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

关于三氮唑的分子和细胞基础的知识有很大的差距。 重要的真菌病原菌光滑假丝酵母的抗药性以及如何克服这种抗药性。 我们的长期目标是通过克服对三氮唑的耐药性来改善念珠菌感染的治疗 一类抗真菌药物。我们在目前应用中的总体目标是直接识别目标基因 受该转录因子、其蛋白相互作用伙伴以及与UPC2A相互作用的基因的调控 病原真菌光滑假丝酵母。我们的初步数据表明，在这两种情况下，Upc2A功能的丧失 野生型和三氮唑耐药菌株导致对类固醇生物合成抑制剂的敏感性增加， 包括降低氟康唑的最低抑菌浓度和最低杀菌浓度 用时间-杀灭分析法测定氟康唑活性。我们的发现表明，Upc2A是麦角甾醇的关键调节因子 生物合成以及其他未知的过程，是光滑念珠菌对氟康唑耐药所必需的。 因此，upc2a通路是增强氟康唑活性的潜在联合治疗靶点。 并恢复和保存这类抗真菌药物用于治疗 侵袭性念珠菌病。在目标1中，我们将使用转录图谱(rna-seq)来鉴定upc2a靶基因。 和芯片序列，然后我们将确定哪些靶基因影响对氟康唑的敏感性 有针对性的基因破坏。在目标2中，我们将使用串联亲和力鉴定Upc2A相互作用伙伴蛋白 纯化(TAP)，并将确定其中哪些是在氟康唑作用下Upc2A活性所必需的 暴露以及这些因素中的哪一个使用靶向基因破坏来影响氟康唑的敏感性。在目标3中 这项建议我们将进行转座子插入突变体文库的筛选以及最近的 开发了与甾醇激活Upc2相互作用并需要的基因的缺失突变体库 生物合成抑制，以识别和表征Upc2A遗传相互作用网络。这个 建议的研究是创新的，因为他们独特地关注转录因子活性的干扰 Upc2a作为规避光滑拟青霉对氟康唑耐药性的策略。此外，我们的方法是 尽管我们将首次利用一套全面的基因组工具和技术进行创新 专为酵母菌研究而设计，并将其应用于真菌病原体光滑拟青霉的临床分离。这个 拟议的研究具有重要意义，因为它将提供最终可以被利用来克服 三氮唑耐药念珠菌的这种固有耐药种，并恢复和保存这一用途 严重念珠菌感染的抗真菌类药物。

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.