Genetic analysis of pleiotropic drug resistance

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 DESCRIPTION (provided by applicant): The close biological similarity of fungi to mammals has made these organisms one of the best model systems for understanding eukaryotic biology. However, this similarity has also limited development of selective antifungal therapies. Candidemias are the third most common blood stream infection (BSI). The species Candida glabrata (Cg) now constitutes the second most common Candida species associated with BSI. Part of this increased incidence of Cg driven candidemia comes from the ability of this species to acquire resistance to the major antifungal drug fluconazole. 30% of drug resistant candidemias are fatal making understanding the molecular physiology of acquired azole resistance of high priority. The most common genetic change leading to azole resistant Cg occurs via substitution mutations in a transcription factor called Pdr1. These mutations produce a hyperactive form of Pdr1 that drives strongly induced levels of downstream gene expression such as the ATP- binding cassette transporter-encoding gene called CDR1. Others have demonstrated that hyperactive forms of Pdr1 also exhibit hypervirulence in a disseminated model of infection along with elevated azole resistance. Recent chromatin immunoprecipitation-next generation sequencing (ChIP-seq) experiments from my lab have identified new genes uniquely regulated by Pdr1 in Cg when compared to the previously characterized Saccharomyces cerevisiae Pdr1 protein. The goal of this proposal is to determine the role of these new genes in drug resistance and virulence in Cg. We will use a new assay for virulence employing a rodent model for central venous catheter infections. These type of infections are a serious clinical complication (mortality approaches 40%) and has not previously been used to evaluate the genetic basis of Cg infection. We will also perform further ChIP-seq analyses to determine the target gene spectrum of hyperactive alleles of PDR1 as well as azole-induced Pdr1. Disruption of key target genes identified by ChIP-seq will allow us to identify the Pdr1-regulated target genes that contribute to drug resistance and virulence in the clinical setting. We have also used mass spectrometry to identify a deubiquitinase subunit (Bre5) that interacts with and regulates Pdr1. Preliminary data suggest that loss of Bre5 leads to an accumulation of a higher molecular weight form of Pdr1. We will use biochemical and genetic analyses to determine the role of ubiquitin in Pdr1 regulation and investigate the regulatory role of other proteins that co-purify with Pdr1. Finally, we will carry out forward genetic analyses to functionally identify proteins that regulate Pdr1 activity. This multidisciplinary approach will provide a detailed picture of Pdr1 regulation, a central modulator of azole resistance and virulence in Cg.