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. Author manuscript; available in PMC: 2009 Aug 1.
Published in final edited form as: Mol Microbiol. 2008 May 27;69(4):827–840. doi: 10.1111/j.1365-2958.2008.06309.x

Mutations in the multidrug resistance regulator MRR1, followed by loss of heterozygosity, are the main cause of MDR1 overexpression in fluconazole-resistant Candida albicans strains

Nico Dunkel 1, Julia-Blaß 1, P David Rogers 2, Joachim Morschhäuser 1,*
PMCID: PMC2678921  NIHMSID: NIHMS65491  PMID: 18577180

Summary

Overexpression of the MDR1 gene, encoding a multidrug efflux pump of the major facilitator superfamily, is a major cause of resistance to the widely used antifungal agent fluconazole and other toxic substances in the fungal pathogen Candida albicans. We found that all tested clinical and in vitro generated C. albicans strains that had become fluconazole-resistant by constitutive MDR1 upregulation contained mutations in the MRR1 gene, which encodes a transcription factor that controls MDR1 expression. Introduction of the mutated alleles into a drug-susceptible C. albicans strain resulted in activation of the MDR1 promoter and multidrug resistance, confirming that the amino acid substitutions in Mrr1p were gain-of-function mutations that rendered the transcription factor constitutively active. The majority of the MDR1 overexpressing strains had become homozygous for the mutated MRR1 alleles, demonstrating that the increased resistance level conferred by two gain-of-function alleles provides sufficient advantage to select for the loss of heterozygosity in the presence of fluconazole both in vitro and within the human host during therapy. Loss of heterozygosity usually occurred by mitotic recombination between the two chromosome 3 homologues on which MRR1 is located, but evidence for complete loss of one chromosome and duplication of the chromosome containing the mutated MRR1 allele was also obtained in two in vitro generated fluconazole-resistant strains. These results demonstrate that gain-of-function mutations in MRR1 are the major, if not the sole, mechanism of MDR1 overexpression in fluconazole-resistant strains and that this transcription factor plays a central role in the development of drug resistance in C. albicans.

Keywords: Candida albicans, efflux pump, fluconazole, gain-of-function mutation, multidrug resistance, transcription factor

Introduction

Infections by the opportunistic pathogenic yeast Candida albicans are frequently treated with the antimycotic agent fluconazole, which inhibits the biosynthesis of ergosterol, the major sterol in the fungal cell membrane. C. albicans can develop resistance to fluconazole, especially during long-term treatment of oropharyngeal candidiasis, a condition that affects many individuals infected with the human immunodeficiency virus (HIV) and AIDS patients (White et al., 1998). Fluconazole resistance can be caused by different mechanisms, including alterations in the sterol biosynthesis pathway, increased expression of the ERG11 gene encoding the drug target enzyme, sterol 14α-demethylase (Erg11p), mutations in Erg11p that result in reduced affinity to fluconazole, and overexpression of multidrug efflux pumps that actively transport fluconazole and many other, structurally unrelated toxic compounds out of the cell. The analysis of serial isolates from individual patients has shown that several of these mechanisms are often combined to result in a stepwise development of clinically relevant fluconazole resistance (Morschhäuser, 2002; White et al., 1998).

C. albicans possesses two types of efflux pumps whose overexpression is responsible for fluconazole resistance in many clinical isolates, the highly homologous ATP-binding cassette (ABC) transporters Cdr1p and Cdr2p and the major facilitator Mdr1p (Sanglard et al., 1995; Sanglard et al., 1996, 1997; Wirsching et al., 2000). The molecular basis for the constitutive upregulation of the genes encoding these efflux pumps has recently been elucidated. Expression of CDR1 and CDR2 is controlled by the zinc cluster transcription factor Tac1p (transcriptional activator of CDR genes), which binds to a drug response element that is present in the promoter region of both of these genes and induces their expression in response to steroids and certain toxic chemicals (Coste et al., 2004; de Micheli et al., 2002). Mutations in Tac1p result in constitutive activity of this transcription factor and, consequently, CDR1 and CDR2 overexpression (Coste et al., 2006; Coste et al., 2007; Znaidi et al., 2007). Although other transcription factors have also been reported to regulate CDR1 expression (Chen et al., 2004; Gaur et al., 2004), so far only mutations in Tac1p have been found as the cause of drug resistance mediated by this efflux pump in clinical C. albicans isolates.

MDR1 expression is also controlled by a transcription factor of the zinc cluster family, termed Mrr1p (multidrug resistance regulator) (Morschhäuser et al., 2007). MRR1 was identified as one of the genes that were commonly upregulated in three different MDR1 overexpressing, fluconazole-resistant C. albicans isolates as compared with genetically related, fluconazole-susceptible isolates from the same patients. MRR1 inactivation in two fluconazole-resistant isolates resulted in the loss of MDR1 expression and increased susceptibility to fluconazole and other metabolic inhibitors that are supposed to be substrates of the Mdr1p efflux pump, like cerulenin and brefeldin A. Deletion of MRR1 from the drug-susceptible C. albicans wild-type model strain SC5314 also abolished MDR1 expression in the presence of inducing chemicals, like benomyl and hydrogen peroxide, demonstrating that Mrr1p mediates both inducible MDR1 expression in drug-susceptible strains and constitutive MDR1 overexpression in drug-resistant strains. The two MDR1 overexpressing clinical isolates that were studied in detail had become homozygous for mutated MRR1 alleles containing a P683S or a G997V mutation. Introduction of these mutated alleles into a drug-susceptible strain resulted in constitutive MDR1 overexpression and multidrug resistance. These results showed that, similar to CDR1/CDR2 upregulation by Tac1p, MDR1 overexpression is also caused by gain-of-function mutations in its regulator, Mrr1p. However, additional transcription factors have been implicated in the control of MDR1 expression, namely, Cap1p and Mcm1p, both of which have binding sites in the MDR1 promoter (Harry et al., 2005; Hiller et al., 2006; Riggle and Kumamoto, 2006; Rognon et al., 2006). Therefore, it seemed possible that MDR1 overexpression in drug-resistant C. albicans isolates might also be due to mutations in regulatory factors other than MRR1. To investigate how frequently MDR1 overexpression is caused by alterations in MRR1, we analyzed a panel of well-documented matched pairs of drug-susceptible and drug-resistant C. albicans isolates in which fluconazole resistance was associated with MDR1 overexpression for the presence of mutations and genomic rearrangements involving MRR1.

