4. Replacement of the EcoRI-PstI fragment in this plasmid by the corresponding region from pZCF36CG5A generated pZCF36K5, which contains the mutated MRR1 allele from isolate G5. Plasmid pMPG2S, which contains a PMDR1-GFP reporter fusion, was constructed by substituting the caSAT1 selection marker from pSAT1 [30] for the URA3 marker in the previously described plasmid pMPG2 [25]. C. albicans strains were transformed by electroporation [48] with gel-purified inserts from the plasmids described above. Nourseothricin-resistant transformants were selected on YPD agar plates containing 200 μg/ml nourseothricin (Werner Bioagents) as described previously [30]. The correct genomic integration of all constructs was confirmed by Southern hybridization. Genomic DNA from C. albicans was isolated as described previously [49]. 10 μg of DNA was digested with appropriate restriction enzymes, separated on a 1% agarose gel and, after ethidium bromide staining, transferred by vacuum blotting onto a nylon membrane and fixed by UV cross-linking. Southern hybridization with enhanced chemiluminescence-labeled probes was performed with the Amersham ECLTM Direct Nucleic Acid Labeling and Detection System (GE Healthcare) according to the instructions of the manufacturer. Stock solutions of the drugs were prepared as follows. Fluconazole (1 mg/ml) and diamide (20 mg/ml) were dissolved in water, while cerulenin (5 mg/ml) and brefeldin A (5 mg/ml) were dissolved in DMSO. In the assays, serial 2-fold dilutions in the assay medium were prepared from the following initial concentrations: cerulenin, 200 μg/ml; brefeldin A, 200 μg/ml; diamide, 800 μg/ml; fluconazole, 200 μg/ml. Susceptibility tests were carried out in high resolution medium (14. 67 g HR-Medium [Oxoid GmbH], 1 g NaHCO3,0. 2 M phosphate buffer [pH 7. 2]), using a previously described microdilution method [50]. Readings were done after 48 h. Fluorescence-activated cell sorter (FACS) analysis was performed with a FACSCalibur cytometry system equipped with an argon laser emitting at 488 nm (Becton Dickinson). Fluorescence was measured on the FL1 fluorescence channel equipped with a 530-nm band-pass filter. Twenty thousand cells were analyzed per sample and were counted at low flow rate. Fluorescence and forward scatter data were collected by using logarithmic amplifiers. The mean fluorescence values were determined with CellQuest Pro (Becton Dickinson) software. The nucleotide sequences corresponding to 6,165 ORFs for C. albicans were downloaded from the Galar Fungail European Consortium (Assembly 6, http: //www. pasteur. fr/Galar_Fungail/CandidaDB). Following the Affymetrix Design Guide, we designed two separate probe sets for each ORF, each consisting of 13 perfect match and 13 mismatch overlapping 25 bp oligonucleotides, to the 3′ 600 bp region. For ORFs less than 600 bp in length, the sequence was divided in two equal segments for subsequent design procedures. For quality control and normalization purposes, we made 2–3 additional probe sets spanning the entire sequence of the C. albicans 18S rRNA (GenBank Accession M60302), genes encoding GAPDH, actin and Mdr1p (Bmr1p) in addition to the standard Affymetrix controls (BioB, C, D, cre, DAP, PHE, LYS, THR). The probe selection was performed by the Chip Design group at Affymetrix, Inc. using their proprietary algorithm to calculate probe set scores, which includes a probe quality metric, cross-hybridization penalty, and gap penalty. The probe sets were then examined for cross-hybridization against all other sequences in the C. albicans genome as well as a number of constitutively expressed genes and rRNA from other common organisms. Consequently, for some target regions we were not able to design high quality probe sets. In the end, the GeneChip contained 10,736 probe sets including 9 controls, 6,123 unique ORFs, and duplicate probe sets for 4,604 ORFs. The duplicate probe sets are made to distinct regions of the ORF, thereby allowing 2 independent measurements of the mRNA level for that particular gene. The C. albicans custom Affymetrix NimbleExpress Arrays (CAN04a530004N) were manufactured by NimbleGen Systems [51] per our specification. Total RNA was isolated using the hot SDS-phenol method [52]. Frozen cells were suspended in 12 ml of 50 mM sodium acetate (pH 5. 