Genetic Analysis of Pleiotropic Drug Resistance

描述（由申请人提供）：S。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，因为两者都可以通过升高水平的ABC转运蛋白（S.将药物排出细胞外。致病性念珠菌属，白色念珠菌和光滑念珠菌，表达ScPdr5的同源物，可以获得相似的多药耐药表型。我们一直在研究S.酿酒酵母作为这种生物体提供了一个简单的实验背景，解剖真核多药耐药性。我们的长期目标是了解多药耐药的生理学基础。多药耐药性在抗真菌药物的应用中造成了一个特殊的问题，因为这些化合物的数量非常有限。了解酵母细胞控制多药耐药性的逻辑将允许使用策略来防止这种表型的出现。 S. cerevisiae和C. glabrata包含目前已知的真菌中最密切相关的多药耐药途径。ScPdr5是主要的ABC转运蛋白，而C. glabrata表达一种非常相似的蛋白质，称为CgCdr1。PDR S.酿酒酵母细胞表达高水平的ScPDR5转录本，这是由于相关的含锌簇的转录调节因子ScPdr1和ScPdr3的活性变化。多药耐药C. glabrata细胞通过增加S.glabrata的同源物CgPdr1的活性而过量产生CgCDR1 mRNA。酿酒酵母多药转录因子。先前的工作已经暗示转录介导复合物是ScPDR 5和CgCDR 1表达的关键决定因素。我们直接研究了C. glabrata的相似性，发现其与S.酿酒，差异也存在。我们建议调查选定的调解员组成部分，在C。glabrata，以确定哪些是重要的转录激活控制通过CgPdr1通过基因破坏。将进行染色质免疫沉淀，以确定目标介体组分对CgCDR 1转录直接作用的可能性。我们还构建了一个全功能的串联亲和纯化（TAP）标记的CgPDR1等位基因。这将用于从C中纯化CgPdr1。glabrata细胞与不同水平的CgCDR1转录，以确定和表征蛋白质参与控制这一关键的转录调节。最后，我们已经开始在S.酿酒酵母细胞，以鉴定参与ScPDR 5表达调控的所有非必需基因。使用这种技术，我们已经发现了一个新的调节输入从蛋白激酶A信号通路调节ScPDR5的表达。我们将使用这个屏幕来识别在药物存在和不存在的情况下ScPDR5表达的重要组成部分。利用S. cerevisiae告诉我们的重要参与者在C. glabrata将使我们能够迅速发现调节这种真菌病原体耐药性的调节网络。

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.