Genetic Analysis of Pleiotropic Drug Resistance

多效耐药性的遗传分析

基本信息

批准号：
8471707
负责人：
W Scott Moye-Rowley
金额：
$ 33.49万
依托单位：
UNIVERSITY OF IOWA
依托单位国家：
美国
项目类别：
财政年份：
1993
资助国家：
美国
起止时间：
1993-08-01 至 2016-02-29
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8471707
关键词：
ABCB1 gene ATP-Binding Cassette Transporters Address Affinity Chromatography Alleles Animal Model Antibiotics Antifungal Agents Candida Candida albicans Candida glabrata Carrier Proteins Cell membrane Cells Communicable Diseases Complex Cyclic AMP-Dependent Protein Kinases DNA Binding Domain Development Drug Efflux Drug Resistance, Multiple, Fungal Drug resistance Eukaryotic Cell Fungal Drug Resistance Gene Expression Gene Targeting Genes Genetic Genetic Screening Genetic Transcription Goals Homologous Gene Infection Laboratories Logic Mammalian Cell Mediator of activation protein Membrane Transport Proteins Messenger RNA Modeling Modification Molecular Multi-Drug Resistance Multidrug Resistance Gene Mutation Organism Outcome Participant Pathway interactions Pharmaceutical Preparations Phenotype Physiology Proteins Regulation Resistance Saccharomyces cerevisiae Sepsis Signal Pathway Signal Transduction Source Specificity Structure Techniques Time Transcript Transcription Coactivator Transcription factor genes Transcriptional Activation Work Yeasts Zinc Cluster base cancer therapy candidemia chromatin immunoprecipitation drug development efflux pump fungus gain of function mutation genetic analysis interest mutant neoplastic cell pathogen prevent research study transcription factor

项目摘要

DESCRIPTION (provided by applicant): S. cerevisiae pleiotropic drug resistance (PDR) is directly analogous to multidrug resistance in mammalian cells as both can be triggered by elevated levels of ABC transporter proteins (ScPdr5 in S. cerevisiae) that efflux drugs out of the cell. The pathogenic Candida species, Candida albicans and Candida glabrata, express homologues of ScPdr5 and can acquire similar multidrug resistant phenotypes. We have been studying PDR in S. cerevisiae as this organism provides a facile experimental background with which to dissect eukaryotic multidrug resistance. Our long term goal is to understand the physiology underlying development of multidrug resistance. Multidrug resistance poses a special problem in deployment of antifungal drugs as these compounds are quite limited in number. Understanding the logic used by a yeast cell to control the multidrug resistance will allow use of strategies to prevent this phenotype from arising. S. cerevisiae and C. glabrata contain the most closely related multidrug resistance pathways currently known in fungi. ScPdr5 is the major ABC transporter protein while C. glabrata expresses a very similar protein called CgCdr1. PDR S. cerevisiae cells express high levels of ScPDR5 transcript owing to changes in activity of the related zinc cluster-containing transcriptional regulators ScPdr1 and ScPdr3. Multidrug resistant C. glabrata cells overproduce CgCDR1 mRNA via increased activity of CgPdr1, a homologue of the S. cerevisiae multidrug transcription factors. Previous work has implicated the transcription Mediator complex as being a key determinant of expression of ScPDR5 and CgCDR1. We have directly studied Mediator mutants in C. glabrata and found that while there are similarities with S. cerevisiae, differences also exist. We propose to investigate selected Mediator components in C. glabrata to determine which are important in transcriptional activation controlled via CgPdr1 via gene disruption. Chromatin immunoprecipitation will be carried out to address the likelihood of direct action of Mediator components of interest on CgCDR1 transcription. We have also constructed a fully-functional tandem affinity purification (TAP)-tagged allele of CgPDR1. This will be used to purify CgPdr1 from C. glabrata cells with different levels of CgCDR1 transcription in order to identify and characterize proteins involved in control of this key transcriptional regulator. Finally, we have begun to use a high throughput genetic screen in S. cerevisiae cells to identify all non-essential genes involved in regulation of ScPDR5 expression. Using this technique, we have found a new regulatory input from the protein kinase A signaling pathway modulating ScPDR5 expression. We will use this screen to identify components important in ScPDR5 expression in the presence and absence of drugs. Using the experimental facility of S. cerevisiae to inform us of important participants in C. glabrata will allow us to rapidly uncover regulatory networks modulating drug resistance in this fungal pathogen.

描述（由申请人提供）：酿酒酵母多效性耐药性（PDR）直接类似于哺乳动物细胞中的多药耐药性，因为这两者都可以由ABC转运蛋白水平升高（SCPDR5）（在酿酒酵母中的SCPDR5）触发，从而使细胞中排出药物。致病性念珠菌物种，白色念珠菌和念珠菌的Glabrata表达SCPDR5的同源物，可以获取类似的多药耐药表型。我们一直在研究酿酒酵母中的PDR，因为该生物提供了一种简便的实验背景，可以通过该背景来剖析真核多药耐药性。我们的长期目标是了解多药耐药性的生理发展。多药抗性在部署抗真菌药物方面构成了一个特殊的问题，因为这些化合物的数量非常有限。了解酵母细胞用于控制多药电阻的逻辑将允许使用策略来防止这种表型产生。 S. cerevisiae和C. glabrata包含当前在真菌中已知的最紧密相关的多药剂阻力途径。 SCPDR5是主要的ABC转运蛋白，而C. glabrata表示非常相似的蛋白质，称为CGCDR1。 PDR S.酿酒酵母细胞由于相关含锌簇的转录调节剂SCPDR1和SCPDR3的活性而表达了高水平的SCPDR5转录本。多药抗性梭状芽胞杆菌细胞通过CGPDR1的活性增加了CGCDR1 mRNA，CGPDR1的活性是酿酒酵母多果转录因子的同源物。先前的工作暗示转录介质复合物是SCPDR5和CGCDR1表达的关键决定因素。我们已经直接研究了Glabrata中的介体突变体，发现尽管与酿酒酵母有相似之处，但也存在差异。我们建议研究glabrata中选定的介体成分，以确定哪些在通过基因破坏通过CGPDR1控制的转录激活中很重要。将进行染色质免疫沉淀，以解决感兴趣的介体成分在CGCDR1转录上的直接作用的可能性。我们还构建了一个完整的串联亲和力纯化（TAP）标记的CGPDR1等位基因。这将用于纯化具有不同水平CGCDR1转录的Glabrata细胞中的CGPDR1，以识别和表征与该关键转录调节剂控制有关的蛋白质。最后，我们已经开始使用酿酒酵母细胞中的高吞吐量遗传筛选来鉴定参与SCPDR5表达调节的所有非必需基因。使用此技术，我们发现了来自蛋白激酶A信号通路调节SCPDR5表达的新调节输入。我们将使用此屏幕来识别在存在和不存在药物的情况下对SCPDR5表达重要的组件。使用S. cerevisiae的实验设施，告知我们glabrata中的重要参与者将使我们能够迅速发现该真菌病原体中耐药性的监管网络。