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.

Public Health Relevance

Understanding fungal multidrug resistance is crucial because relatively few antifungal compounds exist. Candidemia is the 4th leading cause of bloodstream infections and Candida glabrata is emerging as an important source of these infections, likely due to its propensity to acquire multidrug resistance. This project is directed towards understanding the molecular basis for multidrug resistance in C. glabrata.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM049825-23
Application #
9435126
Study Section
Drug Discovery and Mechanisms of Antimicrobial Resistance Study Section (DDR)
Program Officer
Okita, Richard T
Project Start
1993-08-01
Project End
2020-02-29
Budget Start
2018-03-01
Budget End
2019-02-28
Support Year
23
Fiscal Year
2018
Total Cost
Indirect Cost
Name
University of Iowa
Department
Physiology
Type
Schools of Medicine
DUNS #
062761671
City
Iowa City
State
IA
Country
United States
Zip Code
52242
Hagiwara, Daisuke; Miura, Daisuke; Shimizu, Kiminori et al. (2017) A Novel Zn2-Cys6 Transcription Factor AtrR Plays a Key Role in an Azole Resistance Mechanism of Aspergillus fumigatus by Co-regulating cyp51A and cdr1B Expressions. PLoS Pathog 13:e1006096
Paul, Sanjoy; Diekema, Daniel; Moye-Rowley, W Scott (2017) Contributions of both ATP-Binding Cassette Transporter and Cyp51A Proteins Are Essential for Azole Resistance in Aspergillus fumigatus. Antimicrob Agents Chemother 61:
Khakhina, Svetlana; Johnson, Soraya S; Manoharlal, Raman et al. (2015) Control of Plasma Membrane Permeability by ABC Transporters. Eukaryot Cell 14:442-53
Paul, Sanjoy; Doering, Tamara L; Moye-Rowley, W Scott (2015) Cryptococcus neoformans Yap1 is required for normal fluconazole and oxidative stress resistance. Fungal Genet Biol 74:1-9
Moye-Rowley, W S (2015) Multiple mechanisms contribute to the development of clinically significant azole resistance in Aspergillus fumigatus. Front Microbiol 6:70
Paul, Sanjoy; Bair, Thomas B; Moye-Rowley, W Scott (2014) Identification of genomic binding sites for Candida glabrata Pdr1 transcription factor in wild-type and ?0 cells. Antimicrob Agents Chemother 58:6904-12
Paul, Sanjoy; Moye-Rowley, W Scott (2014) Multidrug resistance in fungi: regulation of transporter-encoding gene expression. Front Physiol 5:143
Paul, Sanjoy; Moye-Rowley, W Scott (2013) Functional analysis of an ATP-binding cassette transporter protein from Aspergillus fumigatus by heterologous expression in Saccharomyces cerevisiae. Fungal Genet Biol 57:85-91
Paul, Sanjoy; Diekema, Daniel; Moye-Rowley, W Scott (2013) Contributions of Aspergillus fumigatus ATP-binding cassette transporter proteins to drug resistance and virulence. Eukaryot Cell 12:1619-28
Gulshan, Kailash; Thommandru, Bernice; Moye-Rowley, W Scott (2012) Proteolytic degradation of the Yap1 transcription factor is regulated by subcellular localization and the E3 ubiquitin ligase Not4. J Biol Chem 287:26796-805

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