Mutations in genes encoding zinc cluster transcription factors (ZCFs) such as TAC1, MRR1, and UPC2 play a key role in Candida albicans azole antifungal resistance. Artificial activation of the ZCF Mrr2 has shown increased expression of the gene encoding the Cdr1 efflux pump and resistance to fluconazole.
KEYWORDS: Candida albicans, MRR2, antifungal drug resistance, fluconazole, transcription factors, transcriptional activation, zinc
ABSTRACT
Mutations in genes encoding zinc cluster transcription factors (ZCFs) such as TAC1, MRR1, and UPC2 play a key role in Candida albicans azole antifungal resistance. Artificial activation of the ZCF Mrr2 has shown increased expression of the gene encoding the Cdr1 efflux pump and resistance to fluconazole. Amino acid substitutions in Mrr2 have recently been reported to contribute to fluconazole resistance in clinical isolates. In the present study, 57 C. albicans clinical isolates with elevated fluconazole MICs were examined for mutations in MRR2 and expression of CDR1. Mutations in MRR2 resulting in 15 amino acid substitutions were uniquely identified among resistant isolates, including 4 substitutions (S466L, A468G, S469T, T470N) previously reported to reduce fluconazole susceptibility. Three additional, novel amino acid substitutions (R45Q, A459T, V486M) were also discovered in fluconazole-resistant isolates. When introduced into a fluconazole-susceptible background, no change in fluconazole MIC or CDR1 expression was observed for any of the mutations found in this collection. However, introduction of an allele leading to artificial activation of Mrr2 increased resistance to fluconazole as well as CDR1 expression. Moreover, Mrr2 amino acid changes reported previously to have the strongest effect on fluconazole susceptibility and CDR1 expression also exhibited no differences in fluconazole susceptibility or CDR1 expression relative to the parent strain. While all known fluconazole resistance mechanisms are represented within this collection of clinical isolates and contribute to fluconazole resistance to different extents, mutations in MRR2 do not appear to alter CDR1 expression or contribute to resistance in any of these isolates.
INTRODUCTION
Candida spp. are among the most common pathogen groups implicated in nosocomial bloodstream infections in the United States (1, 2). Candida albicans is the most common Candida sp. in invasive candidiasis and the primary cause of mucosal Candida infections such as oropharyngeal candidiasis (2–6). Fluconazole and other azoles are mainstays of treatment in invasive candidiasis and oropharyngeal candidiasis, and despite the emergence of other antifungal drug classes, there has been no substantial decline in fluconazole resistance rates in C. albicans (3, 7–9). Moreover, the high mortality rate for C. albicans in invasive fungal disease represents a compelling reason to further investigate how we can better utilize the available antifungal drugs in order to overcome treatment failure (4, 10, 11). Therefore, it is imperative that we achieve a comprehensive understanding of the mechanisms of azole resistance within this organism.
Current understanding of fluconazole resistance within C. albicans involves sterol biosynthesis gene mutations as well as zinc-cluster transcription factor-mediated changes in efflux pump activity and drug target abundance (12). Zinc-binding proteins play a crucial role in the transcriptional regulation of a vast number of genes in eukaryotes, and in particular, the zinc cluster transcription factors (ZCFs) within the fungal kingdom make up one of the largest regulatory protein families in yeast (13, 14). Regarding azole antifungal resistance in Candida spp., ZCFs play a major role in almost all species where azole resistance mechanisms have been elucidated, including C. glabrata and C. parapsilosis (12). Changes in the zinc-cluster transcription factors Tac1, Mrr1, and Upc2 are thought to be responsible for the bulk of fluconazole resistance in C. albicans (12, 15–22). The presence of gain-of-function mutations in the transcription factor genes TAC1 and MRR1 results in increased expression of the efflux pump genes CDR1/CDR2 and MDR1, respectively, while gain-of-function mutations discovered in UPC2 lead to overexpression of the gene encoding the azole target, Erg11 (14α-sterol demethylase) (19, 21, 22). Other mechanisms of azole resistance include changes in the azole target 14α-sterol demethylase through mutations in its encoding gene ERG11 and less commonly through mutations in the sterol-C5(6)-desaturase-encoding gene ERG3 (23–26).
There is evidence strongly suggesting that other mechanisms of azole resistance in Candida spp. exist. For example, it has been previously demonstrated that other ZCFs can alter susceptibility to fluconazole when transcriptionally activated (14). In particular, decreased fluconazole susceptibility was observed in a laboratory strain expressing artificially activated Mrr2. The hyperactive ZCF Mrr2 was noted to activate the CDR1 promoter but not the CDR2 promoter, in contrast to activated Tac1, which is known to increase expression of both CDR1 and CDR2 (27). Additionally, fluconazole sensitivity was restored after deletion of the CDR1 gene in the hyperactive Mrr2 strain (14). More recently, potential activating mutations in Mrr2 which increased expression of CDR1 but not CDR2 have been described in fluconazole-resistant clinical isolates of C. albicans, indicating not only that these mechanisms can manifest in vitro but also that they are clinically relevant (28). Mrr2 is a relatively understudied ZCF whose role in clinical azole resistance has not yet been completely defined, but the potential significance of Mrr2 as a contributing azole resistance determinant in C. albicans cannot be overlooked.
