Genetic analysis of pleiotropic drug resistance

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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.