A gain-of-function mutation in the transcription factor Upc2p causes upregulation of ergosterol biosynthesis genes and increased fluconazole resistance in a clinical Candida albicans isolate

Abstract

In the pathogenic yeast Candida albicans, the zinc cluster transcription factor Upc2p has been shown to regulate the expression of ERG11 and other genes involved in ergosterol biosynthesis upon exposure to azole antifungals. ERG11 encodes lanosterol demethylase, the target enzyme of this antifungal class. Overexpression of UPC2 reduces azole susceptibility, whereas its disruption results in hypersusceptibility to azoles and reduced accumulation of exogenous sterols. Overexpression of ERG11 leads to the increased production of lanosterol demethylase, which contributes to azole resistance in clinical isolates of C. albicans, but the mechanism for this has yet to be determined. Using genome-wide gene expression profiling, we found UPC2 and other genes involved in ergosterol biosynthesis to be coordinately upregulated with ERG11 in a fluconazole-resistant clinical isolate compared with a matched susceptible isolate from the same patient. Sequence analysis of the UPC2 alleles of these isolates revealed that the resistant isolate contained a single-nucleotide substitution in one UPC2 allele that resulted in a G648D exchange in the encoded protein. Introduction of the mutated allele into a drug-susceptible strain resulted in constitutive upregulation of ERG11 and increased resistance to fluconazole. By comparing the gene expression profiles of the fluconazole-resistant isolate and of strains carrying wild-type and mutated UPC2 alleles, we identified target genes that are controlled by Upc2p. Here we show for the first time that a gain-of-function mutation in UPC2 leads to the increased expression of ERG11 and imparts resistance to fluconazole in clinical isolates of C. albicans.

FIG. 1.

Construction of upc2Δ mutants and strains expressing the UPC2^S1-1 and UPC2^S2-1 alleles. (A) Structure of the deletion cassette from plasmid pUPC2M2 (top), which was used to delete the UPC2 ORF in strain SC5314, and genomic structure of the UPC2 locus in the parental strain (bottom). The UPC2 coding region is represented by the white arrow and the upstream (UPC2_up) and downstream (UPC2_down) regions by the solid lines. The SAT1 flipper cassette (SAT1-FLIP) is represented by the gray rectangle bordered by FRT sites (black arrows). The 34-bp FRT sites are not drawn to scale. The probes used for Southern hybridization analysis of the mutants are indicated by the black bars. (B) Structure of the DNA fragments from plasmids pUPC2K2 and pUPC2K3 (top), which were used for integration of the UPC2^S1-1 and UPC2^S2-1 alleles, respectively, into the remaining wild-type UPC2 locus (not shown) or the disrupted upc2Δ locus of the heterozygous upc2 mutants (bottom) using the caSAT1 selection marker (gray arrow). T_ACT1, transcription termination sequence of the ACT1 gene. A, ApaI; Bg, BglII; N, NdeI; P, PstI; S, SpeI; ScI, SacI; ScII, SacII; X, XhoI. Only relevant restriction sites are given for panels A and B. The SpeI site shown in parentheses is present only on the chromosome containing the UPC2-1 allele in strain SC5314. (C) Southern hybridization of SpeI-digested genomic DNA of the wild-type strain SC5314 (lane 1), heterozygous (lanes 2 and 3) and homozygous (lanes 4 and 5) upc2Δ mutants, and strains with reinserted UPC2 alleles (lanes 6 to 13) with the UPC2-specific probe 1. The sizes (in kb) of the 1-kb ladder (lane M), which was also labeled, are shown on the left side of the blot. The positions of the original wild-type UPC2 alleles and the inactivated upc2Δ alleles are shown on the right side of the blot.

FIG. 2.

Quantitative real-time RT-PCR for genes of interest. Shown are the relative n-fold changes in gene expression in the clinical isolates (S2 versus S1) and in two pairs of transformants expressing the UPC2^S2-1 or UPC2^S1-1 alleles in the SC5314 background (UPC2M2K31A versus UPC2M2K21A and UPC2M2K31B versus UPC2M2K21B). Error bars represent the standard error of the mean. Asterisks denote statistical significance by Student's t test (P ≤ 0.05). vs., versus.

FIG. 3.

Susceptibilities to fluconazole and terbinafine of the wild-type strain SC5314 (UPC2/UPC2), heterozygous (UPC2/upc2Δ) and homozygous (upc2Δ/upc2Δ) upc2Δ mutants, and strains expressing the mutated UPC2^S2-1 allele or the nonmutated UPC2^S1-1 allele in the absence (upc2Δ/UPC2^S2-1 and upc2Δ/UPC2^S1-1) or presence (UPC2/UPC2^S2-1 and UPC2/UPC2^S1-1) of a wild-type UPC2 allele. Serial 10-fold dilutions of YPD overnight cultures of the strains were spotted on YPD plates without or with 2 μg ml⁻¹ fluconazole or 2 μg ml⁻¹ terbinafine and incubated for 2 days at 30°C.