In the present study, we investigated the contributions to fluconazole resistance of mutations in MRR2 oberved among isolates within a collection of azole-resistant clinical C. albicans isolates. To accomplish this, we sequenced the MRR2 open reading frame (ORF) in a collection of 57 clinical isolates with reduced susceptibility to fluconazole (MIC range, 4 to >64 μg/ml; data not shown). Screening of the strains revealed multiple polymorphisms in MRR2, several of which had been previously described to impact fluconazole MICs. Clinically derived MRR2 alleles were introduced in a fluconazole-susceptible background to generate strains containing multiple single nucleotide polymorphisms (SNPs) in the MRR2 gene. In contrast to a previous report (28), we found no changes in fluconazole MIC or CDR1 expression upon introduction of any of the MRR2 SNPs observed in clinical isolates in our collection or that had been described previously.
RESULTS
SNPs in MRR2 among fluconazole-resistant clinical isolates of C. albicans.
To identify clinically occurring amino acid substitutions in Mrr2, we sequenced the MRR2 gene of 67 C. albicans clinical isolates (including 10 fluconazole-susceptible and 57 fluconazole-resistant isolates) that we had previously described (20, 29). This collection of isolates had been previously characterized for known resistance mechanisms and collectively possessed every known mechanism of azole resistance (20, 29). Overall, 25 (44%) of 57 fluconazole-resistant isolates possessed SNPs that result in amino acid changes in Mrr2 (see Table S1 in the supplemental material); however, many of these SNPs were also observed in fluconazole-susceptible isolates and would not be expected to influence fluconazole MICs. Seven amino acid substitutions resulting from SNPs in MRR2 were present only among fluconazole-resistant isolates within our collection. Three of these substitutions (R45Q, A459T, V486M) had not previously been described in the literature. The remaining four substitutions (S466L, A468G, S469T, T470N) had been previously found in resistant clinical C. albicans isolates and were reported to have an effect on fluconazole MIC and CDR1 expression (28). Within our collection, these four substitutions appeared in four clinical isolates and exclusively co-occurred with each other. Interestingly, constitutive CDR1 expression levels were less than 2-fold greater than that of a composite of fluconazole-susceptible isolates in five clinical isolates containing either the A459T substitution or the combination of substitutions S466L, A468G, S469T, and T470N (Table 1).
TABLE 1.
Mrr2 amino acid substitutions found in azole-resistant clinical isolates expressing CDR1
Fluconazole susceptibility in MRR2 mutant strains.
To determine if the newly discovered amino acid changes in Mrr2 affected fluconazole susceptibility, we introduced two copies of the MRR2 allele from fluconazole-resistant isolates 23, 20, 28, and 29 containing, respectively, the R45Q substitution, the A459T substitution, the V486M substitution, and combined amino acid substitutions S466L, A468G, S459T, and T470N into the mrr2Δ/Δ strain (SCZCF34M4A) via homologous recombination using the SAT1 flipper technique. This subsequently yielded homozygous mutant strains MRR2R45Q, V451A (SCMRR2R2S2), MRR2S143P, L144V, T145A, S165N, A459T, S480P (SCMRR2R4S4), MRR2T83A, A459T, S480P, V486M (SCMRR2R5S5), and MRR2S466L, A468G, S469T, T470N, S480P (SCMRR2R6S6), which we refer to here as MRR2R45Q, MRR2A459T, MRR2A459T, V486M, and MRR2S466L, A468G, S469T, T470N, respectively. An additional mutant, MRR2T83A, V451A, V582L (SCMRR2R3S3), here referred to as MRR2V582L, was similarly constructed from the MRR2 allele of fluconazole-susceptible isolate 22 as it contained a V582L substitution not previously described in the literature. For comparison, we created a MRR2WT (MRR2 wild type; SCMRR2R1S1) strain by inserting wild-type MRR2 amplified from fluconazole-susceptible clinical isolate SC5314 into the mrr2Δ/Δ strain and included the previously described hyperactive Mrr2 strain (SCZCF34GAD1), which highly expresses artificially activated Mrr2 (14). Surprisingly, none of the MRR2 mutant strains showed any change in fluconazole susceptibility compared to SC5314 or MRRWT as measured by CLSI standard methods (Table 2). Those strains included MRR2S466L, A468G, S469T, T470N, whose MRR2 alleles contained four of six mutations previously reported to have an effect on fluconazole susceptibility. As expected, hyperactive Mrr2 exhibited elevated fluconazole MICs. Strain susceptibilities measured on RPMI agar using Etest strips also did not show reduced fluconazole susceptibility for any polymorphism-containing MRR2 strain compared to SC5314. However, via Etest, we observed that the mrr2Δ/Δ strain was slightly hypersusceptible to fluconazole compared to SC5314, as has been established previously (Table 2) (14).
TABLE 2.
