Genome-wide Expression Profiling of the Response to Polyene, Pyrimidine, Azole, and Echinocandin Antifungal Agents in Saccharomyces cerevisiae

Abstract

Antifungal compounds exert their activity through a variety of mechanisms, some of which are poorly understood. Novel approaches to 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 array in response to ketoconazole, amphotericin B, caspofungin, and 5-fluorocytosine (5-FC), respectively. Quantitative real time reverse transcriptase-PCR was used to confirm the microarray analyses. Genes responsive to ketoconazole, caspofungin, and 5-FC were indicative of the drug-specific effects. 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 reflecting 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 antifungal drug development.

Modulation of Yeast Sln1 Kinase Activity by the Ccw12 Cell Wall Protein

“…In addition, conserved sequences (36% identity over 74 residues of the 133 amino acids Ccw12 (41)) in the two proteins suggest possible functional redundancy. Unlike CCW12, SED1 is most highly expressed in stationary phase (32) and is regulated by the PKC pathway (42) in response to cell wall stress (43,44). Our finding that SED1 overexpression partially suppresses both the wall phenotypes of the ccw12 mutant and the SLN1-SKN7 pathway activation phenotype indicates that these two phenotypes are inter-dependent and that SLN1-SKN7 pathway activation may therefore be attributable to the wall defects of the ccw12 mutant.…”

Polyene antibiotic that inhibits membrane transport proteins

“…Further, the amphotericin B methyl ester inhibited the replication, assembly, and release of HIV-1 by interfering with the ion channel viral protein U (VPU) (18,21). Microarray studies with S. cerevisiae clearly show an increase of expression of transport proteins on treatment with the polyenes amphotericin B and nystatin (22,23). However, membrane permeabilization by these polyene antibiotics does mask these effects as a result of the fast collapse of vital ion or substrate gradients.…”

The TRK1 Potassium Transporter Is the Critical Effector for Killing of Candida albicans by the Cationic Protein, Histatin 5

“…TaqMan RT-PCR Assay-Quantitative real time RT-PCR experiments were carried out as described previously for yeast (43). Primers and probes (forward primer EFB1, 5Ј-AAG ACT TGC CAG CCG GTA AAG-3Ј; reverse primer EFB1, 5Ј-TCA GCT TCT TCA TCA ACT TCA TCA-3Ј; forward primer TRK1, 5Ј-CGT CTT CAT CTC CTC AGT CAT CTT-3Ј; reverse primer TRK1, 5Ј-GGC TCA CTA TGG TGC TCT ATA TCA-3Ј; probe EFB1, 5Ј-CCG CTT CTG GTT CTG CTG CTG CCG-3Ј; probe TRK1, 5Ј-AAC AAG CCA GCC GGT TCT GAC AGC-3Ј) were designed via the Primer 3 Input program (available on the World Wide Web at frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi) and confirmed via Amplify 1.2 Software for PCR.…”