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的重要临床障碍。我们使用酵母酿酒酵母作为真核生物系统的模型，该系统具有一组描述良好的多药耐药位点，称为多致耐药基因（PDR）。病原菌白色念珠菌和光念珠菌在酿酒酵母多药耐药过程中表现出显著的调控基因和靶基因的保守性。Pdr3p是一种含锌簇的转录因子，它能感知线粒体基因组的缺失，并诱导PDR5等多药耐药基因的表达。PDR5编码atp结合盒转运蛋白，作为广泛特异性药物外排泵。我们最近发现，参与磷脂酰乙醇胺生成的线粒体酶Psd1p的过量产生也会升高PDR5的表达。Psd1p信号通路靶向转录中介成分Gal11p。我们将构建缺乏Gal11p的致病性念珠菌的突变菌株，以确定这种共激活剂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中过量产生假丝酵母Psd1p同源物，以评估这一新的信号通路的保护程度。遗传分析将用于鉴定连接线粒体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的负调节因子。Ssa1p与Pdr3p结合，在Pdr3p活性升高的状态下，这种结合降低。我们将绘制Ssa1p控制所需的Pdr3p区域，并确定已知的Hsp70调节蛋白如何影响Ssa1p对Pdr3p的控制。将对Pdr3p进行生化纯化以确定该因子的调节因子，并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，多个耐药基因可以仅通过单个基因改变就赋予对许多不同化合物的耐受性。了解真菌中调控多药耐药的基因网络是朝着能够降低病原真菌逃避抗真菌药物治疗的能力迈出的重要一步，这是一个在医院环境中对患者越来越重要的问题。在美国，真菌是致死性血流感染的第四大常见原因，由于抗真菌药物数量有限，这种情况变得更加复杂。本应用程序以酿酒酵母菌为模型真菌，探索真菌耐药的分子基础。

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.