Controlling Drug Resistance in Fungal Systems by Zinc Cluster Proteins

Abstract

Drug resistance is a serious complication for patients with an infection, and unfortunately, bacteria and fungi are quickly rendering many therapeutic drugs ineffective. In Saccharomyces cerevisiae, this mechanism is defined as pleiotropic drug-resistance (PDR). Overexpression of drug efflux pumps, which transport drugs out of the cell, is controlled by two major transcriptional regulators, Pdr1p and Pdr3p. Homologous PDR pathways have been identified in other pathogenic species, such as Canadida albicans. Pdr1p and Pdr3p are zinc cluster transcription factors that recognize a pair of CGG repeats in the promoter region of target PDR genes. Interestingly, these transcriptional regulators tolerate sequence variation within the binding site and recognize a small group of variant sites. Pdr1p and Pdr3p have been shown to selectively regulate PDR genes through canonical and variant binding sites. This is observed in the differential regulation of two PDR genes: PDR5, the prominent ABC transporter and HXT11, a hexose transporter. Pdr1p and Pdr3p recognize a variant binding site in the promoter region of PDR5; however, the transcriptional regulators do not recognize the same variant site in the promoter of HXT11. The regulatory mechanism for how canonical and variant binding sites lead to differential PDR gene expression has yet to be explored. These protein/DNA interactions have been studied using two approaches, binding affinity and specificity studies. Fluorescence anisotropy and surface plasmon resonance were used to determine the individual binding affinities of the DNA-binding domain (DBD) for canonical and variant binding sites, and x-ray crystallography was utilized to resolve the molecular interactions between the DBD complex with canonical and variant binding sites. Differences in the binding motifs and in the DBDs of Pdr1p and Pdr3p must allow for differential regulation, making this a unique system for investigation and a possible drug target.