Fluconazole susceptibilities of MRR2 mutant strains
| Strain | Strain description | 24H FLU MIC (μg/ml) |
|
|---|---|---|---|
| CLSI | Etest | ||
| SC5314 | WT | 0.25 | 0.125 |
| SCZCF34M4A | mrr2Δ/Δ | 0.25 | 0.064 |
| SCZCF34GAD1A | overexpressed MRR2-GADa | 0.5 | 0.75 |
| SCMRR2R1S1 | MRR2WT | 0.25 | 0.094 |
| SCMRR2R2S2 | MRR2S143P,L144V,T145A,S165N,A459T,S480P | 0.25 | 0.125 |
| SCMRR2R3S3 | MRR2T83A,V451A,V582L | 0.25 | 0.125 |
| SCMRR2R4S4 | MRR2R45Q,V451A | 0.25 | 0.125 |
| SCMRR2R5S5 | MRR2T83A,A459T,S480P,V486M | 0.25 | 0.094 |
| SCMRR2R6S6 | MRR2S466L,A468G,S469T,T470N,S480P | 0.25 | 0.094 |
| SCMRR2R8S8 | MRR2C9 | 0.25 | 0.125 |
| SCMRR2GAD1R1S1 | MRR2-GAD | 0.5 | 0.19 |
| SCΔmrr2PADH1MRR2WT A | Overexpressed MRR2WT | 0.25 | 0.125 |
| SCΔmrr2PADH1MRR2WT B | Overexpressed MRR2WT | 0.25 | 0.125 |
| SCΔmrr2PADH1MRR2C9 A | Overexpressed MRR2C9 | 0.25 | 0.125 |
| SCΔmrr2PADH1MRR2C9 B | Overexpressed MRR2C9 | 0.25 | 0.125 |
GAD, 3× HA-tagged GAL4 activation domain.
MRR2 mutant strains do not overexpress CDR1 when expressed from the MRR2 locus.
To investigate whether any of the MRR2 mutant strains could constitutively increase expression of efflux pump gene CDR1, we measured the CDR1 mRNA abundance for each MRR2 strain compared to SC5314 (Fig. 1A) in triplicate via reverse transcription–quantitative real-time PCR (RT-qPCR). Expression of CDR1 was not significantly increased compared to SC5314 in any of the created strains containing polymorphic MRR2 alleles. MRR2R45Q, MRR2A459T, and MRR2A459T, V486M, which contained novel Mrr2 substitutions found only in fluconazole-resistant isolates, did not appear to express CDR1 to a greater degree than MRR2WT or SC5314. MRR2S466L, A468G, S469T, T470N, containing four mutations thought to influence CDR1 expression, also showed no increases in expression over MRRWT or SC5314, in line with the results obtained with our clinical isolates containing these substitutions in Mrr2 but not overexpressing CDR1. The strain expressing hyperactive Mrr2 showed an approximately 8-fold increase in CDR1 expression over the level seen with the control, consistent with its reduced susceptibility to fluconazole.
FIG 1.
(A) Fold change in expression levels compared to SC5314 of CDR1 for the mrr2Δ/Δ strain (SCZCF34M4A); the mutant MRR2 strains containing MRR2WT (SCMRR2R1S1), MRR2A459T (SCMRR2R2S2), MRR2V582L (SCMRR2R3S3), MRR2R45Q (SCMRR2R4S4), MRR2A459T, V486M (SCMRR2R5S5), MRR2S466L, A468G, S469T, T470N (SCMRR2R6S6), and MRR2C9 (SCMRR2R8S8); and artificially activated Mrr2 strains expressed from either the ADH1 promoter (SCZCF34GAD1A) or the native MRR2 promoter (SCMRR2GAD1R1S1). (B) Fold change in MRR2 expression levels compared to SC5314 for the mrr2Δ/Δ strain (SCZCF34M4A), the mutant MRR2C9 strain (SCMRR2R8S8), and the artificially activated Mrr2 strains expressed from either the ADH1 promoter (SCZCF34GAD1A) or the native MRR2 promoter (SCMRR2GAD1R1S1). Expression was obtained in technical and biological triplicate for each strain tested, and error bars reflect standard errors of the means.
Amino acid substitutions S466L, A468G, S469T, and T470N do not influence fluconazole MICs or CDR1 expression.
The four amino acid substitutions (S466L, A468G, S469T, and T470N) that were introduced into MRR2S466L, A468G, S469T, T470N failed to alter fluconazole susceptibility or change CDR1 expression from the level seen with SC5314. However, since we created this strain by inserting the entire MRR2 allele taken from a resistant isolate, these substitutions were accompanied by other polymorphisms within the MRR2 allele; thus, we could not rule out the possibility of an interaction between these polymorphisms and the Mrr2 substitutions S466L, A468G, S469T, and T470N that could mask the increased fluconazole MICs previously associated with these substitutions. Therefore, we created the mutant MRRS143P, L144V, T145A, H358N, E439K, V451A, S466L, A468G, S469T, T470N, S480P strain (SCMRR2R8S8, here referred to as MRR2C9) by introducing a mutant MRR2 allele containing 13 nucleotide changes corresponding to 11 amino acid substitutions in Mrr2 (Table 3) in order to verify the phenotype associated with these changes described in the literature (28). Surprisingly, the levels of MRR2C9 fluconazole MICs (Table 2) and CDR1 expression (Fig. 1A) were not different from those seen with SC5314, suggesting that these amino acid changes did not impact fluconazole resistance or CDR1 expression when MRR2 was expressed from the native promoter.
TABLE 3.
