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

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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.