cis-Acting elements within the Candida albicans ERG11 promoter mediate the azole response through transcription factor Upc2p.

The azole antifungal drugs are used to treat infections caused by Candida albicans and other fungi. These drugs interfere with the biosynthesis of ergosterol, the major sterol in fungal cells, by inhibiting an ergosterol biosynthetic enzyme, lanosterol 14 alpha-demethylase, encoded by the ERG11 gene. In vitro, these drugs as well as other ergosterol biosynthesis inhibitors increase ERG11 mRNA expression by activation of the ERG11 promoter. The signal for this activation most likely is the depletion of ergosterol, the end product of the pathway. To identify cis-acting regulatory elements that mediate this activation, ERG11 promoter fragments have been fused to the luciferase reporter gene from Renilla reniformis. Promoter deletions and linker scan mutations localized the region important for azole induction to a segment from bp -224 to -251 upstream of the start codon, specifically two 7-bp sequences separated by 13 bp. These sequences form an imperfect inverted repeat. The region is recognized by the transcription factor Upc2p and functions as an enhancer of transcription, as it can be placed upstream of a heterologous promoter in either direction, resulting in the azole induction of that promoter. The promoter constructs are not azole inducible in the upc2/upc2 homozygous deletion, demonstrating that Upc2p controls the azole induction of ERG11. These results identify an azole-responsive enhancer element (ARE) in the ERG11 promoter that is controlled by the Upc2p transcription factor. No other ARE is present in the promoter. Thus, this ARE and Upc2p are necessary and sufficient for azole induction of ERG11.

The Candida albicans mating type like locus [MTL] is not involved in chlamydospore formation.

Candida albicans produces chlamydospores, which can be used as a diagnostic tool for species identification. It has been suggested that these chlamydospores are degenerate spores. If so, then their production might be linked to the mating loci, and clinical strains that are homozygous for the C. albicans mating locus MTL may be altered in chlamydospore formation, which could cause problems in diagnostics and species identification. In Saccharomyces cerevisiae diploid cells, the heterodimeric transcriptional repressor formed by the products of the mating genes MATa1 and MATalpha2 is an important regulator of sporulation. It was therefore of interest to determine if the disruptions of the MATa1 and MATalpha2 homologs in C. albicans, MTLa1 and MTLalpha2, result in inhibition of chlamydospore formation. Laboratory strains containing disruptions of either the entire MTL locus or specific genes within the locus were assayed for their ability to form chlamydospores. Clinical strains that are homozygous for one of the two MTL loci were also assayed. No change in chlamydospore formation was seen in these strains compared to the standard laboratory strain.

Drug-induced regulation of the MDR1 promoter in Candida albicans.

Resistance of Candida albicans to azole antifungal drugs is mediated by two types of efflux pumps, encoded by the MDR1 gene and the CDR gene family. MDR1 mRNA levels in a susceptible clinical isolate are induced by benomyl (BEN) but not by other drugs previously shown to induce MDR1. To monitor MDR1 expression under several conditions, the MDR1 promoter was fused to the Renilla reniformis luciferase reporter gene (RLUC). The promoter was monitored for its responses to four oxidizing agents, five toxic hydrophobic compounds, and an alkylating agent, all shown to induce major facilitator pumps in other organisms. Deletion constructs of the MDR1 promoter were used to analyze the basal transcription of the promoter and its responses to the toxic compound BEN and the oxidizing agent tert-butyl hydrogen peroxide (T-BHP). The cis-acting elements in the MDR1 promoter responsible for induction by BEN were localized between -399 and -299 upstream of the start codon. The cis-acting elements responsible for MDR1 induction by T-BHP were localized between -601 and -500 upstream of the start codon. The T-BHP induction region contains a sequence that resembles the YAP1-responsive element (YRE) in Saccharomyces cerevisiae. This Candida YRE was placed upstream of a noninducible promoter in the luciferase construct, resulting in an inducible promoter. Inversion or mutation of the 7-bp YRE eliminated induction. Many of the drugs used in this analysis induce the MDR1 promoter at concentrations that inhibit cell growth. These analyses define cis-acting elements responsible for drug induction of the MDR1 promoter.