Strains used in this study
| Strain | Genotypea | Source or reference |
|---|---|---|
| SC5314 | Wild type | ATCC |
| SCZCF34M4 A/B | Δmrr2::FRT/Δmrr2::FRT | 14 |
| SCZCF34GAD1 A/B | ADH1/adh1::PADH1-MRR2-GAL4AD-3xHA-caSAT1 | 14 |
| SCMRR2R1S1 A/B | MRR2WT::FRT/MRR2WT::FRT | This study |
| SCMRR2R2S2 A/B | MRR2S143P,L144V,T145A,S165N,A459T,S480P::FRT/MRR2S143P,L144V,T145A,S165N,A459T,S480P::FRT | This study |
| SCMRR2R3S3 A/B | MRR2T83A,V451A,V582L::FRT/MRR2T83A,V451A,V582L::FRT | This study |
| SCMRR2R4S4 A/B | MRR2R45Q,V451A::FRT/MRR2R45Q,V451A::FRT | This study |
| SCMRR2R5S5 A/B | MRR2T83A,A459T,S480P,V486M::FRT/MRR2 T83A,A459T,S480P,V486M::FRT | This study |
| SCMRR2R6S6 A/B | MRR2S466L,A468G,S469T,T470N,S480P::FRT/MRR2S466L,A468G,S469T,T470N,S480P::FRT | This study |
| SCMRR2R8S8 A/B/C | MRR2C9::FRT/MRR2C9::FRT | This study |
| SCMRR2GADR1S1 A/B | MRR2-GAL4AD::FRT/MRR2-GAL4AD-3xHA::FRT | This study |
| SCΔmrr2PADH1MRR2WT A/B | Δmrr2::FRT/Δmrr2::FRT, ADH1/adh1::PADH1-MRR2WT | This study |
| SCΔmrr2PADH1MRR2C9 A/B | Δmrr2::FRT/Δmrr2::FRT, ADH1/adh1::PADH1-MRR2C9 | This study |
Tentative activating mutations in MRR2 that were described in reference 28 are underlined. Amino acid changes in MRR2 indicated in bold were discovered in this study. “MRR2C9” refers to the MRR2 allele of isolate C9 as described previously (28) containing S143P, L144V, T145A, H358N, E439K, V451A, S466L, A468G, S469T, T470N, and S480P mutations.
Our tested strains utilized homologous recombination to reintroduce polymorphic MRR2 alleles into the mrr2Δ/Δ strain at the native MRR2 locus. In order to substantiate our method of strain creation, we used the SAT1/FLP strategy to introduce two copies of the MRR2 allele in which the C-terminal end of the gene was fused to the GAL4 activation domain and 3× hemagglutinin (HA) tagged, creating the mutant strain MRR2-GAL4AD-3xHA (SCMRR2GAD1R1S1). Fusion of the HA-tagged Gal4 activation domain to full-length ZCFs had been previously shown to constitutively activate fungal ZCFs such as Tac1, Upc2, and Mrr1 when expressed from the ADH1 promoter (14). Here, our purpose was to create a constitutively active Mrr2 strain in which both copies of MRR2 were natively expressed. CDR1 expression increased ∼2.5-fold from the level seen with SC5314 in this strain, which resulted in fluconazole MICs that were slightly increased over those seen with SC5314 as well, showing that homozygous replacement of the MRR2 allele in the native locus using the SAT1/FLP method was able to produce changes in CDR1 expression levels and fluconazole MICs without foreign promoter-driven overexpression of MRR2. This change was smaller than the increase in CDR1 expression and fluconazole MIC seen with the hyperactivated MRR2 strain, in which MRR2 is expressed from the strong ADH1 promoter.
Activated Mrr2 does not appear to regulate MRR2 expression.
To determine if Mrr2 autoregulates its expression level, we measured the MRR2 mRNA abundance of our created strains relative to SC5314 (Fig. 1B). Mutant strain MRR2C9 and artificially activated MRR2 strain MRR2-GAL4AD-3xHA did not appear to have increased expression of MRR2 compared to SC5314. As expected, strains with hyperactive MRR2 expressed MRR2 at levels ∼14-fold higher than those seen with SC5314. Therefore, it appears that only the relative overexpression of MRR2 from the ADH1 promoter and not activation of Mrr2 itself had an effect on increased MRR2 expression.
Overexpression of wild-type or mutant MRR2 in C. albicans strain did not affect fluconazole susceptibility or CDR1 expression.
In order to investigate whether changes in CDR1 expression and fluconazole susceptibility required strong constitutive expression of MRR2, we constructed strains which lacked both native copies of MRR2 but possessed a single copy of the open reading frame of either MRR2WT or MRR2C9 fused to the ADH1 promoter. In both cases, MRR2 mRNA expression was increased >30-fold compared to SC5314 in the independently created mutant strains mrr2Δ/Δ adh1Δ::PADH1-MRR2WT/ADH1 (SCΔmrr2PADH1MRR2WT A/B) and mrr2Δ/Δ adh1Δ::PADH1-MRR2C9/ADH1 (SCΔmrr2PADH1MRR2C9 A/B) (Fig. 2B). However, compared to SC5314, CDR1 expression was not markedly different for any of the strains, with no increase in constitutive CDR1 mRNA greater than 1.2-fold (Fig. 2A). As expected, the strains also did not show any change in fluconazole susceptibility by either MIC or Etest (Table 2). Thus, overexpression of either MRR2WT or MRR2C9 does not appear to influence CDR1 expression or fluconazole susceptibility in C. albicans.
FIG 2.
