Gene Disruption in Aspergillus fumigatus Using a PCR-Based Strategy and In Vivo Recombination in Yeast
Aspergillus fumigatus is a ubiquitous, filamentous fungal saprophyte and is the causative agent of the vast majority of aspergillosis in that invasive aspergillosis is the life-threatening form of infection by this fungus. The study of gene function using
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duction Aspergillus fumigatus is a saprophytic ubiquitous filamentous fungus with a key role in carbon and nitrogen recycling in soil. However, it is also the causative agent of the vast majority of aspergillosis cases, although the mechanisms whereby this species becomes one of the most prevalent opportunistic pathogens are currently not fully understood. Apparently, the factors involved in the survival of this organism in the environment outside the host, e.g., in compost pile or soil under conditions of limited oxygen, thermal variation, and competition for nutrients, might contribute to its success as an opportunistic pathogen in humans. A. fumigatus colonizes the lungs of immunocompromised individuals, leading to invasive aspergillosis, the life-threatening form of infection. The mortality
Alexandra C. Brand and Donna M. MacCallum (eds.), Host-Fungus Interactions: Methods and Protocols, Methods in Molecular Biology, vol. 845, DOI 10.1007/978-1-61779-539-8_7, © Springer Science+Business Media, LLC 2012
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I. Malavazi and G.H. Goldman
rates for invasive aspergillosis are as high as 95% in patients receiving hematopoietic stem cell transplants under immunosuppressive regimens (1). For this reason, the study of gene function in this opportunistic pathogen has become essential to elucidate the genes and pathways that orchestrate the host interaction, the adaptation of the fungus to the mammalian environment, and, as a consequence, the virulence of A. fumigatus. DNA-mediated transformation of exogenous deletion cassettes targeted to genes of interest has been used successfully in this organism. The ability to introduce an engineered mutation in an organism is vital for both forward and reverse genetic analysis. Reverse genetics, in which the analysis of gene function starts with a cloned uncharacterized gene, relies substantially on methods that interfere in the function of the gene product. Forward genetics, where the analysis of gene function analysis starts with the isolation of a mutant, also employs gene replacement methodologies to confirm predicted phenotypes, for example, by the generation of gainof-function mutants. The generation of deletion cassettes for A. fumigatus gene disruption using a PCR-based strategy and “in vivo” recombination in yeast is based on the procedures first described by Baudin et al. (2) where PCR was used to create gene disruption cassettes for Saccharomyces cerevisiae. However, an important difference between A. fumigatus and S. cerevisiae gene disruption lies in the conditions required by these two organisms for homologous recombination to occur. S. cerevisiae is widely known for its capability to undergo homologous recombination with efficiency rates as high as 95%. Hence, very short DNA sequences are required and an ordinary gene disruption cassette for S. cerevisiae would present about 50–80 bp of homology to the target gene. In contrast, A. fumigatus (and other Aspergillus species) requires sequences of about 1,500–2,000 bp of homology to maximize the frequency of homol
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