2), 10 mM EDTA at room temperature, after which 1 ml of 20% sodium dodecyl sulphate and 12 ml of acid phenol (Fisher Scientific) were added. This mixture was incubated 10 min at 65 °C with mixing each minute, cooled on ice for 5 min, and centrifuged for 15 min at 12,000g. Supernatants were transferred to new tubes containing 15 ml of chloroform, mixed, and centrifuged at 200g for 10 min. The aqueous layer was removed to new tubes, RNA was precipitated with 1 vol isopropanol and 0. 1 vol 2 M sodium acetate (pH 5. 0), and then collected by centrifugation at 17,000g for 35 min at 4 °C. The RNA pellet was suspended in 10 ml of 70% ethanol, collected again by centrifugation, and suspended in nuclease-free water. Immediately prior to cDNA synthesis, the purity and concentration of RNA samples were determined from A260/A280 readings, and RNA integrity was determined by capillary electrophoresis using the RNA 6000 Nano Laboratory-on-a-Chip kit and Bioanalyzer 2100 (Agilent Technologies) as per the manufacturer' s instructions. First and second strand cDNA was synthesized from 15 μg total RNA using the SuperScript Double-Stranded cDNA Synthesis Kit (Invitrogen) and oligo-dT24-T7 primer (PrOligo) according to the manufacturer' s instructions. cRNA was synthesized and labeled with biotinylated UTP and CTP by in vitro transcription using the T7 promoter-coupled double-stranded cDNA as template and the Bioarray HighYield RNA Transcript Labeling Kit (ENZO Diagnostics). Double-stranded cDNA synthesized from the previous steps was washed twice with 70% ethanol and suspended in 22 μl of RNase-free water. The cDNA was incubated as recommended with reaction buffer, biotin-labeled ribonucleotides, dithtiothreitol, RNase inhibitor mix, and T7 RNA polymerase for 5 h at 37 °C. The labeled cRNA was separated from unincorporated ribonucleotides by passing through a CHROMA SPIN-100 column (Clontech) and ethanol precipitated at −20 °C overnight. The cRNA pellet was suspended in 10 μl of RNase-free water and 10 μg was fragmented by ion-mediated hydrolysis at 95 °C for 35 min in 200 mM Tris-acetate (pH 8. 1), 500 mM potassium acetate, 150 mM magnesium acetate. The fragmented cRNA was hybridized for 16 h at 45 °C to the C. albicans NimbleExpress GeneChip arrays. Arrays were washed at 25 °C with 6 × SSPE, 0. 01% Tween 20 followed by a stringent wash at 50 °C with 100 mM MES, 0. 1 M NaCl, 0. 01% Tween 20. Hybridizations and washes employed the Affymetrix Fluidics Station 450 using their standard EukGE-WS2v5 protocol. The arrays were then stained with phycoerythrein-conjugated streptavidin (Molecular Probes) and the fluorescence intensities were determined using the GCS 3000 high-resolution confocal laser scanner (Affymetrix). The scanned images were analysed using software resident in GeneChip Operating System v2. 0 (Affymetrix). Sample loading and variations in staining were standardized by scaling the average of the fluorescent intensities of all genes on an array to a constant target intensity (250). The signal intensity for each gene was calculated as the average intensity difference, represented by [Σ (PM − MM) / (number of probe pairs) ], where PM and MM denote perfect-match and mismatch probes. The scaled gene expression values from GeneChip Operating System v2. 0 software were imported into GeneSpring 7. 2 software (Agilent Technologies) for preprocessing and data analysis. Probe sets were deleted from subsequent analysis if they were called absent by the Affymetrix criterion and displayed an absolute value below 20 in all experiments. The expression value of each gene was normalized to the median expression of all genes in each chip as well as the median expression for that gene across all chips in the study. Pairwise comparison of gene expression was performed for each matched experiment. Among direct comparisons between matched clinical isolates, genes were considered to be differentially expressed if their change in expression was ≥ 1. 