Results

MDR1 overexpressing clinical C. albicans isolates contain mutations in the transcription factor MRR1

To investigate whether MDR1 overexpression in other fluconazole-resistant, clinical C. albicans isolates is also caused by mutations in the transcription factor Mrr1p, we cloned and sequenced the MRR1 alleles from seven additional resistant isolates and genetically related susceptible isolates from the same patients which had been described in the literature. One of these matched pairs of isolates (B3/B4) came from our own group (Franz et al., 1999), three pairs (1442/2271, 1490/1587 and 5044/5052) were provided by Thomas Patterson (Lopez-Ribot et al., 1998; Perea et al., 2001), two pairs (DSY291/DSY292 and DSY2285/DSY2286) by Dominique Sanglard (Calabrese et al., 2000; Sanglard et al., 1995), and one isolate pair (5833/6692) was obtained from Martine Raymond (Saidane et al., 2006) (see Table S1). The nucleotide sequences of all MRR1 alleles found in these isolates have been deposited in GenBank (see Experimental procedures for accession numbers) and amino acid differences in the encoded proteins are listed in Table 1.

TABLE 1.

Allelic polymorphisms and mutations resulting in amino acid differences in Mrr1p

Strain Allele Amino acid polymorphisms
SC5314 -
CAPR510 T381I
CAPR513 1 -
2 A880E
CAPR514 W893R
CAPR515 R873T
CAPR517 L998F
F2 1 -
2 S171P, V341E, E1020Q
F5 P683S
G2 1 E1020Q
2 S171P, V341E, E1020Q
G5 S171P, V341E, G997V, E1020Q
B3 1 -
2 V27I, S171P, L248V, V341E, E1020Q, 5x NPQS
B4 V27I, S171P, L248V, V341E, G878E, E1020Q, 5x NPQS
1442 1 -
2 V27I, S171P, L248V, V341E, E1020Q, 5x NPQS
2271 Q350L
1490 1 -
2 -
1587 N803D
5044 S171P, V341E, E1020Q
5052 S171P, V341E, P683H, E1020Q
DSY291 1 -
2 S16I, T73K, S171P, E1020Q, 2x NPQS
DSY292 1 P683H
2 S16I, T73K, S171P, E1020Q, 2x NPQS
DSY2285 E1020Q
DSY2286 T896I, E1020Q
5833 1 S171P, L248V, V341E, 5x NPQS
2 V27I, S171P, L248V, V341E, E1020Q, 5x NPQS
6692 1 S171P, L248V, V341E, T360I, 5x NPQS
2 V27I, S171P, L248V, K335N, V341E, E1020Q, 5x NPQS
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Only amino acids that differ from those of Mrr1p of strain SC5314 are listed, gain-of-function mutations in resistant strains are highlighted in bold. Matched isolates are grouped together in gray blocks, as are the in vitro generated strains CAPR510-CAPR517 and their parent SC5314. The previously analyzed isolate pairs F2/F5 and G2/G5 (Morschhäuser et al., 2007) are included for completeness. The number of tandem repeats of the amino acid sequence NPQS (amino acids 165–168 in Mrr1p of strain SC5314) is indicated for alleles that contain more than one copy of this sequence.

Two polymorphic MRR1 alleles were found in the susceptible isolate B3. Allele 1 was identical to the MRR1 allele of C. albicans strain SC5314 (orf19.7372), which was used for genome sequencing. Allele 2 differed from it at 41 nucleotide positions, five of which resulted in amino acid exchanges. In addition, allele 2 encoded five tandem repeats of the amino acids NPQS (positions 165–168 in Mrr1p). Only one MRR1 allele was obtained from the resistant isolate B4, which was identical to allele 2 of the matched isolate B3 except for a G2633A substitution that resulted in a G878E exchange in the encoded protein.

The susceptible isolate 1442 also contained two polymorphic MRR1 alleles. Allele 1 corresponded to MRR1 of strain SC5314 but contained ten silent nucleotide exchanges. Allele 2 differed from allele 1 by 31 nucleotide substitutions, five of which resulted in amino acid exchanges, and also contained five tandem repeats of the NPQS sequence. The matched resistant isolate 2271 contained only one MRR1 allele, which was identical to allele 1 of isolate 1442 except for a single A1049T substitution that resulted in a Q350L exhange in Mrr1p.

Two polymorphic MRR1 alleles were found in the susceptible isolate 1490, which differed from one another by 27 silent nucleotide exchanges. The matched resistant isolate 1587 contained only allele 2 with an additional A2407G exchange that caused an N803D mutation in the encoded protein.

The MRR1 alleles obtained from the susceptible isolate 5044 were identical and differed from MRR1 of strain SC5314 by 26 nucleotide exchanges, three of which resulted in amino acid exchanges. The matched resistant isolate 5052 contained the same allele, but with a C2048A mutation in both copies that resulted in a P683H exchange in Mrr1p.

The susceptible isolate DSY291 contained two polymorphic MRR1 alleles, one of which was identical to MRR1 from SC5314. Allele 2 differed from allele 1 at 28 positions, which resulted in four amino acid exchanges, and encoded two tandem repeats of the NPQS sequence. The matched resistant isolate DSY292 contained the same two alleles, however, allele 1 exhibited an additional C2048A exchange that resulted in a P683H mutation in the encoded protein. An identical mutation had been found in isolate 5052, but in an otherwise different MRR1 allele (see above).

The MRR1 alleles in the susceptible isolate DSY2285 were identical and differed from MRR1 of strain SC5314 only by a G3058C exchange that resulted in an E1020Q substitution. In the matched resistant isolate DSY2286 both alleles contained an additional C2687T mutation that caused a T896I exchange in Mrr1p.

The suceptible isolate 5883 contained two polymorphic MRR1 alleles that differed from one another at 14 positions, with two of the polymorphisms resulting in amino acid differences. Both alleles encoded five tandem repeats of the NPQS sequence. These two alleles were also found in the matched resistant isolate 6692, however, each of them exhibited a single nucleotide substitution. Allele 1 contained a C1079T exchange that caused a T360I mutation and allele 2 had an A1005C substitution that resulted in a K335N mutation in Mrr1p.

