Genetic analysis of pleiotropic drug resistance

多效性耐药的遗传分析

• 批准号: 7942226
• 负责人: W Scott Moye-Rowley
• 金额: $ 3.13万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7942226
• 关键词: 11p; ATP-Binding Cassette Transporters; Adult; Affinity Chromatography; Antifungal Agents; Azole resistance; Azoles; Binding; Biochemical; Candida; Candida albicans; Candida glabrata; Carrier Proteins; Cell membrane; Cells; Chemical Structure; Childhood; Clinical; Collaborations; Complex; Disease; Drops; Drug Efflux; Drug Resistance, Multiple, Fungal; Drug resistance; Elements; Employee Strikes; Environment; Enzymes; Eukaryota; Exhibits; Frequencies; Fungi Model; Future; Gene Targeting; Genes; Genetic; Genetic Transcription; Goals; Homologous Gene; Hospitals; Incidence; Infection; Link; Maps; Mediator of activation protein; Mitochondria; Modeling; Molecular; Molecular Probes; Multi-Drug Resistance; Multidrug Resistance Gene; Mutation; Nosocomial Infections; Nuclear; Organism; Pathway interactions; Patients; Pharmaceutical Preparations; Pharmacotherapy; Phenotype; Phosphatidylethanolamine; Phospholipids; Play; Production; Protein Binding; Proteins; RNA Polymerase II; Regulation; Regulator Genes; Resistance; Response Elements; Role; Saccharomyces cerevisiae; Sepsis; Sequence-Specific DNA Binding Protein; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Specificity; Structure; System; Time; Toxic effect; Transcription Initiation; Transcriptional Activation; United States; Work; Yeasts; Zinc Cluster; base; candidemia; chemosensitizing agent; chemotherapy; efflux pump; fungus; genetic analysis; genetic regulatory protein; microbial; mitochondrial genome; mortality; mutant; phosphatidylethanolamine; prevent; promoter; protein complex; prototype; public health relevance; research study; resistance mechanism; transcription factor

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.

描述（由申请人提供）：多药抵抗是使用各种化学疗法的重要临床障碍。我们将酿酒酵母的酵母菌用作模型真核系统，该系统具有一组被描述的多药耐药性基因座，称为多效性耐药性基因（PDR）。致病性酵母白色念珠菌和念珠菌的念珠菌表现出对参与酿酒酵母多种耐药性的调节剂和靶基因的惊人保守性。 PDR3P是一个含有转录因子的锌簇，它感应了线粒体基因组的丧失，并诱导了像PDR5这样的多药抗性基因的表达。 PDR5编码ATP结合的盒式转运蛋白，该蛋白用作广泛的特异性药物外排泵。我们最近发现，参与磷脂酰乙醇胺产生的线粒体酶PSD1P的过量生产也升高了PDR5的表达。 PSD1P信号通路靶向转录介质组件GAL11P。我们将构建缺乏GAL11P的致病念珠菌物种的突变菌株，以确定该共激活因子在这些引起疾病的真菌中的重要性是否保守。我们还将过量生产这些生物体中的念珠菌PSD1P同源物，以评估这种新信号通路的保护程度。遗传分析将用于鉴定将线粒体PSD1P与核PDR5连接的PSD1P信号传导途径的组件。我们的初步实验已将SSA1P HSP70蛋白确定为PDR3P的负调节剂。 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的区域，并确定已知的HSP70调节蛋白如何影响PDR3P的SSA1P对照。将进行PDR3P的生化纯化，以识别该因素的调节因子，并将进行遗传分析，以识别在信号转导途径中作用于将PSD1P与PDR5转录联系起来的组件。这项工作是针对理解较低真核生物中多种耐药性的分子基础的。抗真菌药物的范围相对有限，多种耐药性基因可以赋予许多不同化合物的耐受性，并且只有单个遗传变化。了解调节真菌中多药耐药性的基因网络是能够降低致病真菌逃避抗真菌药物疗法的能力的重要一步，这是医院环境中患者重要性增加的问题。在美国的公共卫生相关性，真菌是致命性血液感染的第四最常见原因，这种情况因有限数量的抗真菌药物而复杂化。该应用使用酿酒酵母的真菌模型来探测真菌耐药性的分子基础。