RNA-mediated gene silencing in non-pathogenic and pathogenic fungi.

Many fungal genomes have now been sequenced and thousands of genes are being discovered. Gene disruption or inactivation technology offers an important tool not only for elucidating the function of the many unknown genes but also for the identification of genes essential for fungal growth and pathogenesis. A variety of gene-silencing methods that inhibit genes at the post-transcriptional level are now being used in both non-pathogenic and human pathogenic fungi. We focus on the recent advances in RNA-mediated gene silencing technologies and their potential for functional genomics studies in fungi.

Progress in functional genomics approaches to antifungal drug target discovery.

Antifungal drug discovery is starting to benefit from the enormous advances in the genomics field, which have occurred in the past decade. As traditional drug screening on existing targets is not delivering the long-awaited potent antifungals, efforts to use novel genetics and genomics-based strategies to aid in the discovery of novel drug targets are gaining increased importance. The current paradigm in antifungal drug target discovery focuses on basically two main classes of targets to evaluate: genes essential for viability and virulence or pathogenicity factors. Here we report on recent advances in genetics and genomics-based technologies that will allow us not only to identify and validate novel fungal drug targets, but hopefully in the longer run also to discover potent novel therapeutic agents. Fungal pathogens have typically presented significant obstacles when subjected to genetics, but the creativity of scientists in the anti-infectives field and the cross-talk with scientists in other areas is now yielding exciting new tools and technologies to tackle the problem of finding potent, specific and non-toxic antifungal therapeutics.

Functional genomics approaches for the identification and validation of antifungal drug targets.

So far, antifungal drug discovery seems to have benefited little from the enormous advances in the field of genomics in the last decade. Although it has become clear that traditional drug screening is not delivering the long-awaited novel potent antifungals, little has been reported on efforts to use novel genome-based methodologies in the quest for new drugs acting on human pathogenic fungi. Although the market for a novel systemic and even topical broad-spectrum antifungal appears considerable, many large pharmaceutical companies have decided to scale back their activities in antifungal drug discovery. Here we report on some of the recent advances in genomics-based technologies that will allow us not only to identify and validate novel drug targets but hopefully also to discover active therapeutic agents. Novel drug targets have already been found by 'en masse' gene inactivation strategies (e.g. using antisense RNA inhibition). In addition, genome expression profiling using DNA microarrays helps to assign gene function but also to understand better the mechanism of action of known drugs (e.g. itraconazole) and to elucidate how new drug candidates work. No doubt, we have a long way to go just to catch up with the advances made in other therapeutic areas, but all tools are at hand to derive practical benefits from the genomics revolution. The next few years should prove a very exciting time in the history of antifungal drug discovery.

Gene-Expression - Based Responses to Drug Treatment.

Perfect drugs are potent, specific and nontoxic. Many compounds fail because of unexpected toxicity and lack of efficacy in later stages of clinical development. Therefore, more complete knowledge and understanding of the properties of a drug is needed at an earlier stage of drug development. DNA microarrays can yield gene expression profiles from cells or tissues treated with a compound. Such "expression fingerprints" are used in drug discovery for drug target identification and validation and for elucidating the mode of action of novel compounds during lead identification and optimization. Moreover, during drug development, DNA microarrays help in the discovery of new diagnostic and prognostic biomarkers, as well as in the prediction of resistance and toxic side effects. This review aims to assess to what extent the promise of gene expression profiling has already materialized for the different stages of drug discovery and development. (c) 2002 Prous Science. All rights reserved.

Single allele knock-out of Candida albicans CGT1 leads to unexpected resistance to hygromycin B and elevated temperature.

Almost all eukaryotic mRNAs are capped at their 5'-terminus. Capping is crucial for stability, processing, nuclear export and efficient translation of mRNA. We studied the phenotypic effects elicited by depleting a Candida albicans strain of mRNA 5'-guanylyltransferase (mRNA capping enzyme; CGT1). Construction of a Cgt1-deficient mutant was achieved by URA-blaster-mediated genetic disruption of one allele of the CGT1 gene, which was localized on chromosome III. The resulting heterozygous mutant exhibited an aberrant colony morphology resembling the 'irregular wrinkle' phenotype typically obtained from a normal C. albicans strain upon mild UV treatment. Its level of CGT1 mRNA was reduced two- to fivefold compared to the parental strain. Proteome analysis revealed a large number of differentially expressed proteins confirming the expected pleiotropic effect of CGT1 disruption. The disrupted strain was significantly more resistant to hygromycin B, an antibiotic which decreases translational fidelity, and showed increased resistance to heat stress. Proteome analysis revealed a 50-fold overexpression of Ef-1alphap and a more than sevenfold overexpression of the cell-wall heat-shock protein Ssa2p. Compared to a reference strain, the cgt1/CGT1 heterozygote was equally virulent for mice and guinea pigs when tested in an intravenous infection model of disseminated candidiasis.