In summary, all analyzed MDR1 overexpressing clinical C. albicans isolates contained a mutation in the transcription factor MRR1. Like the two previously described isolates F5 and G5 (Morschhäuser et al., 2007), five of the seven resistant isolates analyzed in the present study (B4, 2271, 1587, 5052, DSY2286) had become homozygous for the mutated MRR1 allele. One isolate (6692) had aquired independent mutations in both MRR1 alleles and one resistant isolate (DSY292) had retained a wild-type allele in addition to the mutated allele.

In vitro generated, MDR1 overexpressing C. albicans strains have aquired MRR1 mutations

Riggle and Kumamoto recently isolated five fluconazole-resistant derivatives of C. albicans laboratory strains, which themselves were generated from the wild-type strain SC5314, that overexpressed MDR1 after passage in medium containing fluconazole (Riggle and Kumamoto, 2006). We investigated whether MDR1 upregulation in these in vitro generated strains was also caused by mutations in MRR1. Sequencing of the MRR1 alleles of these strains showed that all of them indeed had aquired single point mutations in this transcription factor, resulting in the amino acid substitutions T381I in strain CAPR510, A880E in CAPR513, W893R in CAPR514, R873T in CAPR515, and L998F in CAPR517. Strains CAPR510, CAPR514, CAPR515, and CAPR517 had become homozygous for the mutated alleles while strain CAPR513 was found to be heterozygous and also retained one original MRR1 allele of the parental strain SC5314.

Table 1 summarizes all polymorphisms that were found in the MRR1 alleles of the investigated strains and which resulted in amino acid differences in Mrr1p. The fact that all MDR1 overexpressing, fluconazole-resistant strains contained MRR1 alleles that differed from the corresponding alleles in the matched susceptible isolates (for clinical isolates) or the susceptible parental strain (for in vitro generated strains) by a single nucleotide exchange strongly suggested that the resulting amino acid substitutions in Mrr1p rendered the transcription factor constitutively active and were responsible for MDR1 overexpression in these strains.

MRR1 gain-of-function mutations cause constitutive MDR1 overexpression and multidrug resistance

To verify that the MRR1 mutations found in the fluconazole-resistant strains were indeed gain-of-function mutations causing MDR1 overexpression, we introduced each of the mutated alleles into a derivative of the drug-susceptible C. albicans strain SC5314 from which the endogenous MRR1 alleles had been deleted and which contained a PMDR1-GFP reporter gene fusion that could be used to monitor MDR1 promoter activity (see Fig. 1). The amino acid sequences of Mrr1p encoded by the mutated MRR1 alleles of strains CAPR510 (MRR1T381I), CAPR513 (MRR1A880E), CAPR514 (MRR1W893R), CAPR515 (MRR1R873T), CAPR517 (MRR1L998F), 2271 (MRR1Q350L), 1587 (MRR1N803D), and DSY292 (MRR1P683H) were identical to that of Mrr1p of strain SC5314, except for the putative gain-of-function mutations, and the corresponding transformants could therefore directly be compared with control strains into which the wild-type allele had been reintroduced in the same way. As the mutated MRR1 alleles from isolates B4, DSY2286, and 6692 contained additional amino acid differences not found in Mrr1p of strain SC5314, we incorparated only the relevant part of these MRR1 alleles into the expression cassette to generate the MRR1G878E, 1020Q, MRR1T896I, E1020Q, MRR1S171P, L248V, V341E, T360I, and MRR1S171P, L248V, K335N, V341E alleles (see Experimental procedures). Expression cassettes containing the corresponding parts of the nonmutated alleles (MRR1E1020Q and MRR1S171P, L248V, V341E) served as controls in these cases. The mutated MRR1 allele from isolate 5052 was not tested because the identical P683H mutation was present in the MRR1P683H allele from isolate DSY292. Two independent transformants were kept in each case and used to quantify MDR1 promoter activity by FACS analysis. As can be seen in Fig. 2, expression of all mutated MRR1 alleles resulted in constitutive activation of the MDR1 promoter, while no MDR1 promoter activity was observed in the control transformants carrying the nonmutated MRR1 alleles. These results demonstrated that the MRR1 mutations that had occurred in the fluconazole-resistant strains resulted in constitutive activity of the transcription factor and caused MDR1 overexpression.

Fig. 1.

Fig. 1

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Structure of the DNA cassettes (top) used for integration of different MRR1 alleles into mrr1Δ mutants (bottom) in which the endogenous MRR1 copies had been deleted and replaced by the target sequence of the Flp recombinase (FRT, black arrow). The MRR1 coding region is represented by the white arrow and the upstream (MRR1up) and downstream regions (MRR1down) by the solid lines. The transcription termination sequence of the ACT1 gene (TACT1) is indicated by the filled circle and the caSAT1 selection marker by the grey arrow. Only relevant restriction sites are shown: A, ApaI; E, EcoRI; N, NsiI; Nd, NdeI; P, PstI; ScI, SacI. The nucleotide positions of the NdeI, EcoRI, and PstI sites, which were used to replace specific fragments within the MRR1 coding region, are given. The MRR1 upstream region is identical in all plasmids and corresponds to that of strain SC5314.

Fig. 2.

Fig. 2

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MDR1 promoter activity in an mrr1Δ mutant carrying a PMDR1-GFP reporter fusion (mrr1Δ) and in transformants into which MRR1 alleles with the gain-of-function mutations (highlighted in bold) from strains CAPR510 (T381I), CAPR513 (A880E), CAPR514 (W893R), CAPR515 (R873T), CAPR517 (L998F), 2271 (Q350L), 1587 (N803D), 5052 and DSY292 (P683H), B4 (G878E, E1020Q), DSY2286 (T896I, E1020Q), and 6692 (S171P, L248V, V341E, T360I and S171P, L248V, K335N, V341E) or the corresponding control alleles without the mutations (wild type, E1020Q, and S171P, L248V, V341E) were reintroduced, as outlined in Fig. 1. The strains were grown to log phase in YPD medium and the mean fluorescence of the cells was determined by flow cytometry. Results from two independent transformants are shown in each case.