(A) Fold change in expression levels compared to SC5314 of CDR1 for strains possessing either MRR2WT (SCΔmrr2PADH1MRR2WT A and B) or mutant MRR2C9 (SCΔmrr2PADH1MRR2C9 A and B) expressed via the ADH1 promoter. (B) Fold change in MRR2 expression levels compared to SC5314 for the strains indicated in panel A. Expression was obtained in technical and biological triplicate for each strain tested, and error bars reflect standard errors of the means.
DISCUSSION
Gain-of-function mutations in the genes encoding fungus-specific ZCFs Tac1, Mrr1, and Upc2 have long been known to contribute to azole resistance in C. albicans. However, was it reported only recently that nonsynonymous mutations found in the MRR2 gene of clinical isolates impacted fluconazole susceptibility through increased Cdr1 efflux pump expression, indicating that MRR2 may be a clinically relevant mechanism of azole resistance. Here, we sequenced a collection of predominantly fluconazole-resistant clinical isolates in order to uncover additional mutations in the MRR2 gene that would contribute to fluconazole resistance. In total, mutations resulting in 15 amino acid substitutions were identified across these clinical isolates. Three (R45Q, A459T, and V486M) were found uniquely in fluconazole-resistant clinical isolates and had not been previously reported. The remaining were described in a previous report or were present in fluconazole-susceptible isolates and therefore not likely directly involved in azole resistance. Our results indicate that none of the tested SNPs in MRR2 have an impact on susceptibility to fluconazole. Similarly, CDR1 expression also did not appear to be affected by the presence of any of the tested SNPs.
In particular, four mutations identified in isolates in our collection (S466L, A468G, S469T, and T470N) had been previously shown to impact fluconazole susceptibility. The amino acid substitutions S466L and T470N had also been reported to increase CDR1 expression by at least 2-fold compared to a control strain. In our hands, the amino acid substitutions S466L, A468G, S469T, and T470N did not appear to have any effect on susceptibility to fluconazole and CDR1 expression did not increase in the mutant strains containing these Mrr2 amino acid substitutions compared to SC5314. This finding is consistent with those seen with our clinical isolates that possess these changes in Mrr2 but do not highly express CDR1.
Since the publication of the original report first describing them, mutations in MRR2 have been referenced as a clinically relevant mechanism of fluconazole resistance (30–32). While our results confirm that artificial activation of the Mrr2 ZCF indeed results in CDR1 upregulation and a consequent increase in fluconazole resistance as previously described (14), we were unable to replicate the changes attributed to these mutations in a previous report (28). We were unsuccessful in our efforts to obtain isolate C9 from that previous report, which contained the MRR2 mutations in question, and as such could not make a direct assessment of this clinical isolate. One possible explanation for the disparity seen between our results and those reported previously could be differences in methodology. We expressed MRR2 from its native promoter, including replacement of both ORFs in the MRR2 locus, in order to mimic natural Mrr2 expression. This was in contrast to the methods used previously whereby MRR2 was placed in the ADE2 locus utilizing a single copy of the ORF under the control of the ADH1 promoter. This could possibly have caused altered metabolic burdens in these strains compared to ours. Furthermore, the ADH1 promoter has been described previously as having variability in activity over time under different conditions (33), and therefore Mrr2 protein levels can be different depending on the time at which cells were harvested or observed for assays. Lastly, we used 50% inhibition from the fluconazole-free control well for identifying the MIC of our strains, as directed by the CLSI reference method for broth microdilution antifungal susceptibility testing of yeast (34, 35). The MICs presented in the previous report were identified by 80% inhibition of cell growth in the control well, which could explain the increased MICs observed in MRR2 mutant strains, especially if the trailing growth phenomenon was observed in these strains.
Given the lower level of expression of MRR2 that we observed in our strains using the native MRR2 promoter compared to hyperactive Mrr2, which overexpressed MRR2 via fusion to the ADH1 promoter, we considered the possibility that the level of MRR2 expression in our strains was not sufficient to allow detection of changes in CDR1 expression or fluconazole susceptibility. However, the results obtained with the MRR2-GAL4AD-3xHA strain expressing artificially activated Mrr2 via its native promoter demonstrated that native MRR2 expression levels are sufficient for observation of changes in fluconazole MIC and CDR1 expression. Moreover, we believe that our strains, which tested the effects of the homozygous SNPs in MRR2 at the native locus, more closely approximate the effects that these nucleotide changes would have in a clinical isolate. Lastly, we constructed our own PADH1-MRR2-overexpressing strains to test whether there were differences in CDR1 expression or fluconazole susceptibility with respect to strong, constitutive expression of either MRR2WT or MRR2C9 and were unable to discern any phenotype differences between the strains. Overall, in all strains and under all conditions tested here, we were unable to detect any change in CDR1 expression or fluconazole susceptibility between the strains containing mutant MRR2 and those containing wild-type MRR2.
The ATP-binding cassette transporter Cdr1 has been shown to be regulated not only by Tac1 but also by Ndt80. Furthermore, it has been shown that through artificial activation and overexpression, the zinc cluster transcription factors Mrr2, Znc1, and Stb5 also can influence CDR1 expression levels. Thus, we speculate that there remain additional determinants of efflux pump upregulation that have clinical relevance. However, our experiments indicated that the nucleotide changes observed here, and reported previously, in MRR2 do not impact fluconazole susceptibility or CDR1 expression and are not clinically relevant to fluconazole resistance in C. albicans. Given that azole resistance cannot be fully explained by known mechanisms, further investigation of other ZCFs in azole resistance is warranted.
