Genetic analysis of pleiotropic drug resistance

疟疾是第四种最常见的血液感染类型， 常用的抗真菌药物可用，治疗方案受到耐药性的威胁。 与光滑念珠菌相关的感染代表第二种最常见的念珠菌血症， 自21世纪初以来一直在增加。C. glabrata表现出对唑类药物产生耐药性的强大能力 药物，最常用的抗真菌化合物类。唑类耐药分离株为 通常发现PDR 1基因中含有功能获得性（GOF）突变，编码转录 这是耐药性的核心调节因子。这些GOF形式的Pdr 1驱动组成型高 靶基因的转录水平。其中的核心是ATP结合盒转运蛋白- 编码CDR 1基因座。cdr 1是一种药物外排泵，可防止体内毒性唑类药物水平的积累。 牢房唑类药物的靶点是由ERG 11编码的羊毛甾醇β-14脱甲基酶 基因通过Upc 2A的作用，唑类药物激发诱导ERG 11转录 转录因子由Pdr 1和Upc 2A定义的转录调节回路先前已经被证实是一种转录调节回路， 被视为唑类耐药的单独途径。我们最近发现Upc 2A控制着 PDR 1和CDR 1的转录，表明这些之间存在生理联系。 监管网络。在本提案中，我们计划剖析Pdr 1激活 基因表达，并在靶基因DNA水平上剖析Pdr 1和Upc 2A之间的相互作用。 结合和转录。在目标1中，我们将确定辅活化剂的功能贡献 我们通过质谱法发现的与Pdr 1共纯化的蛋白质。表位标记与基因 破坏等位基因将允许确定这些蛋白质如何相互作用和调节的能力， Pdr 1调控基因表达。目的2将鉴定与Pdr 1 C-末端相互作用的蛋白质靶标 转录激活结构域，负责募集转录介体 复合物和诱导基因表达。我们还将使用交联方法来鉴定蛋白质 与该结构域以及Pdr 1内的潜在负调控结构域相互作用。最后我们 将使用染色质免疫沉淀结合高通量测序， RNA测序以确定Pdr 1和Upc 2A对共调节基因的相互影响。我们将 也破坏由这两个因子调控的其他转录因子基因，以鉴定更大的一组转录因子。 参与唑类药物攻击的转录应答的基因。我们发现的 Pdr 1和Upc 2A之间的关系表明，这些因素协同作用，作为 他们的正常功能。了解这种合作的分子基础将有助于深入了解 利用新的弱点来削弱耐药性。

Candidemias represent the 4th most common type of bloodstream infection and with only three commonly used antifungal drugs available, treatment options are threatened by drug resistance. Infections linked to Candida glabrata represent the second most common type of candidemia and have been increasing since the early 2000s. C. glabrata exhibits a robust ability to acquire resistance to azole drugs, the most commonly used class of antifungal compounds. Isolates that are azole resistant are routinely found to contain gain-of-function (GOF) mutations in the PDR1 gene, encoding a transcription factor that is a central regulator of drug resistance. These GOF forms of Pdr1 drive constitutively high levels of transcription of target genes. Central among these is the ATP-binding cassette transporter- encoding CDR1 locus. Cdr1 is a drug efflux pump that prevents the accumulation of toxic azole levels in the cell. The target of azole drugs is the lanosterol -14 demethylase enzyme encoded by the ERG11 gene. ERG11 transcription is induced by azole drug challenge through the action of the Upc2A transcription factor. The transcriptional regulatory circuits defined by Pdr1 and Upc2A have previously been treated as separate pathways to azole resistance. We recently discovered that Upc2A controls transcription of both PDR1 and CDR1, indicating the presence of a physiological tie between these regulatory networks. In this proposal, we plan on dissecting the mechanism(s) used by Pdr1 to activate gene expression and dissect the interaction between Pdr1 and Upc2A at the levels of target gene DNA- binding and transcription. In aim 1, we will determine the functional contribution made by coactivator proteins that we have found by mass spectrometry to co-purify with Pdr1. Epitope-tagging and gene disruption alleles will allow determination of how these proteins interact with and modulate the ability of Pdr1 to regulate gene expression. Aim 2 will identify protein targets interacting with the Pdr1 C-terminal transcriptional activation domain that are responsible for recruitment of the transcriptional Mediator complex and induction of gene expression. We will also use cross-linking approaches to identify proteins that interact with this domain as well as potential negative regulatory domains within Pdr1. Finally, we will use a combination of chromatin immunoprecipitation coupled with high-throughput sequencing and RNA-sequencing to determine the mutual impact Pdr1 and Upc2A have on co-regulated genes. We will also disrupt other transcription factor genes regulated by these two factors to identify the larger suite of genes involved in the transcriptional response to azole drug challenge. Our finding of the connections between Pdr1 and Upc2A indicates that these factors cooperate to confer azole resistance as part of their normal function. Understanding of the molecular basis of this cooperation will provide insight into new vulnerabilities to use to attenuate drug resistance.