In a complementary approach to investigate the effect of the MRR1 mutations on drug resistance, the same alleles described above were also introduced into mrr1Δ mutants of the wild type strain SC5314 and the susceptibility of the transformants to fluconazole and other metabolic inhibitors to which MDR1 overexpression confers resistance was tested (see Fig. 3). As previously reported (Morschhäuser et al., 2007), reintroduction of the original wild-type allele (MRR1) did not confer increased resistance to cerulenin, brefeldin A, or fluconazole, and the same result was obtained with the two control alleles MRR1E1020Q and MRR1S171P, L248V, V341E which contained amino acid exchanges that were commonly found in susceptible isolates. In contrast, all gain-of-function alleles conferred increased resistance to cerulenin and brefeldin A, two compounds that are good substrates of the Mdr1p efflux pump (Hiller et al., 2006). Expression of a single copy of most of the alleles was also sufficient to increase fluconazole resistance of the strains, except for the MRR1W893R and MRR1R873 alleles, which also had the weakest effect on MDR1 promoter activity (see Fig. 2) and probably have to be present in two copies to result in significantly increased fluconazole resistance, as in strains CAPR514 and CAPR515 in which they were found. These results confirmed that all the mutated MRR1 alleles confer multidrug resistance.

Fig. 3.

Fig. 3

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MICs (in μg ml−1) of cerulenin, brefeldin A, and fluconazole for two independently constructed homozygous mrr1Δ mutants of the wild-type parental strain SC5314 and transformants carrying the indicated MRR1 alleles (see legend to Fig. 2).

Contribution of mutated MRR1 alleles to drug resistance in heterozygous and homozygous C. albicans strains

As described above, in addition to the aquisition of point mutations in MRR1, most of the fluconazole-resistant C. albicans strains had also undergone genomic changes and become homozygous for the mutated MRR1 allele. To assess the contribution of the MRR1 alleles to drug resistance in clinical isolates, we deleted one or both MRR1 alleles in the two heterozygous resistant isolates DSY292 and 6692 as well as in three additional isolates that had become homozygous for a mutated MRR1 allele (isolates B4, 2271, DSY2286). Two independent series of heterozygous and homozygous mrr1Δ mutants were generated from each parental strain using the SAT1-flipping strategy (Reuβ et al., 2004). In all cases, two rounds of gene deletion were sufficient to obtain homozygous mrr1Δ mutants, demonstrating that MRR1 gene amplification was not involved in resistance development in these strains. The susceptibilities of the mutants to fluconazole, cerulenin, and brefeldin A were then compared with those of the respective resistant parental isolates and matched susceptible isolates. As can be seen in Fig. 4, the increased resistance to these inhibitors observed in the parental resistant isolates was completely or almost completely lost in all homozygous mrr1Δ mutants (i.e., the MIC decreased to the level of the matched susceptible isolate or was only one dilution step higher), except for the fluconazole resistance of isolates DSY292 and 6692 (see below and discussion). Deletion of one MRR1 copy in the strains that had become homozygous for a mutated MRR1 allele (B4, 2271, DSY2286) resulted in an intermediate phenotype, demonstrating that both alleles contributed to the resistance of the clinical isolates. The same result was seen in the heterozygous mutants generated from the clinical isolate 6692, which contained two different mutated MRR1 alleles. Sequencing showed that both randomly selected heterozygous deletion mutants had retained the MRR1 allele with the K335N mutation. The partial loss of drug resistance in the heterozygous mutants demonstrated that both mutated MRR1 alleles contributed to the resistant phenotype in this clinical isolate. In contrast, the two heterozygous deletion mutants of isolate DSY292 behaved differently. One of the heterozygous MRR1/mrr1Δ mutants (strain A) displayed the same resistance as the parental isolate (resistance to cerulenin was even slightly increased) and sequencing showed that in this strain the nonmutated MRR1 allele was deleted and the allele with the P683H mutation was retained. The other heterozygous MRR1/mrr1Δ mutant (strain B) exhibited the same sensitivity to cerulenin and brefeldin A as the homozygous mrr1Δ mutants and sequencing demonstrated that in this strain the MRR1P683H allele was deleted and the nonmutated allele was retained. This result showed that only the MRR1 allele with the P683H gain-of-function mutation contributed to the increased drug resistance of isolate DSY292 as compared with the matched susceptible isolate DSY291. Interestingly, while the resistance to cerulenin and brefeldin A was strongly reduced in the mrr1Δ mutants of DSY292, fluconazole resistance of this strain was not or only slightly affected by the MRR1 deletion. This is probably due to the fact that DSY292 contains mutations in ERG11 that confer fluconazole resistance (see discussion). In some cases, we observed minor differences in the drug susceptibilities of the two independently generated mrr1Δ mutants of a particular strain, indicating that unspecific alterations which slightly affected drug resistance had also occurred during the construction of the mutants. However, in all cases the importance of MRR1 in drug resistance of the clinical isolates was clearly evident.

Fig. 4.

Fig. 4

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Susceptibilities of the matched fluconazole-susceptible (S) and resistant (R) clinical C. albicans isolate pairs and the two independent series (A and B) of heterozygous (MRR1/Δ) and homozygous (mrr1Δ/Δ) mrr1 deletion mutants generated from the resistant isolates to cerulenin, brefeldin A, and fluconazole. The names of the resistant parental isolates and the Mrr1p gain-of-function mutations found in these isolates are given to the left of the respective panels. MICs of each compound are given in μg ml−1.

