Date of Award

5-2008

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Interdisciplinary Program

Research Advisor

P. David Rogers, Pharm.D., Ph.D.

Committee

George Hilliard, Ph.D. Ramin Homayouni, Ph.D. Richard Lee, Ph.D. Bernd Meibohm, Ph.D.

Keywords

azole resistance, CDR1, CDR2, TAC1, MDR1, MRR1, ERG11, UPC2, transcription factor

Abstract

Candida albicans is a pathogenic fungi found in the mucosa, gastrointestinal, and urogenital tracts of humans. Oropharyngeal candidiasis (OPC), an opportunistic mucosal infection caused by C. albicans, occurs most frequently in patients infected with human immunodeficiency virus (HIV). OPC is usually treated with azole antifungals, a class of antifungals that target ergosterol biosynthesis, at low doses over long periods of time. This course of treatment allows for the development of azole resistance.

Two major mechanisms of azole resistance exist in C. albicans, the up-regulation of genes encoding efflux pumps and the up-regulation of ERG11, a gene encoding the azole drug target lanosterol demethylase. The overexpression of efflux pumps remain the major contributor of azole resistance. The ATP binding cassette (ABC) transporter genes CDR1 and CDR2 have been shown to be regulated by a zinc cluster transcription factor Tac1p. A gain-of-function mutation in the gene encoding this transcription factor is responsible for the overexpression of CDR1 and CDR2. To identify the Tac1p regulon, we analyzed four matched sets of clinical isolates with gain-of-function mutations in TAC1 representing the development of CDR1- and CDR2-mediated azole resistance, using gene expression profiling. We identified genes that were consistently up-regulated coordinately with CDR1 and CDR2, including TAC1 itself. When a resistant strain disrupted for TAC1 was similarly examined, the expression of almost all of these genes returned to levels similar to those in the matched azole-susceptible isolate. Using chromatin Immunoprecipitation microarray (ChIP-chip) analysis, we found genes whose promoters were bound by Tac1p in vivo, including CDR1 and CDR2. Sequence analysis identified genes whose promoters contain the previously reported Tac1p drug-responsive element (DRE; CGGN4CGG), including TAC1. Our results show that Tac1p is constitutively bound to the promoter 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.

Constitutive overexpression of the MDR1 gene, which encodes an efflux pump of the major facilitator superfamily, is another cause of resistance to fluconazole in clinical C. albicans strains. The regulator of MDR1 gene expression has not been identified or characterized. Using genome-wide gene expression profiling, we have identified a gene encoding a zinc cluster transcription factor, designated as MRR1 (multidrug resistance regulator), that was coordinately up-regulated with MDR1 in drug-resistant clinical C. albicans isolates. The disruption of MRR1 in two resistant clinical isolates abrogated MDR1 expression and multidrug resistance. MRR1 alleles were also sequenced and two gain-of-function mutations were identified (P683S and G997V). The integration of the mutated alleles into the genome strain SC5314 resulted in the overexpression of MDR1 and in the increase of drug resistance. Microarray analysis of the resistant isolates, the disruption mutants and the reintegrated gain-of-function mutants allowed us to define a core group of genes regulated by Mrr1p. In addition to conferring multidrug resistance, Mrr1p also regulates genes that encode oxidoreductases that play a role in the response to oxidative stress.

The second major resistance mechanism is the constitutive up-regulation of ERG11. ERG11 encodes lanosterol demethylase, a cytochrome P-450 enzyme that is the target of the azole antifungals. In C. albicans, the zinc cluster transcription factor Upc2p has been shown to regulate the expression of ERG11 and other ergosterol biosynthesis genes. Overexpression of UPC2 increases azole resistance while disruption of UPC2 results in hypersusceptibility. Using genome-wide gene expression profiling, we found UPC2 and ergosterol biosynthesis genes to be coregulated with ERG11 in a fluconazole resistant clinical isolate. Sequence analysis of the susceptible and resistant UPC2 alleles revealed that the resistant isolate contained one UPC2 allele harboring a single nucleotide substitution, resulting in a G648D exchange. Introduction of the mutated UPC2 allele into the genome strain resulted in constitutive up-regulation of ERG11 and increased resistance to fluconazole. By comparing the gene expression profiles of the fluconazole resistant isolate and of strains carrying wild-type and mutated UPC2 alleles, we identified target genes that are controlled by Upc2p. We show for the first time that a gain-of-function mutation in UPC2 leads to increased expression of ERG11 and imparts resistance to fluconazole in clinical isolates of C. albicans.

By studying genetically matched sets of azole susceptible and resistant clinical isolates, we identified genes co-regulated with known resistance genes. These core genes specific to particular resistance mechanisms allowed us to identify previously characterized but unknown transcriptional regulators that control this process. Gain-of-function mutations in these trans-regulatory elements are responsible for the development of azole resistance in these clinical isolates. The target genes of each of these transcription factors provide insight into their function in addition to their role in azole resistance.

DOI

10.21007/etd.cghs.2008.0186

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