The Transposon impala Is Activated by Low Temperatures: Use of a Controlled Transposition System To Identify Genes Critical for Viability of Aspergillus fumigatus

Abstract

Genes that are essential for viability represent potential targets for the development of anti-infective agents. However, relatively few have been determined in the filamentous fungal pathogen Aspergillus fumigatus. A novel solution employing parasexual genetics coupled with transposon mutagenesis using the Fusarium oxysporum transposon impala had previously enabled the identification of 20 essential genes from A. fumigatus; however, further use of this system required a better understanding of the mode of action of the transposon itself. Examination of a range of conditions indicated that impala is activated by prolonged exposure to low temperatures. This newly identified property was then harnessed to identify 96 loci that are critical for viability in A. fumigatus, including genes required for RNA metabolism, organelle organization, protein transport, ribosome biogenesis, and transcription, as well as a number of noncoding RNAs. A number of these genes represent potential targets for much-needed novel antifungal drugs.

Site-Directed and Random Insertional Mutagenesis in Medically Important Fungi

“…It is estimated that only 61% of the essential genes in S. cerevisiae are also essential in C. albicans. There may be even more differences in filamentous fungi (Carr et al, 2010). The advent of improved genetic techniques and whole-genome sequencing has dramatically improved the ability to perform forward screens in the medically important fungi.…”

“…One major drawback has been diploidism. Ways to circumvent the problem of diploidy have included parasexual genetics (non-meiotic conversion of a diploid to a haploid) (Carr et al, 2010;Firon et al, 2003) and haploid insufficiency (a phenotype resulting from the loss of one allele in a diploid) (Uhl et al, 2003). Additional requirements that are species-specific include a 'mutagen' and an appropriate screen/phenotype.…”

“…The technology has been adapted for diploid organisms using the parasexual cycle, haploid insufficiency, and homologous recombination (Carr et al, 2010;Davis et al, 2002;Firon et al, 2003;Juarez-Reyes et al, 2011;Spreghini et al, 2003;Uhl et al, 2003). The use of transposons has superseded auxotrophic and STM approaches.…”

“…Although an insertion site did not depend on genome sequence or chromosomal location, there did appear to be a bias toward noncoding regions (34%) (Firon et al, 2003). Carr et al (2010), who observed that transposon mobilization could be induced at 10 °C, improved upon this approach. Therefore, using the same screen, 96 additional essential loci were identified.…”

“…The reverse primer is immediately 3' (antisense strand) to the insertion-specific primer used in the first PCR reaction to guarantee that the desired DNA is amplified (Chun et al, 1997). This method was originally defined in Saccharomyces and was successfully used to identify transposon insertions in A. fumigatus (Carr et al, 2010;Firon et al, 2003).…”