A comparison of the effects of MRR1 deletion in the resistant clinical isolates and expression of the corresponding MRR1 allele in the highly susceptible strain SC5314 showed that the contribution of a specific MRR1 gain-of-function allele to drug resistance was largely comparable in the different strain backgrounds. For example, expression of a single copy of the MRR1G878E allele from isolate B4 in strain SC5314 resulted in 16-fold increased resistance to cerulenin, 8-fold increased resistance to brefeldin A, and 4-fold increased resistance to fluconazole (Fig. 3), while deletion of the remaining copy of the same allele from the heterozygous mutants of isolate B4 decreased resistance to these drugs in the homozygous mutants by 16- to 32-fold, 16-fold, and 4-fold, respectively (Fig. 4, top panels). Similarly, expression of the MRR1K335N allele from isolate 6692 in strain SC5314 increased the resistance of this strain to cerulenin, brefeldin A, and fluconazole by 16-fold, 16-fold, and 4-fold, respectively (Fig. 3), while deletion of the same allele from the heterozygous mutants of isolate 6692 decreased resistance to these drugs by 16-fold, 8-fold, and 4-fold, respectively (Fig. 4, bottom panels). In the few cases where major differences were observed, these are presumably related to the different genetic backgrounds. For example, the contribution of the MRR1P683H allele to fluconazole resistance in isolate DSY292 is probably masked by the ERG11 mutations in this isolate (see above and discussion), and the strong effect of deletion of the MRR1T896I allele on fluconazole resistance in isolate DSY2286 could be explained by strain-specific differences in other Mrr1p target genes that contribute to fluconazole resistance (Morschhäuser et al., 2007).

Loss of heterozygosity in C. albicans strains containing MRR1 gain-of-function mutations is caused by mitotic recombination and chromosome loss

Loss of MRR1 heterozygosity in the resistant strains could have occurred by mitotic recombination between the two homologues of chromosome 3, on which MRR1 is located, or by loss of one chromosome 3 homologue and duplication of the other copy containing the mutated MRR1 allele. Both types of events have been found as a mechanism of loss of heterozygosity for genes located on chromosome 5 (Coste et al., 2006; Coste et al., 2007; Wu et al., 2005; Wu et al., 2007). Loss of MRR1 heterozygosity by mitotic recombination must involve at least one cross over event between the centromere and the MRR1 locus on the right arm of chromosome 3. In the absence of additional recombination events this would result in homozygosity of the region between the cross over site and the telomere, but polymorphisms on the other chromosome arm would be retained. In contrast, loss of one chromosome 3 homologue would result in the loss of all polymorphisms on chromosome 3. Several allelic polymorphisms in genes located on chromosome 3 have been reported for strain SC5314 (Jones et al., 2004). To distinguish between chromosome loss and mitotic recombination, we first analyzed the four in vitro generated derivatives of strain SC5314 that had become homozygous for a mutated MRR1 allele for the presence of polymorphisms in CAP1, a gene that is located on the left arm of chromosome 3 (see Fig. 5). The two CAP1 alleles in strain SC5314 can easily be distinguished by restriction site polymorphisms, one allele containing an EcoRI site and the other allele an SphI site. As illustrated in Fig. 5A, strains CAPR515 and CAPR517 retained these polymorphisms, thus excluding chromosome loss as the cause of MRR1 homozygosity in these strains. In contrast, strains CAPR510 and CAPR514 contained only the CAP1 allele with the SphI site but had lost the allele with the EcoRI site, pointing to chromosome loss or multiple recombination events on both arms of chromosome 3 in these strains. We, therefore, looked for the presence of additional polymorphic markers on chromosome 3. Strains CAPR515 and CAPR517 contained the known polymorphisms of strain SC5314 in MEP3 and SAP9 on the left arm and AAP1 on the right arm of chromosome 3, demonstrating that the recombination event that resulted in homozygosity for the mutated MRR1 allele occurred between AAP1 and MRR1. Strains CAPR510 and CAPR514 had lost heterozygosity for all these markers. As SAP9 and AAP1 are located on both sides of and very close to the centromere on chromosome 3, we consider it unlikely that homozygosity was caused by two independent recombination events in the small regions between the centromere and SAP9 on the left arm and between the centromere and AAP1 on the right arm. These two strains most probably became homozygous for the mutated MRR1 allele by loss of the chromosome containing the wild-type allele.

Fig. 5.

Fig. 5

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Mechanism of loss of MRR1 heterozygosity in MDR1 overexpressing, fluconazole-resistant C. albicans strains. (A) shows the position of known polymorphic markers (gray boxes) on chromosome 3, on which MRR1 (white arrow) is located, in strain SC5314. The in vitro generated, fluconazole-resistant derivatives CAPR515 and CAPR517 have retained these polymorphisms whereas CAPR510 and CAPR514 have become homozygous for all tested polymorphic alleles, as indicated by the white boxes. The location of the centromere is indicated by the black oval. (B) shows the results of a similar analysis of six clinical isolate pairs in which the resistant isolate had become homozygous for a mutated MRR1 allele. Gray shading: allelic polymorphisms detected, white: no allelic polymorphisms found.

To investigate by which mechanism the fluconazole-resistant, clinical C. albicans isolates F5 and G5 described in a previous study (Morschhäuser et al., 2007) and isolates B4, 2271, 1587, DSY2286, and 5052 analyzed in the present study had become homozygous for their mutated MRR1 alleles, we first tested whether the matched susceptible isolates contained any of the heterozygous markers on chromosome 3 described above. Isolate 5044 was homozygous for all tested markers, therefore, the mechanism of loss of MRR1 heterozygosity in the matched resistant isolate 5052 could not be investigated. The other susceptible isolates contained one or more polymorphic alleles on chromosome 3, as illustrated in Fig. 5B. In all these cases, the matched resistant isolates retained the polymorphisms, which shows that the loss of MRR1 heterozygosity was caused by mitotic recombination and not by chromosome loss. Altogether, these results demonstrate that mitotic recombination is the major cause of loss of heterozygosity for MRR1 on chromosome 3, but chromosome loss can also occur, at least during the in vitro selection of MDR1 overexpressing, fluconazole-resistant strains.

Identification of a clinical isolate that represents the missing link from drug susceptibility to homozygosity for a mutated MRR1 allele and drug resistance

Most of the MDR1 overexpressing, drug-resistant C. albicans isolates had become homozygous for mutated MRR1 alleles, while the matched susceptible isolates from the same patients contained two nonmutated alleles. One must assume that a gain-of-function mutation first appeared in one allele and that the advantage gained by becoming homozygous for the mutated allele is sufficient to rapidly select for such an event in the presence of antimycotic therapy. Isolates F2 and F5 are from a series in which the first two described isolates (F1 and F2) did not detectably express MDR1 whereas the last two described isolates (F4 and F5) displayed high MDR1 transcript levels. This series also included an isolate, F3, that exhibited intermediate MDR1 expression levels and intermediate fluconazole resistance (Franz et al., 1998). We speculated that this isolate might represent the missing link between the susceptible isolate F2 with two polymorphic wild-type MRR1 alleles and the resistant isolate F5 that had become homozygous for the MRR1P683S allele and strongly expressed MDR1. We therefore amplified the MRR1 alleles from isolate F3 and sequenced the relevant part of the PCR product. We found that F3 indeed still possessed both MRR1 alleles found in the suscpetible isolate F2, but contained the MRR1P683S mutation in allele 1, while allele 2 remained unchanged. This finding in a clinical isolate confirmed that a gain-of-function mutation in one MRR1 allele results only in intermediate resistance levels and explains why most MDR1 overexpressing, fluconazole-resistant isolates obtained from patients have become homozygous for mutated MRR1 alleles.

