Genetic analysis of pleiotropic drug resistance

描述(由申请人提供)：多药耐药是所有类型化疗使用中的一个重要临床障碍。我们正在使用酿酒酵母作为一个真核模型系统，它有一组被很好地描述的多药耐药基因，称为多效性耐药基因(PDR)。致病酵母菌白色念珠菌和光滑念珠菌显示出与酿酒酵母多药耐药相关的调控基因和靶基因的显著保守性。Pdr3p是一个含有转录因子的锌簇，它可以感知线粒体基因组的丢失，并诱导像PDR5这样的多药耐药基因的表达。PDR5编码一种ATP结合盒转运蛋白，作为一种广泛的特异性药物外排泵。我们最近发现，参与磷脂酰乙醇胺生产的线粒体酶Psd1p的过度生产也会提高PDR5的表达。Psd1p信号通路以转录调节因子Gal11p为靶标。我们将构建缺乏Gal11p的致病假丝酵母菌突变株，以确定这种共激活因子的重要性是否在这些致病真菌中保守。我们还将在这些生物体中过量生产假丝酵母菌Psd1p同源物，以评估这一新信号通路的保守程度。遗传分析将用于确定连接线粒体Psd1p和核PDR5的Psd1p信号通路的组成部分。我们的初步实验已经确定Ssa1p Hsp70蛋白是Pdr3p的负调控蛋白。Ssa1p与Pdr3p结合，这种结合在Pdr3p活性升高的状态下降低。我们将定位Ssa1p调控所需的Pdr3p区域(S)，并确定已知的HSP70调控蛋白如何影响Pdr3p的Ssa1p调控。将对Pdr3p进行生化纯化，以确定该因子的调节因子，并将进行遗传分析，以确定在连接Psd1p和PDR5转录的信号转导途径中起作用的成分。这项工作旨在了解低等真核生物多重耐药的分子基础。抗真菌药物的范围相对有限，多个耐药基因只需一个基因变化就可以对许多不同的化合物产生耐受性。了解调节真菌多药耐药性的基因网络是降低病原真菌逃避抗真菌药物治疗的能力的重要一步，这是医院环境中患者日益重要的问题。在美国，真菌是导致致命血液感染的第四大常见原因，抗真菌药物的数量有限使情况变得更加复杂。本应用以模式真菌酿酒酵母为研究对象，探索真菌耐药性的分子基础。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE 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.