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Genomic profiling of the response of Candida albicans to itraconazole treatment using a DNA microarray.

ABSTRACT: The application of genome-wide expression profiling to determine how drugs achieve their therapeutic effect has provided the pharmaceutical industry with an exciting new tool for drug mode-of-action studies. We used DNA chip technology to study cellular responses to perturbations of ergosterol biosynthesis caused by the broad-spectrum antifungal agent itraconazole. Simultaneous examination of over 6,600 Candida albicans gene transcript levels, representing the entire genome, upon treatment of cells with 10 microM itraconazole revealed that 296 genes were responsive. For 116 genes transcript levels were decreased at least 2.5-fold, while for 180 transcript levels were similarly increased. A global upregulation of ERG genes in response to azole treatment was observed. ERG11 and ERG5 were found to be upregulated approximately 12-fold. In addition, a significant upregulation was observed for ERG6, ERG1, ERG3, ERG4, ERG10, ERG9, ERG26, ERG25, ERG2, IDII, HMGS, NCP1, and FEN2, all of which are genes known to be involved in ergosterol biosynthesis. The effects of itraconazole on a wide variety of known metabolic processes are discussed. As over 140 proteins with unknown function were responsive to itraconazole, our analysis might provide-in combination with phenotypic data-first hints of their potential function. The present report is the first to describe the application of DNA chip technology to study the response of a major human fungal pathogen to drug treatment.

SUBMITTER: De Backer MD

PROVIDER: S-EPMC90529 | biostudies-literature | 2001 Jun

REPOSITORIES: biostudies-literature

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Genomic profiling of the response of Candida albicans to itraconazole treatment using a DNA microarray.

De Backer M D MD Ilyina T T Ma X J XJ Vandoninck S S Luyten W H WH Vanden Bossche H H

Antimicrobial agents and chemotherapy 20010601 6

The application of genome-wide expression profiling to determine how drugs achieve their therapeutic effect has provided the pharmaceutical industry with an exciting new tool for drug mode-of-action studies. We used DNA chip technology to study cellular responses to perturbations of ergosterol biosynthesis caused by the broad-spectrum antifungal agent itraconazole. Simultaneous examination of over 6,600 Candida albicans gene transcript levels, representing the entire genome, upon treatment of ce ...[more]

PMID: 11353609

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Project description:The effects of itraconazole on ergosterol biosynthesis were investigated in a series of 16 matched clinical Candida albicans isolates which had been previously analyzed for mechanisms of resistance to azoles (D. Sanglard, K. Kuchler, F. Ischer, J. L. Pagani, M. Monod, and J. Bille, Antimicrob. Agents Chemother., 39:2378-2386, 1995). Under control conditions, all isolates contained ergosterol as the predominant sterol, except two strains (C48 and C56). In isolates C48 and C56, both less susceptible to azoles than their parent, C43, substantial concentrations (20 to 30%) of 14alpha-methyl-ergosta-8,24(28)-diene-3beta,6alpha-dio l (3, 6-diol) were found. Itraconazole treatment of C43 resulted in a dose-dependent inhibition of ergosterol biosynthesis (50% inhibitory concentration, 2 nM) and accumulation of 3,6-diol (up to 60% of the total sterols) together with eburicol, lanosterol, obtusifoliol, 14alpha-methyl-ergosta-5,7,22,24(28)-tetraene-3betaol, and 14alpha-methyl-fecosterol. In strains C48 and C56, no further increase of 3,6-diol was observed after exposure to itraconazole. Ergosterol synthesis was less sensitive to itraconazole inhibition, as was expected for these azole-resistant isolates which overexpress ATP-binding cassette transporter genes CDR1 and CDR2. In addition to 3,6-diol, substantial amounts of obtusifolione were found after exposure to itraconazole. This toxic 3-ketosteroid was demonstrated previously to accumulate after itraconazole treatment in Cryptococcus neoformans and Histoplasma capsulatum but has not been reported in Candida isolates. Accumulation of obtusifolione correlated with nearly complete growth inhibition in these azole-resistant strains compared to that found in the susceptible parent strain, although the onset of growth inhibition only occurred at higher concentrations of itraconazole. ERG25 and ERG26 are the only genes assigned to the 4-demethylation process, of which the 3-ketoreductase is part. To verify whether mutations in these ERG25 genes contributed to obtusifolione accumulation, their nucleotide sequences were determined in all three related isolates. No mutations in ERG25 alleles of isolates C48 and C56 were found, suggesting that this gene is not involved in obtusifolione accumulation. The molecular basis for the accumulation of this sterol in these two strains remains to be established.

Project description:Azoles are commonly used for the treatment of fungal infections, and the ability of human fungal pathogens to rapidly respond to azole treatment is critical for the development of antifungal resistance. While the roles of genetic mutations, chromosomal rearrangements, and transcriptional mechanisms in azole resistance have been well-characterized, very little is known about post-transcriptional and translational mechanisms that drive this process. In addition, most previous genome-wide studies have focused on transcriptional responses to azole treatment and likely serve as inaccurate proxies for changes in protein expression due to extensive post-transcriptional and translational regulation. In this study, we use ribosome profiling to provide the first picture of the global translational response of a major human fungal pathogen, Candida albicans, to treatment with fluconazole (Flu), one of the most widely used azole drugs. We identify sets of genes showing significantly altered translational efficiency, including genes associated with a variety of biological processes such as the cell cycle, DNA repair, cell wall/cell membrane biosynthesis, transport, signaling, DNA- and RNA-binding activities, and protein synthesis. We observe both similarities and differences among the most highly represented gene categories (as defined by gene ontology) that are regulated by fluconazole at the translational vs transcriptional levels. Importantly, however, very few genes that are translationally regulated by fluconazole are also controlled transcriptionally under this condition. Our findings suggest that C. albicans possesses distinct translational mechanisms that are important for the response to antifungal treatment, which could eventually be targeted by novel antifungal therapies. IMPORTANCE Azoles are one of the most commonly used drug classes to treat human fungal pathogens. While point mutations, chromosomal rearrangements, and transcriptional mechanisms that drive azole resistance have been well-characterized, we know very little about the role of translational mechanisms. In this study, we determined the global translational profile of genes that are expressed in the major human fungal pathogen Candida albicans in response to fluconazole, one of the most widely used azole drugs. We find both similarities and differences among the most highly represented categories of genes regulated by fluconazole at the transcriptional and translational levels. Interestingly, however, many of the specific genes that are regulated by fluconazole at the translational level do not appear to be controlled by transcriptional mechanisms under this condition. Our results suggest that distinct C. albicans translational mechanisms control the response to antifungals and could eventually be targeted in the development of new therapies.

Dataset Information

Genomic profiling of the response of Candida albicans to itraconazole treatment using a DNA microarray.

Publications

Genomic profiling of the response of Candida albicans to itraconazole treatment using a DNA microarray.

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