5-fold in three independent experiments. An aliquot of the RNA preparations from the samples used in the microarray experiments was saved for quantitative real-time RT-PCR follow-up studies. First-strand cDNAs were synthesized from 2 μg of total RNA in a 21-μl reaction volume using the SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen) in accordance with the manufacturer' s instructions. Quantitative real-time PCRs were performed in triplicate using the 7000 Sequence Detection System (Applied Biosystems). Independent PCRs were performed using the same cDNA for both the gene of interest and the 18S rRNA, using the SYBR Green PCR Master Mix (Applied Biosystems). Gene-specific primers were designed for the gene of interest and the 18S rRNA using Primer Express software (Applied Biosystems) and the Oligo Analysis & Plotting Tool (QIAGEN) and are shown in Table S5. The PCR conditions consisted of AmpliTaq Gold activation at 95 °C for 10 min, followed by 40 cycles of denaturation at 95 °C for 15 s and annealing/extension at 60 °C for 1 min. A dissociation curve was generated at the end of each PCR cycle to verify that a single product was amplified using software provided with the 7000 Sequence Detection System. The change in fluorescence of SYBR Green I dye in every cycle was monitored by the system software, and the threshold cycle (CT) above the background for each reaction was calculated. The CT value of 18S rRNA was subtracted from that of the gene of interest to obtain a ΔCT value. The ΔCT value of an arbitrary calibrator (e. g. , untreated sample) was subtracted from the ΔCT value of each sample to obtain a ΔΔCT value. The gene expression level relative to the calibrator was expressed as 2−ΔΔCT.
The coding sequences of the MRR1 alleles described in this study have been deposited in GenBank (http: //www. ncbi. nlm. nih. gov/Genbank/index. html) with the following accession numbers: EU139261 (MRR1F2–1), EU139262 (MRR1F2–2), EU139263 (MRR1F5), EU139264 (MRR1G2–1), EU139265 (MRR1G2–2), EU139266 (MRR1G5).
The Candida albicans MDR1 (multidrug resistance) gene encodes a multidrug efflux pump of the major facilitator superfamily that is constitutively overexpressed in many fluconazole-resistant strains. Although MDR1 overexpression is a major cause of resistance to this widely used antifungal agent and other metabolic inhibitors, so far the molecular basis of MDR1 upregulation in resistant strains has remained elusive. By comparing the transcription profiles of MDR1 overexpressing, clinical C. albicans isolates and matched, drug-susceptible isolates from the same patients, we identified a transcription factor, termed multidrug resistance regulator 1 (MRR1), which was upregulated in all resistant isolates and turned out to be a central regulator of MDR1 expression. Resistant isolates contained point mutations in MRR1, which rendered the transcription factor constitutively active. Introduction of these mutated alleles into a susceptible strain caused MDR1 overexpression und multidrug resistance. Inactivation of MRR1 in clinical isolates abolished MDR1 expression and affected fluconazole resistance even more strongly than deletion of the MDR1 efflux pump itself, indicating that additional Mrr1p target genes, which were identified by genome-wide gene expression analysis, contribute to fluconazole resistance. These findings provide detailed insights into the molecular basis of multidrug resistance in one of the most important human fungal pathogens.
infectious diseases
yeast and fungi
microbiology
eukaryotes
genetics and genomics
saccharomyces
The Transcription Factor Mrr1p Controls Expression of the MDR1 Efflux Pump and Mediates Multidrug Resistance in Candida albicans