Upc2p, a transcription factor of the zinc cluster family, is an important regulator of sterol biosynthesis and azole drug resistance in Candida albicans. To better understand Upc2p function in C. albicans, we used genomewide location profiling to identify the transcriptional targets of Upc2p in vivo. A triple hemagglutinin epitope, introduced at the C terminus of Upc2p, conferred a gain-of-function effect on the fusion protein.Location profiling identified 202 bound promoters (P < 0.05). Overrepresented functional groups of genes whose promoters were bound by Upc2p included 12 genes involved in ergosterol biosynthesis (NCP1, ERG11, ERG2, and others), 18 genes encoding ribosomal subunits (RPS30, RPL32, RPL12, and others), 3 genes encoding drug transporters (CDR1, MDR1, and YOR1), 4 genes encoding transcription factors (INO2, ACE2, SUT1, and UPC2), and 6 genes involved in sulfur amino acid metabolism (MET6, SAM2, SAH1, and others). Bioinformatic analyses suggested that Upc2p binds to the DNA motif 5-VNCGBDTR that includes the previously characterized Upc2p binding site 5-TCGTATA. Northern blot analysis showed that increased binding correlates with increased expression for the analyzed Upc2p targets (ERG11, MDR1, CDR1, YOR1, SUT1, SMF12, and CBP1). The analysis of ERG11, MDR1, and CDR1 transcripts in wild-type and upc2⌬/upc2⌬ strains grown under Upc2p-activating conditions (lovastatin treatment and hypoxia) showed that Upc2p regulates its targets in a complex manner, acting as an activator or as a repressor depending upon the target and the activating condition. Taken together, our results indicate that Upc2p is a key regulator of ergosterol metabolism. They also suggest that Upc2p may contribute to azole resistance by regulating the expression of drug efflux pump-encoding genes in addition to ergosterol biosynthesis genes.Candida albicans is an important human fungal pathogen, in terms of both its clinical significance and its use as an experimental model for scientific investigation. This opportunistic pathogen is a natural component of the human flora, colonizing skin and the gastrointestinal and genitourinary tracts (4). Although many infections involve the colonization of surface mucosal membranes (oral thrush and vaginal candidiasis, for example), immunosuppressed patients can be subject to potentially lethal systemic infections (45).Many antifungal drugs used to treat C. albicans infections function by targeting ergosterol, the analogue of cholesterol in mammalian cells and the major sterol of fungal cell membranes. Polyenes, such as amphotericin B, bind directly to ergosterol and perturb general membrane functions, resulting in low selectivity and high toxicity (2, 35). Azoles, including fluconazole and voriconazole, are more widely used and target the enzyme lanosterol demethylase (Erg11p) in the ergosterol biosynthesis pathway, with the consequence that ergosterol is depleted and replaced by unusual sterols, altering the fluidity of the membrane and the activities of membrane-bound proteins (e.g., enzymes...

A major mechanism of azole resistance in Candida albicans is overexpression of the genes encoding the ATP binding cassette transporters Cdr1p and Cdr2p due to gain-of-function mutations in Tac1p, a transcription factor of the zinc cluster family. To identify the Tac1p regulon, we analyzed four matched sets of clinical isolates representing the development of CDR1-and CDR2-mediated azole resistance by using gene expression profiling. We identified 31 genes that were consistently up-regulated with CDR1 and CDR2, including TAC1 itself, and 12 consistently down-regulated genes. When a resistant strain deleted for TAC1 was examined similarly, expression of almost all of these genes returned to levels similar to those in the matched azolesusceptible isolate. Using genome-wide location (ChIP-chip) analysis (a procedure combining chromatin immunoprecipitation with hybridization to DNA intergenic microarrays), we found 37 genes whose promoters were bound by Tac1p in vivo, including CDR1 and CDR2. Sequence analysis identified nine new genes whose promoters contain the previously reported Tac1p drug-responsive element (CGGN 4 CGG), including TAC1. In total, there were eight genes whose expression was modulated in the four azole-resistant clinical isolates in a TAC1-dependent manner and whose promoters were bound by Tac1p, qualifying them as direct Tac1p targets: CDR1, CDR2, GPX1 (putative glutathione peroxidase), LCB4 (putative sphingosine kinase), RTA3 (putative phospholipid flippase), and orf19.1887 (putative lipase), as well as IFU5 and orf19.4898 of unknown function. Our results show that Tac1p binds under nonactivating conditions to the promoters of its targets, including to its own promoter. They also suggest roles for Tac1p in regulating lipid metabolism (mobilization and trafficking) and oxidative stress response in C. albicans.Candida albicans causes mucosal, cutaneous, and systemic infections, including oropharyngeal candidiasis, the most frequent opportunistic infection among patients with AIDS (25, 40). Azole antifungal agents have proven effective in the management of oropharyngeal candidiasis; however, with increased use of these agents, treatment failures that have been associated with the emergence of azole-resistant strains of C. albicans have occurred (47,52,56,63,82).The azole antifungals target lanosterol demethylase (Erg11p), a key enzyme in the ergosterol biosynthesis pathway (38). Several mechanisms of resistance to the azole antifungal agents have been described for C. albicans, including increased expression of genes encoding multidrug efflux pumps (27,28,47,67,69,80,81). These include the gene encoding a transporter of the major facilitator superfamily (MDR1) and genes encoding two ATP binding cassette (ABC) transporters (CDR1 and CDR2) (27,28,47,69,80). Overexpression of these efflux pumps is presumed to prevent accumulation of effective concentrations of the azole antifungal agents within the fungal cell. Among studies examining multiple matched azole-susceptible and -resistant sets of isolates,...

SummaryMany Candida albicans azole-resistant (A R ) clinical isolates overexpress the CDR1 and CDR2 genes encoding homologous multidrug transporters of the ATP-binding cassette family. We show here that these strains also overexpress the PDR16 gene, the orthologue of Saccharomyces cerevisiae PDR16 encoding a phosphatidylinositol transfer protein of the Sec14p family. It has been reported that S. cerevisiae pdr16 D mutants are hypersusceptible to azoles, suggesting that C. albicans PDR16 may contribute to azole resistance in these isolates. To address this question, we deleted both alleles of PDR16 in an A R clinical strain overexpressing the three genes, using the mycophenolic acid resistance flipper strategy. Our results show that the homozygous pdr16 D / pdr16 D mutant is approximately twofold less resistant to azoles than the parental strain whereas reintroducing a copy of PDR16 in the mutant restored azole resistance, demonstrating that this gene contributes to the A R phenotype of the cells. In addition, overexpression of PDR16 in azole-susceptible (A S ) C. albicans and S. cerevisiae strains increased azole resistance by about twofold, indicating that an increased dosage of Pdr16p can confer low levels of azole resistance in the absence of additional molecular alterations. Taken together, these results demonstrate that PDR16 plays a role in C. albicans azole resistance.