Discussion

The results of this study demonstrate that constitutive MDR1 upregulation in fluconazole-resistant C. albicans strains is tightly linked to mutations in MRR1, a transcription factor that controls MDR1 expression. All nine clinical C. albicans isolates that have been investigated to date contain gain-of-function mutations in MRR1 that render the transcription factor constitutively active and cause upregulation of the MDR1 efflux pump and multidrug resistance. In addition, all five in vitro generated, MDR1 overexpressing strains that were available also contain mutated MRR1 alleles. Therefore, our results strongly suggest that mutations in MRR1 are the main, if not the sole cause of MDR1-mediated multidrug resistance in C. albicans.

It is presently unknown how Mrr1p is activated to induce expression of its target genes in response to environmental signals and how the gain-of-function mutations affect the activity of the transcription factor. Fig. 6 shows the location of all activating mutations that have been identified so far in Mrr1p. There are four major mutational hot spots which are located in different regions of the protein. Four mutations (K335N, Q350L, T360I, T381I) lie between amino acid positions 335 and 381, five mutations (R873T, G878E, A880E, W893R, T896I) cluster in a small stretch between amino acids 873 and 896, and two mutations are located at positions 997 and 998 (G997V and L998F). In addition, the proline at position 683 is mutated in three different MDR1 overexpressing isolates (two P683H substitutions and one P683S exchange). As suggested previously (Morschhäuser et al., 2007), it is likely that these mutations relieve the transcription factor from inhibition by an autoregulatory domain or another repressing factor and render it constitutively active. Apart from the predicted DNA binding domain in the N-terminal part of the protein, which is typical for the zinc cluster transcription factor family, other functional domains of Mrr1p remain to be experimentally defined. It seems reasonable to assume that such a large protein of more than 1100 amino acid residues is subject to multiple regulatory inputs that may affect its interaction with other proteins, binding to the promoters of its target genes, and/or transcriptional activation. The knowledge gained from our present study about activating mutations that have occurred in different regions of Mrr1p in drug-resistant C. albicans strains should help to understand how the activity of the transcription factor is regulated once its functional domains and interacting proteins as well as in vivo DNA binding sites in the C. albicans genome have been identified, which are major aims for future investigations.

Fig. 6.

Fig. 6

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Location of all known gain-of-function mutations in Mrr1p. The Mrr1p protein is represented as a linear bar and the DNA binding domain at the N-terminus is indicated by black shading.

We have previously demonstrated that MRR1 alleles containing gain-of-function mutations can activate MDR1 expression and confer drug resistance in the presence of a wild-type MRR1 allele, although a slight negative effect of a nonmutated allele on the activity of gain-of-function alleles was also observed (Morschhäuser et al., 2007). Similarly, deletion of the wild-type MRR1 allele in strain DSY292 also resulted in a detectable additional increase in cerulenin resistance of the heterozygous mutant that retained only the mutated MRR1P683H allele (Fig. 4). The ability of MRR1 gain-of-function alleles to activate MDR1 expression in the presence of a wild-type MRR1 allele is in contrast to the activation of CDR1 and CDR2 by the transcription factor Tac1p, where TAC1 gain-of-function alleles conferred hyperactive phenotypes only when homozygous (Coste et al., 2006). Nevertheless, the presence of two instead of only one mutated MRR1 allele also results in increased drug resistance. In two clinical C. albicans isolates that were examined in a previous study (Morschhäuser et al., 2007) as well as in four additional isolates investigated in our present work, the deletion of one of the two mutated MRR1 copies resulted in a partial loss of drug resistance. These findings explain why the majority of MDR1 overexpressing, fluconazole-resistant strains had become homozygous for a mutated MRR1 allele. Apparently, the increased fluconazole resistance provides sufficient advantage in the presence of the drug both in vitro and in vivo during therapy to select for the loss of heterozygosity once a gain-of-function mutation has occurred in one of the MRR1 alleles. Further support for this scenario came from the analysis of the serial isolates from patient F, where we identified an intermediate isolate (F3) that contained the P683S mutation in only one of the two MRR1 alleles and displayed intermediate MDR1 expression and fluconazole resistance. Isolate F5, which was obtained from a later infection episode, had become homozygous for the mutated MRR1 allele and exhibited higher MDR1 transcript levels and further increased fluconazole resistance (Franz et al., 1998, and this study).

Only one of the clinical isolates, DSY292, had retained a wild-type MRR1 allele that did not contribute to the drug resistance of this strain. Deletion of the mutated MRR1P683H allele largely abolished the increased resistance of DSY292 to cerulenin and brefeldin A, which in other drug-resistant strains has been shown to be mediated mostly or exclusively by MDR1 overexpression (Wirsching et al., 2001). In contrast, fluconazole resistance of strain DSY292 was not or only slightly affected by deletion of MRR1. It was previously shown that DSY292 has aquired a Y132H mutation in one ERG11 allele, in addition to the G464S and R467K mutations that were already present in both ERG11 alleles of the matched isolate DSY291 (Sanglard et al., 1998). The presence of the Y132H mutation resulted in a more than 8-fold increased fluconazole resistance of cells expressing this ERG11 allele (Sanglard et al., 1998), explaining why DSY292 retained a high level of resistance to fluconazole after deletion of MRR1. We speculate that the P683H mutation may have occurred in one MRR1 allele and been selected before the appearance of the Y132H mutation in ERG11. The high fluconazole resistance conferred by the ERG11 mutations then probably diminished the contribution of the MRR1 mutation and prevented selection for MRR1 homozygosity in DSY292. Mutated MRR1 alleles also contribute only partially to the overall fluconazole resistance of other strains that exhibit additional resistance mechanisms, like isolate G5, which contains a G464S mutation in both ERG11 alleles (Franz et al., 1998; Morschhäuser et al., 2007), or isolate 6692 (see Fig. 4), which exhibits increased resistance to other azoles that are not Mdr1p substrates, although the basis of this resistance has not been identified (Saidane et al., 2006).

