|ROGERS, P. DAVID|
Submitted to: Journal of Biological Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/24/2003
Publication Date: 9/12/2003
Citation: Agarwal, A.K., Baerson, S.R., Rogers, P., Jacob, M.R., Barker, K.S., Cleary, J.D., Walker, L.A., Nagle, D.G., Clark, A.M. 2003. Genome-wide expression profiling of the response to polyene, pyrimidine, azole, and echinocandin antifungal agents in saccharomyces cerevisiae. Journal of Biological Chemistry. 278:34998-35015.
Interpretive Summary: Recent technological advancements in the field of molecular biology have created powerful new approaches for mode of action studies of fungicidal compounds. One such technology is called a "DNA microarray", which enables one to simultaneously monitor the activity of tens of thousands of genes in a single experiment. Gene expression changes that occur in response to fungicide treatments provide important clues about their mode of action. All organisms possess the ability to change gene expression levels when needed to counteract the effects of toxic chemicals. This so-called "feedback response" can be detected in DNA microarray experiments and may directly pinpoint the likely mode of action of a given pesticide. At a minimum, the collective gene expression changes exhibited create a specific "fingerprint" for that mode of action, enabling the comparison of new compounds with previously characterized ones. In this manuscript, gene expression "fingerprints" were determined for four fungicidal compounds using Bakers yeast as a test organism. The effect of polyene, pyrimidine, azole, and echinocandin fungicides were tested for growth inhibition, then microarray experiments were performed to identify gene expression changes that occurred in response to these four fungicide treatments. Fingerprints were obtained for all four, which describe all of the gene expression changes that occur in response to these compounds in yeast. These results will benefit researchers working in the area of new fungicide discovery, and hopefully will help accelerate these efforts. The gene expression fingerprints contributes to a body of knowledge that will enable researchers to rapidly identify the mechanisms related to these four compounds in new compounds. In addition, these results provide new information as to how different biochemical pathways interact to cope with growth inhibitors having these modes of action. This type of understanding also enables one to predict ways that microorganisms can become resistant to specific fungicides.
Technical Abstract: Antifungal compounds exert their activity through a variety of mechanisms, some of which are poorly understood. Novel approaches to identify and characterize the mechanism of action of antifungal agents will be of great use in the antifungal drug development process. The aim of the present study was to investigate the changes in the gene expression profile of Saccharomyces cerevisiae following exposure to representatives of the four currently available classes of antifungal agents used in the management of systemic fungal infections. Microarray analysis indicated differential expression of 0.8%, 4.1%, 3.0% and 2.6% of the genes represented on the Affymetrix S98 Yeast gene set in response to ketoconazole, amphotericin B, caspofungin and 5-fluorocytosine (5-FC) respectively. Quantitative real-time RT-PCR assays were used to confirm the microarray analyses. Genes responsive to ketoconazole, caspofungin, and 5-FC were indicative of the drug-specific mechanism of action. Ketoconazole exposure primarily affected genes involved in ergosterol biosynthesis and sterol uptake, caspofungin exposure affected genes involved in cell wall integrity, and 5-FC affected genes involved in DNA and protein synthesis, DNA damage repair, and cell cycle control. In contrast, amphotericin B elicited changes in gene expression reflective of cell stress, membrane reconstruction, transport, phosphate uptake, and cell wall integrity. Genes with the greatest specificity for a particular drug were grouped together as drug-specific genes, whereas genes with a lack of drug specificity were also identified. Taken together, these data shed new light on the mechanisms of action of these classes of antifungal agents and demonstrate the potential utility of gene expression profiling in the antifungal drug development process.