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. Candida albicans is an opportunistic fungal pathogen that causes mucosal, cutaneous, and systemic infections, including oropharyngeal candidiasis (OPC), the most frequent infection in people with AIDS (9, 13). In the United States, Candida is the fourth-most-common organism isolated from nosocomial bloodstream infections and is associated with a mortality rate approaching 40% (24). Fluconazole and other azole antifungal agents have proven effective in the management of OPC; however, with increased use of these agents, treatment failures have occurred that have been associated with the emergence of azole-resistant strains of C. albicans (25a). The azole class of antifungals work by inhibiting the cytochrome P450 enzyme lanosterol demethylase, a critical enzyme in the synthesis of ergosterol which is encoded by the ERG11 gene (14). The efficacy of fluconazole is decreased in clinical isolates of C. albicans by the interplay of several mechanisms of resistance (17,21,23,32). Overexpression of the efflux transporter genes CDR1, CDR2, and MDR1 is a common mechanism of drug resistance in this organism (10,17,26). Point mutations in the ERG11 gene result in reduced binding affinity of azoles to their target without precluding enzymatic function (31). In addition to point mutations, overexpression of ERG11 has also...

Structural mechanism of ergosterol regulation by fungal sterol transcription factor Upc2

“…The majority of the constitutive activation mutations in S. cerevisiae Upc2 and C. albicans Upc2 are clustered in the C-terminal loop between helix a11 and the predicted a12 helix 21,29 . The crystal structure does not include this C-terminal activation loop of 35 residues.…”

Mechanisms of Antifungal Drug Resistance

“…Molecular epidemiology of azole resistance in a large population of C. albicans suggests that CDR1/CDR2 up-regulation is more prevalent than ERG11 or MDR1 up-regulation, with combinations of CDR1/CDR2 up-regulation and ERG11 alterations as the most common (Goldman et al 2004;Park and Perlin 2005;Coste et al 2007;Siikala et al 2010;Flowers et al 2012). Although CDR1/CDR2 and MDR1 up-regulation can be explained by TAC1 and MRR1 GOF mutations, ERG11 up-regulation is not always associated with UPC2 GOF mutations, implicating additional regulators (Flowers et al 2012). The contribution of individual mutations to resistance of a clinical isolate was recently dissected by sequential replacement of mutated alleles (TAC1/ERG11) with wild-type alleles, confirming the functional importance of each mutation (MacCallum et al 2010).…”

“…First, amplification of the ERG11 gene can occur by the formation of an isochromosome with two copies of the left arm of chromosome 5 (i(5L)), in which ERG11 resides, or by duplication of the entire chromosome . Second, activating mutations in the gene encoding the transcription factor Upc2 up-regulate most ergosterol biosynthesis genes (Silver et al 2004;MacPherson et al 2005;Flowers et al 2012); disruption of C. albicans UPC2 causes hypersusceptibility to azoles.…”

Competitive Fitness of Fluconazole-Resistant Clinical Candida albicans Strains

“…Similarly, GOF mutations in Tac1 confer fluconazole resistance by mediating overexpression of the multidrug efflux pumps CDR1 and CDR2 and other Tac1 target genes (12)(13)(14)(15)(16)(17). Finally, GOF mutations in Upc2 result in upregulation of ERG11 and other ergosterol biosynthesis genes (18)(19)(20)(21). Many fluconazole-resistant clinical C. albicans strains exhibit several of these resistance mechanisms, which results in high levels of drug resistance and therapy failure (22)(23)(24)(25).…”