Characterisation of mariner type transposons in the human opportunistic pathogen Aspergillus fumigatus and development of tagged transposon mutagenesis tools

• Main Author: Hey, Peter
• Published: University of Manchester 2007
• Subjects: 579.5
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.686245

Aspergillus fumigatus is the foremost human pathogen of the genus Aspergillus. Current therapies are only partially effective and mortality rates still exceed 60%. All current anti› aspergillus drugs target cell wall and cell membrane components so the identification of novel drug targets may provide an avenue for development of drugs directed against other fungal cell sites. In this study native and non-native mariner transposable elements were investigated with the intention of using these for random mutagenesis, which when coupled with parasexual genetics can be used to identify essential genes as potential antifungal drug targets. Current mutagenesis techniques generate genomic rearrangements complicating downstream analysis whereas transposon mutagenesis results in precise integration enabling faster identification of essential genes. Six novel mariner type transposons were identified in A. fumigatus by bioinformatic analysis. These were examined for conserved functional domains within the transposase and interstrain variation in distribution both of which suggest potential activity. An insertion of the Fotl/pogo like transposon Aft1 was found to disrupt the expression of a putative G-protein coupled receptor in strain AF293 which was expressed in other strains lacking this insertion. This transposon excised from the pAft I-zeo construct in-situ however the introduction of a selectable marker within this element prevented transposition. The Aspergillus niger transposon Anti was cloned and demonstrated activity in a phenotypic excision assay however the introduction of a selectable marker eliminated this activity. Attempts at tagging Aspergillus transposons proved unsuccessful therefore experiments were conducted using the Fusarium oxysporum transposon impala, which had been demonstrated to transpose in A. fumigatus but had proven problematic in mutagenesis screens. Variations in germination and growth conditions solved reported problems and this element was used in a random mutagenesis screen to identify several putative essential genes. Medium throughput methodologies were developed and were found to be suitable for integration with current F2G Ltd. proprietary Mycobank technology.