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 #
2R01GM049825-17
Application #
8250766
Study Section
Special Emphasis Panel (ZRG1-IDM-B (02))
Program Officer
Okita, Richard T
Project Start
1993-08-01
Project End
2016-02-28
Budget Start
2012-06-01
Budget End
2013-02-28
Support Year
17
Fiscal Year
2012
Total Cost
$351,132
Indirect Cost
$115,132
Name
University of Iowa
Department
Physiology
Type
Schools of Medicine
DUNS #
062761671
City
Iowa City
State
IA
Country
United States
Zip Code
52242
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 74:9-Jan
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; 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
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
Gulshan, Kailash; Moye-Rowley, W Scott (2011) Vacuolar import of phosphatidylcholine requires the ATP-binding cassette transporter Ybt1. Traffic 12:1257-68
Gulshan, Kailash; Lee, Stella S; Moye-Rowley, W Scott (2011) Differential oxidant tolerance determined by the key transcription factor Yap1 is controlled by levels of the Yap1-binding protein, Ybp1. J Biol Chem 286:34071-81
Paul, Sanjoy; Schmidt, Jennifer A; Moye-Rowley, W Scott (2011) Regulation of the CgPdr1 transcription factor from the pathogen Candida glabrata. Eukaryot Cell 10:187-97
Shahi, Puja; Gulshan, Kailash; Naar, Anders M et al. (2010) Differential roles of transcriptional mediator subunits in regulation of multidrug resistance gene expression in Saccharomyces cerevisiae. Mol Biol Cell 21:2469-82
Johnson, Soraya S; Hanson, Pamela K; Manoharlal, Raman et al. (2010) Regulation of yeast nutrient permease endocytosis by ATP-binding cassette transporters and a seven-transmembrane protein, RSB1. J Biol Chem 285:35792-802
Shahi, Puja; Moye-Rowley, W Scott (2009) Coordinate control of lipid composition and drug transport activities is required for normal multidrug resistance in fungi. Biochim Biophys Acta 1794:852-9

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