Genetic analysis of pleiotropic drug resistance

描述（由申请人提供）：多药耐药性是使用所有类型化疗的重要临床障碍。我们使用酿酒酵母作为真核系统模型，该系统具有一组详细描述的多药耐药基因座，称为多效耐药基因 (PDR)。致病性酵母白色念珠菌和光滑念珠菌表现出与酿酒酵母多药耐药性相关的调节因子和靶基因的惊人保守性。 Pdr3p 是一种含有转录因子的锌簇，可感知线粒体基因组的丢失并诱导 PDR5 等多药耐药基因的表达。 PDR5 编码 ATP 结合盒转运蛋白，充当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 的过量产生也会提高 PDR5 的表达。 Psd1p 信号通路以转录介质成分 Gal11p 为目标。我们将构建缺乏 Gal11p 的致病性念珠菌突变株，以确定这种共激活剂的重要性在这些致病真菌中是否保守。我们还将在这些生物体中过量产生念珠菌 Psd1p 同源物，以评估这一新信号通路的保守程度。遗传分析将用于识别连接线粒体 Psd1p 与核 PDR5 的 Psd1p 信号通路的组成部分。我们的初步实验已确定 Ssa1p Hsp70 蛋白是 Pdr3p 的负调节蛋白。 Ssa1p 与 Pdr3p 结合，并且在 Pdr3p 活性升高的状态下，这种结合会降低。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。将进行 Pdr3p 的生化纯化，以鉴定该因子的调节因子，并进行遗传分析，以鉴定在连接 Psd1p 和 PDR5 转录的信号转导途径中发挥作用的成分。这项工作旨在了解低等真核生物多重耐药性的分子基础。抗真菌药物的范围相对有限，多个耐药基因只需一个基因改变即可赋予对许多不同化合物的耐受性。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能力的重要一步，这对于医院环境中的患者来说是一个日益重要的问题。公共卫生相关性 在美国，真菌是致命性血液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。该应用使用模型真菌酿酒酵母来探讨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.