Upc2A: A Central Regulator and "Achilles' Heel" of Fluconazole Resistance in Candida glabrata

关于三唑的分子和细胞基础的知识有一个很大的空白 重要真菌病原体光滑念珠菌的耐药性以及如何克服这种耐药性。 我们的长期目标是通过克服对三唑的耐药性来改善念珠菌感染的治疗 抗真菌药的种类。我们在本申请中的总体目标是直接鉴定靶基因 受该转录因子、其蛋白质相互作用伴侣以及与UPC 2A相互作用的基因调控， 病原真菌光滑念珠菌。我们的初步数据表明，Upc 2A功能的丧失， 野生型和三唑抗性分离株导致对甾醇生物合成抑制剂的敏感性增加， 包括降低氟康唑的最小抑菌浓度和最小杀真菌浓度， 氟康唑活性通过时间杀灭分析。我们的研究结果表明，Upc 2A是麦角固醇的关键调节因子 生物合成以及其他未知的过程，是必不可少的抗药性氟康唑在C。光滑的 因此，Upc 2A途径代表了增强氟康唑活性的潜在共治疗靶点 针对这种固有的耐药物种，恢复和保存这类抗真菌药物， 侵袭性疟原虫病在目标1中，我们将使用转录谱（RNA-seq）鉴定Upc 2A靶基因。 和ChIP-seq，然后我们将确定哪些靶基因影响氟康唑的敏感性， 定向基因破坏在目标2中，我们将使用串联亲和性鉴定Upc 2A相互作用伴侣蛋白 纯化（TAP），并将确定其中哪些是必不可少的Upc 2A活性下氟康唑 暴露和这些影响氟康唑敏感性使用靶向基因破坏。目标3 这个建议，我们将进行转座子插入突变库的筛选，以及最近的 开发了与甾醇激活Upc 2相互作用并为之所需的基因的缺失突变体文库 为了鉴定和表征Upc 2A遗传相互作用网络，需要对Upc 2A生物合成抑制进行鉴定。的 所提出的研究是创新的，因为它们独特地关注转录因子活性的干扰， Upc 2A作为避免念珠菌对氟康唑耐药的策略光滑的此外，我们的方法是 创新，因为我们将首次利用一套全面的基因组工具和技术， 设计用于酵母研究，并将其应用于真菌病原体C的临床分离株。光滑的的 拟议的研究是重要的，因为它将提供新的知识，最终可以利用克服 三唑耐药性在这种固有的耐药念珠菌物种，恢复和保存使用这种 抗真菌类严重念珠菌感染。

There is a significant gap in knowledge concerning the molecular and cellular underpinnings of triazole resistance in the important fungal pathogen Candida glabrata and how such resistance might be overcome. Our long-term goal is to improve the treatment of Candida infections by overcoming resistance to the triazole class of antifungals. Our overall objective in the present application is to identify the target genes directly regulated by this transcription factor, its protein interaction partners, and the genes that interact with UPC2A in the pathogenic fungus Candida glabrata. Our preliminary data demonstrate that loss of Upc2A function in both wild-type and triazole resistant isolates results in increased susceptibility to sterol biosynthesis inhibitors, including a reduction in fluconazole minimum inhibitory and minimum fungicidal concentrations and enhanced fluconazole activity by time-kill analysis. Our findings indicate that Upc2A is a key regulator of ergosterol biosynthesis as well as other unknown processes and is essential for resistance to fluconazole in C. glabrata. The Upc2A pathway therefore represents a potential co-therapeutic target for enhancing fluconazole activity against this inherently resistant species and restoring and preserving this class of antifungal for the treatment of invasive Candidiasis. In Aim 1 we will identify Upc2A target genes using transcriptional profiling (RNA-seq) and ChIP-seq, and we will then determine which target genes influence susceptibility to fluconazole by targeted gene disruption. In Aim 2 we will identify Upc2A interaction partner proteins using tandem affinity purification (TAP) and will determine which of these are essential for Upc2A activity under fluconazole exposure and which of these influence fluconazole susceptibility using targeted gene disruption. In Aim 3 of this proposal we will undertake screens of a transposon insertion mutant library as well as a recently developed deletion mutant library for genes that interact with, and are required for, Upc2 activation by sterol biosynthesis inhibition in order to identify and characterize the Upc2A genetic interaction network. The proposed studies are innovative as they uniquely focus on interference of activity of the transcription factor Upc2A as a strategy for circumventing fluconazole resistance in C. glabrata. Moreover, our approach is innovative as we will for the first time make use of a comprehensive set of genomic tools and techniques designed for yeast research and apply them to clinical isolates of the fungal pathogen C. glabrata. The proposed research is significant as it will provide new knowledge that can ultimately be exploited to overcome triazole resistance in this inherently resistant species of Candida and restore and preserve the use of this antifungal class for serious Candida infections.