MATERIALS AND METHODS
Strains and growth conditions.
Table 2 lists the C. albicans strains used in this study. The C. albicans isolates were obtained from a repository of clinical isolates at the University of Iowa and have been previous reported elsewhere (20). Strains were stored in 40% glycerol frozen stocks at −80°C. Routine growth of cells was performed in YPD liquid media (1% yeast extract, 2% peptone, 2% dextrose) at 30°C. Nourseothricin (200 μg/ml)-containing YPD agar plates were used for selection of strains containing the SAT1 marker. For plasmid propagation, DH5α competent Escherichia coli cells (Invitrogen) were grown in Luria-Bertani (LB) broth or on LB agar plates containing either 100 μg/ml ampicillin or 50 μg/ml kanamycin.
Plasmid construction.
Plasmids were derived from plasmid strain pBSS2, which contains the SAT1 flipper disruption cassette from pSFS2 (36) placed in the pBluescript II KS+ vector. The 3′ flanking region of the MRR2 ORF was amplified from SC5314 genomic DNA using primers CaMRR2C_(NotI) and CaMRR2D_(SacI) and ligated into pBSS2 at the NotI and SacI restriction sites to create pMRR2CD. The 5′ upstream region and MRR2 ORF of isolates SC5314, 20, 22, 23, 28, and 29 were amplified using primers CaMRR2A_(KpnI) and CaMRR2E_(XhoI) and cloned into pMRR2CD to obtain plasmids pBSS2-MRR2WT, pBSS2-MRR220, pBSS2-MRR222, pBSS2-MRR223, pBSS2-MRR228, and pBSS2-MRR229, respectively. For pMRR2_C9.3, which replicated the MRR2 allele of isolate C9 described by Wang et al. (28), primers CaMRR2_P1_F and CaMRR2_C9_R were used to amplify a 5′ portion of the MRR2 ORF from genomic DNA of isolate 33. The resulting amplicon was fused to a synthesized gBlocks fragment (Integrated DNA Technologies) containing the 3′ portion of the MRR2 ORF using CaMRR2_Nested_F_KpnI and MRR2_Nested_DR_XhoI and cloned into pMRR2CD to give pMRR2_C9.2. Final changes of nucleotides were accomplished using short overlapping extension (SOE) PCR on plasmid pMRR2_C9.2 using primer pairs pBSS2_1F.2 and CaMRR2_C9.2_R6, CaMRR2_C9.2_F6 and CaMRR2_C9.2_R7, CaMRR2_C9.2_F7 and CaMRR2_C9.2_R8, and CaMRR2_C9.2_F8 and pBSS2_MAL2_R to generate amplicon fragments of the MRR2 ORF. Fragments were fused together via SOE PCR using CaMRR2_Nested_F_KpnI and CaMRR2_Nested_DR_XhoI and were cloned into pMRR2CD to create pMRR2_C9.3. Plasmid pMRR2GAD1 was generated using genomic DNA from SCZCF34GAD1A and primers CaMRR2GAD1A_F_KpnI and 3XHA_ACT1_R_XhoI to amplify the MRR2 ORF, including the fused GAL4 activation domain and 3× HA-tagged C-terminal region. The resulting amplicon was cloned into pMRR2CD to yield plasmid pMRR2GAD1. Plasmids pPADH1-MRR2WT and pPADH1-MRR2C9 were used to express the open reading frame of MRR2 from the ADH1 locus, utilizing the ADH1 promoter and termination sequences. The MRR2 open reading frame of either strain SC5314 or the C9 isolate was fused to the 3′ ADH1 terminator via overlap extension PCR utilizing primers CaMRR2_2F_EcoRI, CaMRR2_R_ADH1t.2, CaADH1t_F, and ADH1t_R_XhoI. The ADH1 promoter was PCR amplified using primers CaPADH1_AF_KpnI and CaPADH1_2R_EcoI. Both the ADH1 promoter and fused MRR2-ADH1t amplicons were digested either with KpnI and EcoRI or with EcoRI and XhoI, respectively, and subsequently ligated into KpnI-XhoI linearized plasmid pADH1CD, a pBSS2 derivative containing the 342-bp 3′ flanking homology region near the ADH1 locus generated with primers CaADH1_C_SacII and CaADH1_D_NcoI_SacI. Successful transformants were screened on LB agar plates containing 50 μg/ml ampicillin and sequenced for the MRR2 open reading frame corresponding to either strain SC5314 or isolate C9 for plasmid pPADH1-MRR2WT or pPADH1-MRR2C9, respectively.
MRR2 amplification and sequencing.
Table 4 lists the primers used for MRR2 amplification and sequence verification. The MRR2 coding sequence of each isolate was PCR amplified from genomic DNA using primers CaMRR2_F_Amp and CaMRR2_R_Amp. PCR products were purified using a QIAquick PCR purification kit (Qiagen), and products were sequenced on an ABI 3130XL genetic analyzer using MRR2 sequencing primers. Sequencing was accomplished in duplicate in independently grown isolates.
TABLE 4.
