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Chapter 42 : Functional Genomic Approaches to Fungal Pathogenesis, Drug Target Validation, and Antifungal Drug Discovery

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Abstract:

Genomic studies of the nonpathogenic budding yeast are proving to be highly applicable to antifungal drug discovery. Paradigms derived from studies will continue to direct research and contribute to one's understanding of fungal pathogenesis and the identification of antifungal drug targets. Many of these resources and their corresponding technologies have direct applications to antifungal drug discovery, but perhaps the most relevant resource is the yeast deletion mutant set. A discussion of how to exploit the knowledge of both essential genes and biological networks containing nonessential genes to identify drug targets is provided in this chapter. Because chemical-genetic profiling focuses on compounds that impair cell growth, it can also be applied to natural-product extracts, which often contain only one growth-inhibitory active compound. Identification of the set of essential yeast genes is one of the most important results to immediately fall out of the deletion project because it immediately defined ~1,000 candidate antifungal targets. In summary, the chapter highlights the incredible power of combining systematic genetics with antifungal drug target identification. In particular, the availability of a complete barcoded set of deletion mutants, including conditional alleles of essential genes, for the major fungal pathogens would permit identification of the “universal fungal essential gene set,” allow a broad application of chemical genomics in these organisms, and enable a systematic analysis of phenotypes associated with pathogenicity.

Citation: Parsons A, Bussey H, Boone C. 2006. Functional Genomic Approaches to Fungal Pathogenesis, Drug Target Validation, and Antifungal Drug Discovery, p 627-642. In Heitman J, Filler S, Edwards, Jr. J, Mitchell A (ed), Molecular Principles of Fungal Pathogenesis. ASM Press, Washington, DC. doi: 10.1128/9781555815776.ch42
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Figure 1

Construction strategy for the yeast deletion mutant collection. Each yeast open reading frame (ORF) is replaced with a deletion cassette consisting of an antibiotic resistance marker, KAN MX4, conferring resistance to kanamycin, and two unique 20-mer molecular barcodes (uptag and downtag). Each barcode is flanked by common primer sites (indicated by half arrows). Incorporation of the cassette is accomplished through homologous recombination of 45-bp regions of homology up- and downstream of the yeast open reading frame.

Citation: Parsons A, Bussey H, Boone C. 2006. Functional Genomic Approaches to Fungal Pathogenesis, Drug Target Validation, and Antifungal Drug Discovery, p 627-642. In Heitman J, Filler S, Edwards, Jr. J, Mitchell A (ed), Molecular Principles of Fungal Pathogenesis. ASM Press, Washington, DC. doi: 10.1128/9781555815776.ch42
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Figure 2

Parallel analysis of large pools of deletion mutants. Populations of pooled mutant cells, each marked with unique molecular barcodes, are grown in the presence or absence of a growth-inhibitory drug. Genomic DNA is extracted from the pool of mutants, and barcodes representing each strain are amplified by PCR with common primers labeled with the fluorescent marker Cy3 or Cy5. Drug-sensitive mutants are identified by competitive hybridization of the barcoded PCR products to a microarray containing oligonucleotides corresponding to each barcode.

Citation: Parsons A, Bussey H, Boone C. 2006. Functional Genomic Approaches to Fungal Pathogenesis, Drug Target Validation, and Antifungal Drug Discovery, p 627-642. In Heitman J, Filler S, Edwards, Jr. J, Mitchell A (ed), Molecular Principles of Fungal Pathogenesis. ASM Press, Washington, DC. doi: 10.1128/9781555815776.ch42
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Figure 3

A compendium of chemical-genetic interaction profiles organizes yeast genes into functional pathways. Here, deletion mutants that are sensitive to a particular compound are indicated by the black boxes and an absence of a chemical-genetic interaction is indicated by a white box. Benomyl and nocodazole are microtubule-depolymerizing agents, hydroxyurea inhibits DNA synthesis, camptothecin inhibits topoisomerase function, and caspofungin is a cell wall inhibitor.

Citation: Parsons A, Bussey H, Boone C. 2006. Functional Genomic Approaches to Fungal Pathogenesis, Drug Target Validation, and Antifungal Drug Discovery, p 627-642. In Heitman J, Filler S, Edwards, Jr. J, Mitchell A (ed), Molecular Principles of Fungal Pathogenesis. ASM Press, Washington, DC. doi: 10.1128/9781555815776.ch42
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Figure 4

Synthetic genetic array analysis. A strain carrying a query mutation (for example, ) linked to a dominant selectable marker, such as the nourseothricin resistance marker , and an reporter is mated to ~5,000 deletion mutants, each linked to a kanamycin resistance marker, . Growth of the resultant diploids is selected for on medium containing nourseothricin and kanamycin. The diploids are pinned onto medium designed to induce spore formation. haploids are recovered using the reporter gene, and double mutants are selected for medium containing nourseothricin and kanamycin. Inviable double mutants are scored as synthetic lethal, and slow-growing double mutants are scored as synthetic sick.

