An A643V Amino Acid Substitution in Upc2p Contributes to Azole Resistance in Well-Characterized Clinical Isolates of Candida albicans

Abstract

). In the present study, UPC2 was sequenced from a matched set of 17 isolates. An A643V substitution was present in all of the isolates in the series that overexpressed ERG11. Azole susceptibility, ergosterol levels, and expression of ERG genes were elevated in the A643V clinical isolates and in reconstructed strains.The ergosterol biosynthesis pathway in budding yeast is tightly regulated by a number of transcriptional activators and repressors (3,5,10,11). In Saccharomyces cerevisiae, the paralogous transcription factors ScUpc2p and ScEcm22p activate the expression of genes required for ergosterol biosynthesis (ERG genes) in response to sterol depletion during aerobic growth (6,17). An early study on ScUPC2 identified a dominant gain-of-function mutation in ScUPC2 that resulted in a glycine-to-aspartic acid substitution at position 888 (G888D) within the protein sequence (5). This mutation, termed UPC2-1, conferred constitutive activity on the transcription factor, resulting in increased aerobic expression of genes required for sterol uptake, as well as increased expression of ERG genes.In Candida albicans, gain-of-function mutations in several transcription factors, such as TAC1 and MRR1, in drug-resistant clinical isolates have been previously documented (4, 7). In fact, these trans-acting mutations have been central to regulation of many of the molecular mechanisms of antifungal resistance, such as CDR1 and MDR1 overexpression. Gain-offunction mutations in UPC2 that result in G648D or A643T substitutions in drug-resistant isolates of C. albicans were recently identified (8,9). In these studies, the UPC2 point mutations were found to confer a decrease in azole susceptibility and increase expression of Upc2p-regulated genes. The two mutations are in close proximity in the C-terminal portion of the protein, which may indicate that this is a hot spot for mutations or that this region of the protein is important for regulation of its functional activity.Previous studies of C. albicans azole-resistant clinical isolates revealed a role for overexpression of ERG11, which encodes the enzyme target of the azole drugs (18,19). In these strains, it is thought that increased expression of the Erg11p target enzyme contributes to a decrease in the susceptibility of the strain to azoles. Genome-wide expression profiling has expanded this view to include overexpression of other ERG genes in association with reduced azole susceptibility (15). Increased ERG gene expression in these strains may result from cis-acting mutations within the promoters of the ERG genes themselves or from alterations in trans-acting factors, such as Upc2p. Because some strains that overexpress ERG11 also show increased expression of other ERG genes, it is likely that alterations in trans-acting factors are responsible for the coordinated upregulation of these genes. Because Upc2p directly activates the transcription of many ERG genes, alterations of the Upc2p activity may be responsible for overexpression of these genes in azole-resistant clinical i...

UPC2 Is Universally Essential for Azole Antifungal Resistance in Candida albicans

“…UPC2 has been well characterized with regard to its impact on fluconazole susceptibility and its role in regulating genes of the ergosterol biosynthesis pathway (19,29,36,37 (19,36). We and others have established that in some azole-resistant isolates, specific mutations render UPC2 constitutively active, resulting in increases in the expression of ERG genes (including ERG11), cellular ergosterol levels, and fluconazole resistance (25,28,29,(38)(39)(40)(41). Moreover, UPC2 disruption results in a reduction in cellular ergosterol content (28).…”

Structural mechanism of ergosterol regulation by fungal sterol transcription factor Upc2

“…The gain-offunction mutation, G888D, in S. cerevisiae Upc2 results in the constitutive activation of the TF with a high level of aerobic sterol uptake 11,12 . These constitutive activation mutations are in close proximity in the C-terminal portion of Upc2, which may indicate that the CTD is important for the regulation of its transcriptional activity 9 . The CTDs of Upc2 and Ecm22 have no recognizable similarities to any characterized protein domains of known structures.…”

“…Many gain-offunction mutations in Upc2 that contribute to the drug resistance were identified in clinical isolates 8 . Mutations in the C-terminal domain (CTD) of Upc2 (A643V, A643T or G648D in C. albicans Upc2) result in the constitutive activation of the transcription factor (TF), resulting in the upregulation of ergosterol synthesis pathway and sterol uptake, which leads to a decreased susceptibility of the strains to azole drugs 9,10 . The gain-offunction mutation, G888D, in S. cerevisiae Upc2 results in the constitutive activation of the TF with a high level of aerobic sterol uptake 11,12 .…”

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).…”