Primers used in this study
| Primer name and purpose | Sequencea |
|---|---|
| Amplification | |
| CaMRR2_F_Amp | 5′-TACGAAATACTTGGAGTTATTCCCTAC-3′ |
| CaMRR2_R_Amp | 5′-CTAGTTTTGTGTCTAGTTCTATTGTTATTG-3′ |
| CaMRR2GAD1A_F_KpnI | 5′-CAGGGTACCAACTTGAAAAATTGCTCAACTCTTATATAGCAAAAA TAAACAACCAATAGCTTCTTCGCCAATGACCAAACGTGATCGTAC-3′ |
| 3XHA_ACT1_R_XhoI | 5′-CTACTCGAGGATTTCCAGAATTTCACTCTTA-3′ |
| CaPADH1_AF_KpnI | 5′-GATGGTACCACTACCACTGCAGCTGCATC-3′ |
| CaMRR2_2R_EcoRI | 5′-CTTTTTGAGTTTTTGGGATTTGTTCGAATTCAATTGTTTTTGTATTTGTTGTTGTTGTTG-3′ |
| CaMRR2_2F_EcoRI | 5′-GATGAATTCATGACCAAACGTGATCGTACAAT-3′ |
| CaADH1t_R_XhoI | 5′-CATCTCGAGTTAACCAAAATCAACGACAAATTG-3′ |
| CaADH1_C_SacII | 5′-GATCCGCGGCATTGATTGTTTGTGTTAGTTTTTCA-3′ |
| CaADH1_D_NcoI_SacI | 5′-GATGAGCTCCCATGGAACACCCAGTTTAATTTCCATGA-3′ |
| Sequencing | |
| CaMRR2SeqA | 5′-GCAGAAGCGAGGGAACTTGAAA-3′ |
| CaMRR2SeqB | 5′-ACTTGGAGAAGCATACATACCGAG-3′ |
| CaMRR2SeqC | 5′-TACTCGCTCGCCTTACATCGA-3′ |
| CaMRR2SeqD | 5′-AATCTCAACTACATCCACCTTGTC-3′ |
| CaMRR2SeqE | 5′-CGAAACTTCTGCCATCCTCAAT-3′ |
| CaMRR2SeqF | 5′-GTACATCGGACGACCGTTCC-3′ |
| CaMRR2SeqG | 5′-CTATACTTTGCTCCATTGGCGG-3′ |
| CaMRR2SeqH | 5′-GAACGATGTTAATGGGTCAGCAAAG-3′ |
| Short, overlapping extension | |
| CaMRR2_P1_F | 5′-CACTGTGATCGGTTATCTTTGTTGCAC-3′ |
| CaMRR2_C9_3R | 5′-GTTGCTTGGGGTTGTTTTCGCCAATG-3′ |
| CaMRR2_Nested_F_KpnI | 5′-AGCGGTACCTTGGACTTTGACTGTTCAGA-3′ |
| CaMRR2_Nested_DR_XhoI | 5′-CAACTCGAGGGGCGATGATTGTTAGTTGTATATT-3′ |
| pBSS2_1F.2 | 5′-CAAGGCGATTAAGTTGGGTAAC-3′ |
| pBSS2_MAL2_R | 5′-CGTGGTTTCAGTGGCTACAAC-3′ |
| CaMRR2_C9.2_F6 | 5′-GGGCCCACAGATCGACGGACATTAC-3′ |
| CaMRR2_C9.2_R6 | 5′-GTAATGTCCGTCGATCTGTGGGCCC-3′ |
| CaMRR2_C9.2_F7 | 5′-CATTAACATCGTTCTTTAGTCTCATCCC-3′ |
| CaMRR2_C9.2_R7 | 5′-GGGATGAGACTAAAGAACGATGTTAATG-3′ |
| CaMRR2_C9.2_F8 | 5′-CAGATTTACCAGTTGCCAAAAGAC-3′ |
| CaMRR2_C9.2_R8 | 5′-GTCTTTTGGCAACTGGTAAATCTG-3′ |
| CaMRR2_2F_EcoRI | 5′-GATGAATTCATGACCAAACGTGATCGTACAAT-3′ |
| CaMRR2_R_ADH1t.2 | 5′-GCTATTTGCTTACGAGATTTTGAGGAAATCCC-3′ |
| CaADH1t_F | 5′-CAAAATCTCGTAAGCAAATAGCTAAATTATATACG-3′ |
| CaADH1t_R_XhoI | 5′-CATCTCGAGTTAACCAAAATCAACGACAAATTG-3′ |
| Real-time qPCR | |
| CaMRR2_qPCR_F | 5′-TCCAAGTAAGTGTGGGTGTCC-3′ |
| CaMRR2_qPCR_R | 5′-ATGTAAGGCGAGCGAGTAGC-3′ |
| CaCDR1-f_qPCR | 5′-ATTCTAAGATGTCGTCGCAAGATG-3′ |
| CaCDR1-R_qPCR | 5′-AGTTCTGGCTAAATTCTGAATGTTTTC |
| CaACT1-FWD_qPCR | 5′-ACGGTGAAGAAGTTGCTGCTTTAGTT-3′ |
| CaACT1-rvs_qPCR | 5′-CGTCGTCACCGGCAAAA-3′ |
Underlined nucleotide indicates introduction of a restriction site sequence.
Candida albicans strain construction.