Citation: Parsons A, Bussey H, Boone C. 2006. Functional Genomic Approaches to Fungal Pathogenesis, Drug Target Validation, and Antifungal Drug Discovery, p 627-642. In Heitman J, Filler S, Edwards, Jr. J, Mitchell A (ed), Molecular Principles of Fungal Pathogenesis. ASM Press, Washington, DC. doi: 10.1128/9781555815776.ch42
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Figure 5

genetic interaction network. Genes are represented by nodes, and spokes between nodes indicate synthetic lethal or synthetic sick relationships. Genes are grouped according to their biological function.

Citation: Parsons A, Bussey H, Boone C. 2006. Functional Genomic Approaches to Fungal Pathogenesis, Drug Target Validation, and Antifungal Drug Discovery, p 627-642. In Heitman J, Filler S, Edwards, Jr. J, Mitchell A (ed), Molecular Principles of Fungal Pathogenesis. ASM Press, Washington, DC. doi: 10.1128/9781555815776.ch42
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Figure 6

Chemical-genetic interactions can be modeled by synthetic lethal interactions. (A) A deletion mutant sensitive to a particular drug should also be synthetically lethal with the drug target. (B) Genome-wide chemical-genetic profiles can be compared to genome-wide synthetic lethal profiles to link compounds with their target protein or pathway.

Citation: Parsons A, Bussey H, Boone C. 2006. Functional Genomic Approaches to Fungal Pathogenesis, Drug Target Validation, and Antifungal Drug Discovery, p 627-642. In Heitman J, Filler S, Edwards, Jr. J, Mitchell A (ed), Molecular Principles of Fungal Pathogenesis. ASM Press, Washington, DC. doi: 10.1128/9781555815776.ch42
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Figure 7

Drug-induced haploinsufficiency. Lowering the gene dosage of a drug target from two copies to one in a diploid cell results in increased sensitivity to drugs acting on the gene product. For example, Alg7 is the target of tunicamycin. At a semi-inhibitory concentration of tunicamycin, wild-type cells are viable. However, lowering the gene dosage of to one copy in the heterozygote deletion mutant results in hypersensitivity to tunicamycin.

Citation: Parsons A, Bussey H, Boone C. 2006. Functional Genomic Approaches to Fungal Pathogenesis, Drug Target Validation, and Antifungal Drug Discovery, p 627-642. In Heitman J, Filler S, Edwards, Jr. J, Mitchell A (ed), Molecular Principles of Fungal Pathogenesis. ASM Press, Washington, DC. doi: 10.1128/9781555815776.ch42
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Figure 8

Construction of an essential gene set. (Step 1) Heterozygote strains are constructed by transforming a wild-type strain with a PCR-generated disruption cassette containing a selectable marker flanked with appropriate homologous sequence to replace one allele of the target gene. Two unique barcodes (uptag and downtag) flanked by primer sites common to all strains are introduced into the disruption cassette during PCR amplification. (Step 2) The endogenous promoter of the remaining wild-type allele is replaced with a PCR-generated tetracycline promoter replacement cassette containing the dominant selectable marker engineered for expression in

Citation: Parsons A, Bussey H, Boone C. 2006. Functional Genomic Approaches to Fungal Pathogenesis, Drug Target Validation, and Antifungal Drug Discovery, p 627-642. In Heitman J, Filler S, Edwards, Jr. J, Mitchell A (ed), Molecular Principles of Fungal Pathogenesis. ASM Press, Washington, DC. doi: 10.1128/9781555815776.ch42
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Figure 9

Synthetic drug combinations as predicted by synthetic lethal relationships can lead to synergistic effects. (Top) Synthetic lethal interaction between hypomorphic alleles of and . (Bottom) The synthetic lethal interaction between and is mimicked by treatment with specific drugs inhibiting the gene products of both genes. While this example describes an interaction between two essential genes, it is equally applicable to interactions between two nonessential genes or one essential gene and one nonessential gene.

Citation: Parsons A, Bussey H, Boone C. 2006. Functional Genomic Approaches to Fungal Pathogenesis, Drug Target Validation, and Antifungal Drug Discovery, p 627-642. In Heitman J, Filler S, Edwards, Jr. J, Mitchell A (ed), Molecular Principles of Fungal Pathogenesis. ASM Press, Washington, DC. doi: 10.1128/9781555815776.ch42
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