Genetic and Proteomic Analysis of Fungal Virulence

Jennifer K. Lodge; Michael C. Lorenz

doi:10.1128/9781555815776.ch43

Chapter 43 : Genetic and Proteomic Analysis of Fungal Virulence

Authors: Jennifer K. Lodge¹, Michael C. Lorenz²

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Affiliations: 1: Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, 1402 S. Grand Blvd., St. Louis, MO 63104; 2: Department of Microbiology and Molecular Genetics, The University of Texas Health Science Center, 6431 Fannin, Houston, TX 77030

Content Type: Monograph

Publication Year: 2006

Category: Fungi and Fungal Pathogenesis

Book DOI: 10.1128/9781555815776

Chapter DOI: 10.1128/9781555815776.ch43

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Abstract:

This chapter explores the genetic and proteomic approaches that are now feasible for many fungal systems. Post-genomic approaches to the analysis of biological function, networks, and processes often include techniques that permit global analysis of gene expression at the protein level. Analysis of protein expression during modulation of external conditions or during particular developmental states can provide useful clues about which genes might be important for a particular function. These proteomic approaches are complementary to techniques, such as serial analysis of gene expression (SAGE) and microarrays, that measure gene expression at the RNA level. Signature-tagged mutagenesis (STM) was also used to identify Cryptococcus neoformans var. grubii mutants with altered virulence in a mouse model of disseminated disease. Restriction enzyme-mediated insertion has also been used with fungi to promote relatively random insertions in fungal genomes. It has been commonly used in phytopathogens, but the only published studies in a human pathogen are those done with C. albicans and Aspergillus fumigatus. Proteomic analysis of the C. albicans response to several antifungal drugs showed that inhibition of specific targets resulted in a unique profile. Mechanisms of antifungal resistance can be elucidated by identification of proteins induced by treatment with antifungal agents. To determine mechanisms of fungal virulence, it is important to study the expression of proteins during the interaction of a pathogen with host cells, either in vitro or in vivo.

Citation: Lodge J, Lorenz M. 2006. Genetic and Proteomic Analysis of Fungal Virulence, p 643-655. In Heitman J, Filler S, Edwards, Jr. J, Mitchell A (ed), Molecular Principles of Fungal Pathogenesis. ASM Press, Washington, DC. doi: 10.1128/9781555815776.ch43

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Genetic and Proteomic Analysis of Fungal Virulence

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Figure 1

Schematic for STM. A unique signature tag (ST) oligonucleotide is synthesized, incorporated into a plasmid vector, and flanked by primer binding sites. Each “tagged” vector is used to transform cells producing many uniquely tagged mutants. One mutant representing each uniquely tagged vector (up to 96 in total) is assembled into an input pool. Tags present in the input pool are PCR amplified, labeled, and used to probe a filter containing dot blots of each tag forming the preinoculum filter. The pooled organisms are inoculated into an animal, and the infection is allowed to proceed for a determined period. Organisms are recovered from tissue at the site of infection, and the tags are amplified using common primers, labeled, and used to probe a duplicate filter (postinoculum filter). Tags missing from the pool are identified by a loss of signal on the blot. Mutants that overproliferate can also be identified by having a much stronger signal compared to the input blot. Mutants that did not have a change in their virulence have a similar signal to that of the input blot.

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Figure 1

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Figure 2

Comparison of lysates from C. neoformans grown at 25 and 37°C by 2D gel electrophoresis. The two thiol peroxidases, Tsa1 and Tsa3, that are up-regulated at higher temperature are indicated by arrows. Reprinted from reference 39 .

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