Chemical genetic analysis of Candida glabrata CDR1 expression

与念珠菌相关的感染导致第四种最常见的血液感染。 目前只有三种有效的抗真菌药物，对抗真菌药物的持续生存造成了真正的威胁。 心理治疗。光滑念珠菌是与念珠菌有关的第二种最常见的分离物种， 对唑类药物的内在敏感度较低，并且很容易对大多数 常用的一类抗真菌化合物。对氮唑耐药的光滑念珠菌分离株几乎 完全由编码一种名为PDR1的转录因子的基因点突变引起。这些 点突变导致Pdr1转录的功能增强(GOF)形式的产生 调节因子，进而导致目标基因的高水平结构性表达。Pdr1的一个关键靶基因是 编码三磷酸腺苷结合盒转运体的CDR1基因被认为具有广泛的特异性 药物外排泵，防止唑类药物在携带这些GOF PDR1等位基因的细胞中积累。 这项应用的目标是使用化学遗传方法来识别能够 抑制GOF形式的Pdr1对CDR1的激活。我们将使用两个不同的化学库来筛选 一株含有CDR1-荧光素酶(CDR1-Luc)基因融合的光滑葡萄球菌菌株，以鉴定 能够在这种致病酵母中抑制获得唑类耐药性的这一中央调控步骤。我们 已经验证了我们使用384井容易地检测CDR1-Luc表达变化的能力 将在化学文库筛选中使用的格式化分析。AIM 1将使用CDR1- 荧光素酶报告菌株筛选两个不同的化学文库以识别 能够阻断在存在PDR1的GOF等位基因时看到的正常的结构性高表达。 荧光素酶的酶反应抑制物和聚集依赖的抑制物将被淘汰。 满足这些初步筛选标准的化合物将被测试其调节天然的能力 RT-qPCR法检测CDR1基因座。目标2将确定调节CDR1的化合物的作用 通过测试它们影响光滑念珠菌临床分离株对唑类耐药性的能力来表达 以及我们之前描述过的一组不同的PDR1GOF等位基因。的相互作用 具有一组转录调节分子复合体突变的候选化合物也将被分析以 深入了解这些分子可能如何影响Pdr1转录调控。RNA-SEQ实验 将在几个最有希望的化合物上进行，以检查转录范围 这些分子引起的变化及其活动之间的相似性。完成本建议书 将提供新的化学试剂，将作为潜在的佐剂与氟康唑和 Pdr1介导的CDR1表达激活的强大新探针。

Infections associated with Candida species cause the 4th most common type of bloodstream infection. Only three effective antifungal drugs exist, causing a real threat to the continued viability of antifungal therapy. C. glabrata is the second most commonly isolated species associated with candidemias and has both low intrinsic susceptibility to azole drugs and can readily acquire robust tolerance to this most commonly used class of antifungal compounds. Azole-resistant C. glabrata isolates are almost exclusively caused by point mutations in the gene encoding a transcription factor called PDR1. These point mutations lead to the production of a gain-of-function (GOF) form of the Pdr1 transcriptional regulator that in turn cause high level constitutive expression of target genes. A key Pdr1 target gene is the CDR1 locus that encodes an ATP-binding cassette transporter thought to act as a broad specificity drug efflux pump, preventing the accumulation of azole drugs in cells carrying these GOF PDR1 alleles. The goal of this application is to use a chemical genetic approach to identify small molecules that can inhibit activation of CDR1 by GOF forms of Pdr1. We will use two different chemical libraries to screen a C. glabrata strain containing a CDR1-luciferase (CDR1-LUC) gene fusion to identify compounds that are able to inhibit this central regulatory step in azole resistance acquisition in this pathogenic yeast. We have already validated our ability to readily detect expression changes in CDR1-LUC using a 384 well format assay that will be employed in the chemical library screen. Aim 1 will employ this CDR1- luciferase reporter strain to screen two different chemical libraries to identify small molecules that are able to block the normal constitutively high expression seen in the presence of a GOF allele of PDR1. Inhibitors of the luciferase enzymatic reaction and aggregation-dependent inhibitors will be eliminated. Compounds that satisfy these initial screening criteria will be tested for the ability to modulate the native CDR1 locus by RT-qPCR assays. Aim 2 will identify the action of compounds that modulate CDR1 expression by testing their ability to impact azole resistance of a collection of C. glabrata clinical isolates as well as a set of different PDR1 GOF alleles that we have previously characterized. The interaction of candidate compounds with a set of transcriptional Mediator complex mutations will also be analyzed to gain insight into how these molecules may impact Pdr1 transcriptional regulation. RNA-seq experiments will be carried out on several of the most promising compounds to examine the range of transcriptional changes these molecules cause and the similarity between their activities. Completion of this proposal will provide new chemical reagents that will be useful as potential adjuvants with fluconazole and powerful new probes for Pdr1-mediated activation of CDR1 expression.