The SAT1/FLP-containing MRR2 replacement cassette was excised from plasmids pBSS2-MRR2WT, pBSS2-MRR220, pBSS2-MRR222, pBSS2-MRR223, and pBSS2-MRR228, and plasmids pBSS2-MRR229, pMRR2_C9.3, and pMRR2GAD1 were digested using KpnI-HF and NcoI-HF restriction enzymes and transformed via electroporation into C. albicans strain SCZCF34M4A to generate strains heterozygous for their respective MRR2 alleles of interest. Figure 3 diagrams the general strain construction method for allelic replacement of MRR2 at the native locus (Fig. 3A) and inserted at the ADH1 locus (Fig. 3B). The nourseothricin marker in all strains was recycled by FLP recombinase induction after 48 h of growth in YPD liquid media. Repeat transformation of the resultant strains generated the homozygous MRR2 allele replacements MRR2WT (SCMRR2R1S1), MRR2S143P, L144V, T145A, S165N, A459T, S480P (SCMRR2R2S2), MRR2T83A, V451A, V582L (SCMRR2R3S3), MRR2R45Q, V451A (SCMRR2R4S4), MRR2T83A, A459T, S480P, V486M (SCMRR2R5S5), MRR2S466L, A468G, S469T, T470N, S480P (SCMRR2R6S6), MRR2C9 (SCMRR2R8S8), and MRR2-GAL4AD-3xHA (SCMRR2GAD1R1S1). MRR2 allelic replacements were confirmed by Southern blotting, and confirmation of polymorphisms or fused domains present in generated strains was accomplished through Sanger sequencing. For strains SCΔmrr2PADH1MRR2WT and SCΔmrr2PADH1MRR2C9, plasmids pPADH1-MRR2WT and pPADH1-MRR2C9 were similarly digested with KpnI-HF and NcoI-HF to excise and purify the MRR2-containing SAT1/FLP cassette targeting the ADH1 locus. Paired double-strand breaks near the 5′ and 3′ ends of the ADH1 gene were induced utilizing clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 technology and previously described methods (37). Briefly, 100 pmol Alt-R CRISPR-Cas9 trans-activating crRNA (tracrRNA) (Integrated DNA Technologies, Inc.) was duplexed with an equal amount of crRNA targeting either the 5′ or 3′ region of the ADH1 locus at 95˚C for 5 min. The duplexed guide RNA was then complexed with 2 μg of Alt-R S.p. HiFi Cas9 Nuclease V3 (Integrated DNA Technologies, Inc.) to form the resulting ribonucleoprotein complex. Approximately 1 μg of purified digests from either pPADH1-MRR2WT or pPADH1-MRR2C9 were transformed via electroporation along with Cas9 ribonucleoprotein complex into SCZCF34M4A to create SCΔmrr2PADH1MRR2WT and SCΔmrr2PADH1MRR2C9 expressing a single copy of the MRR2 open reading frame from the ADH1 locus.
FIG 3.
Representative schematic of MRR2 mutant strain construction in C. albicans. The diagram shows the MRR2/CaSAT1/FLP cassette integration at the native MRR2 gene locus (A) or replacing the ADH1 open reading frame at the ADH1 locus (B). The black line represents the sequence of a single allele of genomic DNA. Open reading frames are depicted as filled arrows along the genomic sequence, with gene names labeled. Promoter regions, terminators, and the FLP recognition target sequences are represented by bent arrows, hairpins, and black triangles, respectively. The double slashes interrupting the genomic DNA sequence as shown in panel B represent the CRISPR-Cas9 cutting sites. The homologous 5′ and 3′ flanking sequences for targeted fragment integration are represented as forward-striped and backward-striped rectangles, respectively.
Azole susceptibility testing.
Fluconazole MICs were determined using the broth microdilution methods described by the Clinical Laboratory and Standards Institute (34, 35), with slight modification by addition of 2% glucose to RPMI 1640 media to reduce trailing growth in wells. Fluconazole MICs ranged from 256 to 0.06 μg/ml. Measurements were read visually at 24 and 48 h after incubation at 35°C for a 50% reduction in growth from drug-free control wells. For fluconazole susceptibility testing using Etest strips (bioMérieux), cells were diluted to an optical density at 600 nm (OD600) of 0.100 and swabbed using sterile cotton tips onto RPMI agar plates. MICs were visually read at the border of the zone of inhibition after 24 and 48 h of incubation at 35°C.
Quantitative real-time reverse transcription-PCR.
First strand cDNAs were synthesized from 1 μg of total RNA using SuperScript VILO for quantitative real-time reverse transcription-PCR (qRT-PCR) (Invitrogen). Real-time PCR primers for ACT1, CDR1, and MRR2 were synthesized by Integrated DNA Technologies (Table 4). Relative levels of mRNA abundance of CDR1 and MRR2 against endogenous control gene ACT1 and reference strain SC5314 were measured in triplicate on a StepOnePlus instrument (Applied Biosystems). To assess statistical significance, standard errors of the means of the average expression levels of each isolate measured in triplicate were calculated.
Data accessibility.
The coding sequences of the MRR2 alleles described in this study have been deposited in GenBank under accession numbers MK332630 through MK332702.
Supplementary Material
ACKNOWLEDGMENTS
We thank Daniel Diekema from the University of Iowa for generously sending us the clinical isolates of C. albicans used in this study.
This work was supported by NIH grant R01 AI058145 to P.D.R.
Footnotes
Supplemental material for this article may be found at https://doi.org/10.1128/AAC.00078-19.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Data Availability Statement
The coding sequences of the MRR2 alleles described in this study have been deposited in GenBank under accession numbers MK332630 through MK332702.