Gain-of-function mutations in UPC2 are a frequent cause of ERG11 upregulation in azole-resistant clinical isolates of Candida albicans

Abstract

In Candida albicans, Upc2 is a zinc-cluster transcription factor that targets genes, including those of the ergosterol biosynthesis pathway. To date, three documented UPC2 gain-of-function (GOF) mutations have been recovered from fluconazole-resistant clinical isolates that contribute to an increase in ERG11 expression and decreased fluconazole susceptibility. In a group of 63 isolates with reduced susceptibility to fluconazole, we found that 47 overexpressed ERG11 by at least 2-fold over the average expression levels in 3 unrelated fluconazole-susceptible strains. Of those 47 isolates, 29 contained a mutation in UPC2, whereas the remaining 18 isolates did not. Among the isolates containing mutations in UPC2, we recovered eight distinct mutations resulting in putative single amino acid substitutions: G648D, G648S, A643T, A643V, Y642F, G304R, A646V, and W478C. Seven of these resulted in increased ERG11 expression, increased cellular ergosterol, and decreased susceptibility to fluconazole compared to the results for the wild-type strain. Genome-wide transcriptional analysis was performed for the four strongest Upc2 amino acid substitutions (A643V, G648D, G648S, and Y642F). Genes commonly upregulated by all four mutations included those involved in ergosterol biosynthesis, in oxidoreductase activity, the major facilitator efflux pump encoded by the MDR1 gene, and the uncharacterized ATP binding cassette transporter CDR11. These findings demonstrate that gain-of-function mutations in UPC2 are more prevalent among clinical isolates than previously thought and make a significant contribution to azole antifungal resistance, but the findings do not account for ERG11 overexpression in all such isolates of C. albicans.

Fig 1

Expression levels of CDR1, CDR2, MDR1, and ERG11 in 63 fluconazole-resistant clinical isolates were measured by qRT-PCR. All gene expression levels were measured in triplicate, and fold expression of genes in resistant isolates was compared to the average of the expression levels in three susceptible isolates. Results for 63 isolates with reduced susceptibility to fluconazole are represented, but only even-numbered isolates' results are labeled. Error bars show standard errors.

Fig 2

ERG11 transcription levels in strains carrying mutant UPC2 alleles are represented as averages from three independent qRT-PCR assays. Error bars show standard errors. ERG11 expression was quantified for each mutant strain listed and is compared to that of wild-type strain SC5314.

Fig 3

Ergosterol quantification in C. albicans laboratory strains expressing mutant UPC2 alleles. Increased ergosterol content was shown for strains expressing 2 UPC2 mutant alleles compared to the expression levels in heterozygote and wild-type strains. The heptane extraction layer from 16-h cultures was scanned spectrophotometrically between 240 and 300 nm. The presence of ergosterol in an extracted sample resulted in a 4-peak curve. A decrease in absorbance peaks correlates to a decrease in ergosterol content. Each panel represents the results for a different Upc2 GOF mutation, as follows: G648S (A), A643V (B), Y642F (C), G648D (D), A646V (E), A643T (F), W478C (G), and G304R (H). UPC2 mutant alleles were expressed as homozygotes with the same GOF mutation (○) or as heterozygotes with the wild-type allele (▲). Their absorbencies were compared to those of the wild-type strain SC5314 (●). The results for the homozygous revertants shown in panels C, D, and G showed statistically significant greater ergosterol contents than were found in the wild type (P < 0.05).