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Category: Clinical Microbiology; Fungi and Fungal Pathogenesis
Essential Genes in Aspergillus fumigatus, Page 1 of 2
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This chapter discusses recent developments in the identification of essential genes and validation of potential antifungal drug targets in Aspergillus fumigatus. Essential genes were identified based on (i) the inability to construct haploid insertional mutants or (ii) identification of temperature-sensitive conditional mutants. The compendium of recently defined conserved essential genes in Saccharomyces cerevisiae, Candida albicans, and other fungi has provided important insights for predicting essential genes in A. fumigatus. Essential genes that are required for fungal survival and growth provide potential antifungal drug targets. A. fumigatus genes which have been experimentally demonstrated to be essential for growth are summarized. This essential gene set includes genes involved in various biological and biochemical functions, such as amino acid, cell wall, ergosterol, heme, and lipid biosynthesis, as well as cell cycle control, cellular metabolism, protein transport, ribosome biogenesis, and RNA splicing. Additional A. fumigatus essential genes involved in ergosterol biosynthesis include ERG10, ERG12, ERG7, ERG8, and ERG20 and as such, provide new targets for therapeutic intervention. Currently, identification of A. fumigatus essential genes largely depends on the following four approaches: conventional gene deletion and disruption, parasexual genetics, RNAi knockdown, and conditional promoter replacement strategies. Completion of the A. fumigatus genome sequence, however, combined with current molecular genetic strategies and their inevitable refinements, has now made large-scale genetic analysis of A. fumigatus possible for the first time, thus expanding our knowledge of its biology, pathogenesis, and potential antifungal targets.
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Outline of the GRACE method of target validation in C. albicans. Step 1: A heterozygote strain of the target gene was constructed by transforming a wild-type C. albicans strain with a PCR-generated HIS3 disruption cassette flanked with homologous sequences to precisely delete one copy of the target gene. Step 2: The heterozygote strain obtained in step 1 was further transformed with a PCR-generated conditional promoter replacement cassette. Each cassette contains a SAT-1 dominant selectable marker and a conditional promoter (pTet) flanked with homologous DNA sequences to precisely replace the endogenous promoter of the remaining wild-type allele with pTet. Gene essentiality of the target gene was directly assessed by comparing the growth phenotype under inducing and repressing conditions ( Roemer et al., 2003 ).
Schematic overview of gene disruption and deletion methods. (A) Schematic representation of a gene disruption event. The open arrow represents the ORF of a gene of interest. A gene disruption plasmid was constructed by cloning a truncated fragment of the target gene into a plasmid containing a selectable marker. Following a single-crossover homologous recombination, the plasmid integrates into the target gene, leading to a disruption of the gene. (B) Schematic representation of the gene deletion method. The open arrow represents the ORF of a gene of interest. A gene deletion cassette containing a selectable marker flanked with appropriate homologous DNA sequence was used to transform A. fumigatus. Following a double-crossover homologous recombination, the ORF of the target gene was precisely replaced by the selectable marker.
Schematic overview of the parasexual strategy. A diploid (heterozygous) A. fumigatus strain was first created by gene disruption or transposon mutagenesis and contains one inactivated allele of the target gene (gene X) as well as a wild-type allele. The heterozygous strain is used to perform haploidization analysis using benomyl as an inducer on selective and nonselective medium in two independent tests. The haploidization process will result in two subpopulations of haploid cells: one bearing the inactivated allele of the target gene and one bearing a wild-type allele. If gene X is essential for A. fumigatus growth, haploid progenies cannot be obtained from selective medium, as haploids with the inactivated allele will not be viable and haploids with the wild-type allele lack the selectable marker. Replicated from Firon and d’Enfert (2002) with permission from the publisher and the authors.
Schematic overview of the RNAi method. (A) Representative example of the RNAi cassette used to silence the target gene in A. fumigatus. The RNAi cassette was constructed with inverted repeats of 500 bp of the coding region of the target gene separated by a spacer segment of GFP sequence. (B) Representative example of the AMA1 -based RNAi cassette.
Conditional promoter replacement strategy. (A) Schematic overview of the pNiiA-CPR strategy. A conditional promoter replacement cassette containing a PyrG selectable marker and a pNiiA conditional promoter flanked with 1.5 kb of homologous DNA sequence (L-arm and R-arm) was used to transform the A. fumigatus CEA17 strain (PyrG -). Following homologous recombination, the endogenous promoter of the target gene was precisely replaced by the pNiiA condition promoter ( Hu et al., 2007 ). (B) Representative example of gene essentiality validation with a pNiiA-MET2 mutant. The pNiiA-MET2 mutant displayed a no-growth phenotype under repressing conditions, suggesting its essential role for growth. Images are reprinted from Hu et al. (2007) with permission from the publisher and the authors.
In vivo validation of gene essentiality using pNiiA-CPR mutants. (A) ICR male mice were immunocompromised by administrating cyclophosphamide at 150 mg/kg of body weight twice prior to infection and then 100 mg/kg twice a week after infection. Approximately 105 viable conidia from individual pNHA-CPR mutants were injected into the tail vein of immunocompromised mice (five mice per group). CEA10 (wild-type) and CEA17 (a PyrG–auxotroph of CEA10) were included as controls for virulence and avirulence, respectively. (B) Genetic inactivation of the ERG11 gene family promotes avirulence in an immunocompromised murine model of systemic infection. Pathogenesis of ergllAA, ergllBA, and an ERG11 double mutant (ergl 1BA pNHA-ERGl 1A) was similarly analyzed but over a longer postinfection period (22 days), and animal survival was compared to CEA10 and CEA17 control strains. Figures are reprinted from Hu et al. (2007) with permission from the publisher and the authors.
Experimentally validated A. fumigatus essential genes