Genetic analysis of pleiotropic drug resistance

描述（由申请方提供）：多药耐药性是所有类型化疗药物使用的重要临床障碍。我们正在使用酵母酿酒酵母作为模型真核系统，具有一个良好的描述集的多药耐药基因座称为多效性耐药基因（PDR）。致病性酵母菌白色念珠菌和光滑念珠菌在S.酿酒酵母多药耐药性Pdr3p是一种含锌簇的转录因子，可感知线粒体基因组的丢失并诱导多药耐药基因（如PDR 5）的表达。PDR5编码一种ATP结合盒转运蛋白，作为一种广泛特异性的药物外排泵。我们最近发现，参与磷脂酰乙醇胺生产Psd1p的线粒体酶的过度生产也提高了PDR 5的表达。Psd1p信号通路靶向转录介质组分Gal11p。我们将构建缺乏Gal 11 p的致病性念珠菌属物种的突变株，以确定这种共激活剂的重要性是否在这些致病真菌中是保守的。我们还将在这些生物体中过量生产念珠菌Psd1p同系物，以评估这种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信号通路的组分。我们的初步实验已经确定Ssa1p Hsp70蛋白作为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。将进行Pdr3p的生化纯化以鉴定该因子的调节剂，并进行遗传分析以鉴定在将Psd1p与PDR5转录连接的信号转导途径中起作用的组分。这项工作是针对了解在低等真核生物的多重耐药的分子基础。抗真菌药物的范围是相对有限的，多个耐药基因可以赋予耐受许多不同的化合物，只有一个单一的遗传变化。了解调节真菌多药耐药性的基因网络是朝着能够降低致病真菌逃避抗真菌药物治疗的能力迈出的重要一步，这是医院环境中患者日益重要的问题。在美国，真菌是致命性血流感染的第四大常见原因，这种情况因抗真菌药物数量有限而变得复杂。本申请使用模式真菌酿酒酵母来探测真菌中耐药性的分子基础。

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.