Aspergillus fumigatus is the major filamentous fungal pathogen in humans. Invasive aspergillosis has up to a 90% mortality rate in particular patient populations, primarily among patient groups with reduced immune function. The high intrinsic resistance of A. fumigatus to currently available antifungal agents, coupled with the limited number of these drugs, makes drug resistance an especially acute issue in clinical treatment of aspergillosis. Previous work in fungal pathogens such as Candida albicans and Candida glabrata has provided clear implication of gene transcription as a key determinant of drug resistance. The goal of this proposal is to analyze the transcriptional contribution to azole tolerance in A. fumigatus. While many azole resistant mutants are caused by changes within the cyp51A gene encoding the target of azole drugs, more recent analyses indicate that an increasing number of azole tolerant mutants have wild-type cyp51A genes. This suggests the presence of alternative means of acquiring resistance to azole drugs. We have generated a luciferase reporter gene system to allow the identification of A. fumigatus promoters that drive azole inducible luciferase enzyme activity. This reporter gene system will be used to test a series of promoter fusions from A. fumigatus for the ability to drive azole inducible luciferase activity. Azole inducible expression will be verified by RNA measurements and transcriptional mapping. From this information, deletion mutagenesis will be carried out to identify regions of the promoter(s) important for the response to azole drug challenge. Identification of these DNA elements will serve as the starting point for future work aimed at discovering the trans-acting factors responsible for increased expression in the presence of this important antifungal drug. Successful completion of this work will provide both a useful series of plasmids for expression analysis in A. fumigatus and important information about control of genes that respond to drug challenge. Interference with control of gene expression of drug resistance loci will prevent normal levels of drug tolerance from developing. Increased sensitization of A. fumigatus to antifungal drugs is an important long term goal of this work.

Public Health Relevance

Aspergillus fumigatus is the major human filamentous fungal pathogen, a situation complicated by the relative resistance of this organism to common antifungal drugs. This work will initiate analysis of the contribution of gene transcription to antifungal drug resistance in A. fumigatus.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Exploratory/Developmental Grants (R21)
Project #
5R21AI092331-02
Application #
8264953
Study Section
Drug Discovery and Mechanisms of Antimicrobial Resistance Study Section (DDR)
Program Officer
Duncan, Rory A
Project Start
2011-06-01
Project End
2013-05-31
Budget Start
2012-06-01
Budget End
2013-05-31
Support Year
2
Fiscal Year
2012
Total Cost
$226,500
Indirect Cost
$76,500
Name
University of Iowa
Department
Physiology
Type
Schools of Medicine
DUNS #
062761671
City
Iowa City
State
IA
Country
United States
Zip Code
52242