Multidrug resistance is an important clinical impediment in the use of chemotherapies of all types. We are using the yeast Saccharomyces cerevisiae as a model eukaryotic system that has a well described set of multidrug resistance loci called pleiotropic drug resistance genes (PDR). The pathogenic yeasts Candida albicans and Candida glabrata exhibit striking conservation of the regulators and target genes involved in S. cerevisiae multidrug resistance. Pdr3p is a zinc cluster containing transcription factor that senses loss of the mitochondrial genome and induces expression of multidrug resistance genes like the PDR5. PDR5 encodes an ATP-binding cassette transporter protein that serves as a broad specificity drug efflux pump. We have recently found that overproduction of the mitochondrial enzyme involved in phosphatidylethanolamine production Psd1p also elevates PDR5 expression. The Psd1p signaling pathway targets the transcriptional mediator component Gal11p. We will construct mutant strains of pathogenic Candida species that lack Gal11p to determine if the importance of this co-activator is conserved in these disease-causing fungi. We will also overproduce Candida Psd1p homologues in these organisms to assess the degree of conservation of this new signaling pathway. Genetic analysis will be used to identify components of the Psd1p signaling pathway connecting the mitochondrial Psd1p with nuclear PDR5. Our preliminary experiments have identified the Ssa1p Hsp70 protein as a negative regulator of Pdr3p. Ssa1p binds to Pdr3p and this binding is lowered in states in which Pdr3p activity is elevated. We will map the region(s) of Pdr3p required for Ssa1p control and determine how known Hsp70 regulatory proteins influence Ssa1p control of Pdr3p. Biochemical purification of Pdr3p will be carried out to identify regulators of this factor and genetic analysis will be performed to identify components that act in the signal transduction pathway linking Psd1p to PDR5 transcription. This work is directed towards understanding the molecular basis of multiple drug resistance in lower eukaryotes. The range of antifungal drugs is relatively limited and multiple drug resistance genes can confer tolerance to many different compounds with only a single genetic change. Understanding the network of genes that regulate multidrug resistance in fungi is an important step towards being able to reduce the ability of pathogenic fungi to evade antifungal drug therapies, a problem of increasing importance in patients in the hospital setting.
In the United States, fungi are the 4th most common cause of fatal bloodstream infection, a situation complicated by the limited number of antifungal drugs. This application uses the model fungus Saccharomyces cerevisiae to probe the molecular basis of drug resistance in fungi. ? ? ?
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