Interestingly, we also found one MDR1 overexpressing clinical isolate that had aquired independent gain-of-function mutations in either of its MRR1 alleles, both of which contributed to the drug resistance of this isolate. A similar finding has been reported by Coste et al., who identified two independent hyperactive TAC1 alleles in a fluconazole-resistant isolate that overexpressed CDR1 and CDR2 (Coste et al., 2007). However, most of the MDR1 overexpressing strains had become homozygous for a mutated MRR1 allele (11 out of 14 strains analysed so far), presumably because genomic alterations that result in homozygosity occur more frequently than the aquisition of an independent mutation in the second allele. A more detailed analysis of these strains showed that loss of heterozygosity was usually caused by mitotic recombination, although duplication of the chromosome containing the mutated MRR1 allele and loss of the other copy of chromosome 3 seems to have occurred in two of the in vitro selected fluconazole-resistant strains. These results are in line with recent findings by Wu et al., who reported that loss of one copy of chromosome 5, followed by duplication of the remaining homolog, is the most common mechanism generating homozygosity for the mating type locus (MTL) in strains cultured in the laboratory, whereas MTL homozygosity in natural C. albicans strains usually occurs by mitotic recombination, which allows the retention of many polymorphic genes and results in a smaller decrease in competitiveness than the complete loss of heterozygosity on that chromosome (Wu et al., 2005; Wu et al., 2007). While we can not exclude the possibility that loss of heterozygosity may have occurred during storage and propagation of the strains in the laboratory, the fact that most susceptible isolates were heterozygous, while most resistant isolates were homozygous for mutated MRR1 alleles indicates that loss of heterozygosity has in most, if not all cases been selected for during fluconazole therapy of the patients.

Genomic alterations involving chromosome 5, which result in aneuploidies that are sometimes combined with an increased copy number of mutated TAC1 and ERG11 alleles have been found in fluconazole-resistant C. albicans strains that overexpress CDR1 and CDR2 (Coste et al., 2007). Trisomy for chromosome 3, on which MRR1 is located, has also been reported in in vitro generated, fluconazole-resistant C. albicans strains, however, no MDR1 overexpression was seen in these strains (Perepnikhatka et al., 1999). Our analyses indicate that MDR1 overexpression is not usually associated with an increase in the copy number of MRR1. All seven MDR1 overexpressing clinical C. albicans isolates in which the MRR1 alleles were inactivated by targeted gene deletion in this and a previous study (Morschhäuser et al., 2007) contained only two copies of MRR1. Additional strains, which had become homozygous for mutated MRR1 alleles but retained heterozygosity for other genes on chromosome 3 (strains 1587, CAPR515, and CAPR517), also had not amplified one of the chromosome 3 homologues, as direct sequencing of the PCR products showed equal signal intensities at heterozygous positions (data not shown). Although we have shown that the presence of two instead of only one mutated MRR1 allele results in increased drug resistance, it seems that the selection pressure was not strong enough to result in a further stable increase in the MRR1 copy number in these strains.

In summary, this study demonstrates that gain-of-function mutations in the transcription factor Mrr1p, followed by loss of heterozygosity, are the main cause of MDR1 overexpression in fluconazole-resistant C. albicans strains. This underscores the important role of Mrr1p in the development of multidrug resistance in C. albicans and illustrates again how the genomic flexibility of this fungal pathogen contributes to its ability to adapt to the selection pressure caused by the presence of fluconazole both in vitro and in the human host during antimycotic therapy.

Experimental procedures

Strains and growth conditions

C. albicans strains used in this study are listed in Table S1. All strains were stored as frozen stocks with 15% glycerol at −80°C and subcultured on YPD agar plates (10 g yeast extract, 20 g peptone, 20 g glucose, 15 g agar per liter) at 30°C. For routine growth of the strains YPD liquid medium was used. Selection of nourseothricin-resistant (NouR) transformants was performed on YPD agar plates containing 200 μg ml−1 nourseothricin (Werner Bioagents, Jena, Germany). To obtain nourseothricin-sensitive (NouS) derivatives in which the SAT1 flipper was excised by FLP-mediated recombination, transformants were grown overnight in YCB-BSA-YE medium (23.4 g yeast carbon base, 4 g bovine serum albumin, 2 g yeast extract per litre, pH 4.0) without selective pressure to induce the SAP2 promoter controlling caFLP expression. One hundred to 200 cells were then spread on YPD plates containing 20 μg ml−1 of nourseothricin and grown for 2 days at 30°C. NouS clones were identified by their small colony size and confirmed by restreaking on YPD plates containing 100 μg ml−1 of nourseothricin as described previously (Reuβ et al., 2004).

