There is a significant lack of knowledge concerning the contribution of the genes encoding the azole target lanosterol demethyase (ERG11) and its transcriptional regulator (UPC2) to azole antifungal resistance in C. albicans. Our long-term goal is to improve the treatment of Candida infections by understanding the molecular basis of antifungal resistance. The overall objective of this proposal is to understand the molecular basis of UPC2- and ERG11-mediated azole resistance in C. albicans. Our central hypothesis is that Upc2p, its interaction partner proteins, and its target genes play a central role along with Erg11p in mediating azole antifungal resistance. We have discovered mutations that activate Upc2p, increase expression of ERG11, and increase azole resistance. We have found that many clinical isolates overexpress ERG11. In several resistant isolates, ERG11 overexpression is not due to activating mutations in UPC2, suggesting undiscovered, novel resistance mechanisms.
Aim 1 of this proposal is to identify mechanisms of ERG11 overexpression in azole resistant isolates of C. albicans by making use of matched pairs of azole susceptible and resistant isolates, as well as unmatched resistant isolates. Novel activating mutations in UPC2 will be identified and characterized through sequencing and standard molecular techniques whereas novel mechanisms of ERG11 overexpression will be identified through a candidate gene approach. Many isolates in our collection carry either novel or characterized ERG11 mutations associated with resistance. While some ERG11 mutations have been associated with or shown to influence azole resistance, their direct effect on this phenotype in C. albicans itself has not been investigated.
Aim 2 is to determine the contribution of specific ERG11 mutations to azole resistance in C. albicans by constructing strains that are heterozygous and homozygous for ERG11 mutations that are associated with azole resistance, measuring their effect on susceptibility to azole antifungals, and characterizing their biochemical effects on the interactio between azole antifungals and their target enzyme. Moreover, activation of zinc cluster transcription factors, such as Upc2p, appears to involve proteins that interact directly with these regulators. Activating mutations may influence interactions between Upc2p and its interaction partner proteins.
Aim 3 is to identify interaction partner proteins required for Upc2-mediated azole resistance in C. albicans using Tandem Affinity Purification (TAP). Finally, we have found that some activating mutations in UPC2 confer significant increases in azole resistance compared to others, yet have similar effects on ERG11 expression. It is therefore likely that other Upc2-targets contribute to azole resistance.
Aim 4 is to determine the role of Upc2-target genes other than ERG11 in Upc2-mediated azole resistance in C. albicans. The proposed research is significant as it will lead to novel strategies for predicting treatment failure, overcoming azole resistance, and improving antifungal therapy. Our approach is innovative as it focuses on novel resistance mechanisms and employs creative strategies to achieve the proposed specific aims.

Public Health Relevance

The proposed research is relevant to public health because the discovery of novel mechanisms of azole antifungal resistance will ultimately contribute to the development of novel strategies for predicting treatment failure, overcoming azole resistance, and improving antifungal therapy. This research is therefore relevant to that part of the National Institute of Allergy and Infectious Diseases'mission that pertains to supporting basic and applied research to better understand, treat, and ultimately prevent infectious diseases, particularly with regard to the emphasis area of antimicrobial resistance.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI058145-07
Application #
8415780
Study Section
Drug Discovery and Mechanisms of Antimicrobial Resistance Study Section (DDR)
Program Officer
Duncan, Rory A
Project Start
2003-12-01
Project End
2017-01-31
Budget Start
2013-02-01
Budget End
2014-01-31
Support Year
7
Fiscal Year
2013
Total Cost
$352,500
Indirect Cost
$117,500
Name
University of Tennessee Health Science Center
Department
Other Health Professions
Type
Schools of Pharmacy
DUNS #
941884009
City
Memphis
State
TN
Country
United States
Zip Code
38163
Flowers, Stephanie A; Colón, Brendan; Whaley, Sarah G et al. (2015) Contribution of clinically derived mutations in ERG11 to azole resistance in Candida albicans. Antimicrob Agents Chemother 59:450-60
Cowen, Leah E; Sanglard, Dominique; Howard, Susan J et al. (2015) Mechanisms of Antifungal Drug Resistance. Cold Spring Harb Perspect Med 5:a019752
Blankenship, Jill R; Cheng, Shaoji; Woolford, Carol A et al. (2014) Mutational analysis of essential septins reveals a role for septin-mediated signaling in filamentation. Eukaryot Cell 13:1403-10
Vasicek, Erin M; Berkow, Elizabeth L; Flowers, Stephanie A et al. (2014) UPC2 is universally essential for azole antifungal resistance in Candida albicans. Eukaryot Cell 13:933-46
Vasicek, Erin M; Berkow, Elizabeth L; Bruno, Vincent M et al. (2014) Disruption of the transcriptional regulator Cas5 results in enhanced killing of Candida albicans by Fluconazole. Antimicrob Agents Chemother 58:6807-18
Whaley, Sarah G; Caudle, Kelly E; Vermitsky, John-Paul et al. (2014) UPC2A is required for high-level azole antifungal resistance in Candida glabrata. Antimicrob Agents Chemother 58:4543-54
Mavrianos, John; Berkow, Elizabeth L; Desai, Chirayu et al. (2013) Mitochondrial two-component signaling systems in Candida albicans. Eukaryot Cell 12:913-22
Caudle, Kelly E; Barker, Katherine S; Wiederhold, Nathan P et al. (2011) Genomewide expression profile analysis of the Candida glabrata Pdr1 regulon. Eukaryot Cell 10:373-83
Hoehamer, Christopher F; Cummings, Edwin D; Hilliard, George M et al. (2010) Changes in the proteome of Candida albicans in response to azole, polyene, and echinocandin antifungal agents. Antimicrob Agents Chemother 54:1655-64
Znaidi, Sadri; Barker, Katherine S; Weber, Sandra et al. (2009) Identification of the Candida albicans Cap1p regulon. Eukaryot Cell 8:806-20

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