S. cerevisiae pleiotropic drug resistance (PDR) is directly analogous to multidrug resistance in mammalian cells as both can be triggered by elevated levels of ABC transporter proteins (ScPdr5 in S. cerevisiae) that efflux drugs out of the cell. The pathogenic Candida species, Candida albicans and Candida glabrata, express homologues of ScPdr5 and can acquire similar multidrug resistant phenotypes. We have been studying PDR in S. cerevisiae as this organism provides a facile experimental background with which to dissect eukaryotic multidrug resistance. Our long term goal is to understand the physiology underlying development of multidrug resistance. Multidrug resistance poses a special problem in deployment of antifungal drugs as these compounds are quite limited in number. Understanding the logic used by a yeast cell to control the multidrug resistance will allow use of strategies to prevent this phenotype from arising. S. cerevisiae and C. glabrata contain the most closely related multidrug resistance pathways currently known in fungi. ScPdr5 is the major ABC transporter protein while C. glabrata expresses a very similar protein called CgCdr1. PDR S. cerevisiae cells express high levels of ScPDR5 transcript owing to changes in activity of the related zinc cluster-containing transcriptional regulators ScPdr1 and ScPdr3. Multidrug resistant C. glabrata cells overproduce CgCDR1 mRNA via increased activity of CgPdr1, a homologue of the S. cerevisiae multidrug transcription factors. Previous work has implicated the transcription Mediator complex as being a key determinant of expression of ScPDR5 and CgCDR1. We have directly studied Mediator mutants in C. glabrata and found that while there are similarities with S. cerevisiae, differences also exist. We propose to investigate selected Mediator components in C. glabrata to determine which are important in transcriptional activation controlled via CgPdr1 via gene disruption. Chromatin immunoprecipitation will be carried out to address the likelihood of direct action of Mediator components of interest on CgCDR1 transcription. We have also constructed a fully-functional tandem affinity purification (TAP)-tagged allele of CgPDR1. This will be used to purify CgPdr1 from C. glabrata cells with different levels of CgCDR1 transcription in order to identify and characterize proteins involved in control of this key transcriptional regulator. Finally, we have begun to use a high throughput genetic screen in S. cerevisiae cells to identify all non-essential genes involved in regulation of ScPDR5 expression. Using this technique, we have found a new regulatory input from the protein kinase A signaling pathway modulating ScPDR5 expression. We will use this screen to identify components important in ScPDR5 expression in the presence and absence of drugs. Using the experimental facility of S. cerevisiae to inform us of important participants in C. glabrata will allow us to rapidly uncover regulatory networks modulating drug resistance in this fungal pathogen.

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 and other fungal species.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM049825-18
Application #
8471707
Study Section
Special Emphasis Panel (ZRG1-IDM-B (02))
Program Officer
Okita, Richard T
Project Start
1993-08-01
Project End
2016-02-29
Budget Start
2013-03-01
Budget End
2014-02-28
Support Year
18
Fiscal Year
2013
Total Cost
$334,855
Indirect Cost
$113,097
Name
University of Iowa
Department
Physiology
Type
Schools of Medicine
DUNS #
062761671
City
Iowa City
State
IA
Country
United States
Zip Code
52242
Moye-Rowley, W S (2015) Multiple mechanisms contribute to the development of clinically significant azole resistance in Aspergillus fumigatus. Front Microbiol 6:70
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
Paul, Sanjoy; Moye-Rowley, W Scott (2014) Multidrug resistance in fungi: regulation of transporter-encoding gene expression. Front Physiol 5:143
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 ρ0 cells. Antimicrob Agents Chemother 58:6904-12
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
Morano, Kevin A; Grant, Chris M; Moye-Rowley, W Scott (2012) The response to heat shock and oxidative stress in Saccharomyces cerevisiae. Genetics 190:1157-95
Paul, Sanjoy; Klutts, J Stacey; Moye-Rowley, W Scott (2012) Analysis of promoter function in Aspergillus fumigatus. Eukaryot Cell 11:1167-77
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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