Plasmid constructions

The coding region and flanking sequences of the MRR1 alleles of the different C. albicans strains was amplified by polymerase chain reaction (PCR) with the primers ZCF36-3 and ZCF36-6, which bind in the MRR1 upstream and downstream regions, respectively (primer sequences are given in Table S2). The PCR products were digested at the introduced SacI and ApaI restriction sites and cloned into the vector pBluescript to generate plasmids pZCF36-B3-1, pZCF36-B3-2, pZCF36-B4, pZCF36-1442-1, pZCF36-1442-2, pZCF36-2271, pZCF36-1490-1, pZCF36-1490-2, pZCF36-1587, pZCF36-5044, pZCF36-5052, pZCF36-DSY291-1, pZCF36-DSY291-2, pZCF36-DSY292-1, pZCF36-DSY2285-1, pZCF36-DSY2285-2, pZCF36-DSY2286, pZCF36-5833-1, pZCF36-5833-2, pZCF36-6692-1, pZCF36-6692-2, pZCF36-CAPR510, pZCF36-CAPR513, pZCF36-CAPR514, pZCF36-CAPR515 and pZCF36-CAPR517. The SacI-PstI fragments from pZCF36-CAPR510, pZCF36-CAPR513, pZCF36-CAPR514, pZCF36-CAPR515, pZCF36-CAPR517, pZCF36-DSY292-1, pZCF36-2271, pZCF36-1587 and pZCF36-DSY2286 were then substituted for the corresponding fragment in the previously described plasmid pZCF36K2 (Morschhäuser et al., 2007) to produce pZCF36K6, pZCF36K7, pZCF36K8, pZCF36K9, pZCF36K10, pZCF36K13, pZCF36K14, pZCF36K15 and pZCF36K19, respectively (see Fig. 1 for illustration). Similarly, the EcoRI-PstI fragments from pZCF36-B3-2 and pZCF36-B4 were substituted for the corresponding fragment in pZCF36K2 to yield pZCF36K11 and pZCF36K12, respectively. For the generation of pZCF36K16 and pZCF36K17, the SacI-EcoRI fragments from pZCF36-5833-1 and pZCF36-6692-1, respectively, were substituted for the corresponding fragment in pZCF36K2. pZCF36K18 was obtained by ligating the SacI-NdeI fragment from pZCF36-6692-1 and the NdeI-EcoRI fragment from pZCF36-6692-2 into the SacI/EcoRI-digested pZCF36K2.

C. albicans transformation

C. albicans strains were transformed by electroporation (Köhler et al., 1997) with the following gel-purified linear DNA fragments: The SacI-ApaI fragments from plasmids pZCF36K6-K19 were used to insert mutated MRR1 alleles and the corresponding nonmutated alleles into one the disrupted mrr1Δ loci of strains SCMRR1M4A and -B and the reporter strain CAG48MRR1M4B. The SacI-ApaI fragment from plasmid pZCF36M2 (Morschhäuser et al., 2007) was used for deletion of MRR1 in the drug-resistant clinical C. albicans isolates. Selection of nourseothricin-resistant transformants was performed as described previously (Reuβ et al., 2004). The correct integration of each construct was confirmed by Southern hybridization of NsiI-digested genomic DNA with MRR1-specific probes.

Isolation of genomic DNA and Southern hybridization

Genomic DNA from C. albicans was isolated as described previously (Millon et al., 1994). 10 μg of DNA was digested with NsiI, 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 ECL™ Direct Nucleic Acid Labelling and Detection System (GE Healthcare, Braunschweig, Germany) according to the instructions of the manufacturer.

DNA sequencing and analysis of allelic polymorphisms

The sequences of the cloned MRR1 alleles were determined using universal forward and reverse primers and the primers listed in Table S2. The loss of heterozygosity in resistant isolates and the absence of the gain-of-function mutations in susceptible isolates was confirmed by direct sequencing of PCR products amplified from genomic DNA of the strains with primers covering the relevant regions. The presence or absence of allelic polymorphisms in the CAP1, MEP1, SAP9, and AAP1 genes in the SC5314 derivatives and in the clinical isolates was determined by amplifying parts of these genes and direct sequencing of the MEP1, SAP9, and AAP1 PCR products with the primers listed in Table S2. Heterozygosity for the CAP1 alleles was tested by digesting the respective PCR products with EcoRI and (separately) SphI and analysing the fragments by agarose gel electrophoresis. For isolates F2 and F5, in which the CAP1 alleles did not exhibit these restriction site polymorphisms, the PCR products were also sequenced. The results of these analyses are described in detail in Table S3.

FACS analysis

Fluorescence-activated cell sorter (FACS) analysis was performed with a FACSCalibur cytometry system equipped with an argon laser emitting at 488 nm (Becton Dickinson, Heidelberg, Germany). 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 data were collected by using logarithmic amplifiers. The mean fluorescence values were determined with CellQuest Pro (Becton Dickinson) software.

Drug susceptibility tests

Stock solutions of the drugs were prepared as follows. Fluconazole (1 mg ml−1) was dissolved in water and cerulenin (5 mg ml−1) and brefeldin A (5 mg ml−1) were dissolved in dimethylsulfoxide (DMSO). In the assays, serial twofold dilutions in the assay medium were prepared from the following initial concentrations: cerulenin, 100 μg ml−1; brefeldin A, 200 μg ml−1; fluconazole, 100 μg ml−1. Susceptibility tests were carried out in high resolution medium (14.67 g HR-Medium [Oxoid GmbH, Wesel, Germany], 1 g NaHCO3, 0.2 M phosphate buffer [pH 7.2]), using a previously described microdilution method (Ruhnke et al., 1994).

Nucleotide sequence accession numbers

The coding sequences of the MRR1 alleles described in the present work and in a previous study (Morschhäuser et al., 2007) have been deposited in GenBank with the following accession numbers: EU139261 (MRR1F2-1), EU139262 (MRR1F2-2), EU139263 (MRR1F5), EU139264 (MRR1G2-1), EU139265 (MRR1G2-2), EU139266 (MRR1G5), EU497768 (MRR1B3-1), EU497744 (MRR1B3-2), EU497745 (MRR1B4), EU497746 (MRR11442-1), EU497747 (MRR11442-2), EU497748 (MRR12271), EU497749 (MRR11490-1), EU497750 (MRR11490-2), EU497751 (MRR11587), EU497752 (MRR15044), EU497753 (MRR15052), EU497754 (MRR1DSY291-1), EU497755 (MRR1DSY291-2), EU497756 (MRR1DSY292-1), EU497757 (MRR1DSY2285), EU497758 (MRR1DSY2286), EU497759 (MRR15833-1), EU497760 (MRR15833-2), EU497761 (MRR16692-1), EU497762 (MRR16692-2), EU497763 (MRR1CAPR510), EU497764 (MRR1CAPR513-2), EU497765 (MRR1CAPR514), EU497766 (MRR1CAPR515), and EU497767 (MRR1CAPR517).

Acknowledgments

We are indebted to our colleagues William Kirkpatrick and Thomas Patterson, Carol Kumamoto, Martine Raymond, and Dominique Sanglard for providing C. albicans strains. Sequence data for Candida albicans was obtained from Candida Genome Database (http://www.candidagenome.org). This study was supported by the Deutsche Forschungsgemeinschaft (DFG grant MO 846/3 and SFB 630) and the National Institutes of Health (NIH grant AI058145).

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