title: Flippase (FLP) recombinase-mediated marker recycling in the dermatophyte Arthroderma vanbreuseghemii.
abstract: Biological processes can be elucidated by investigating complex networks of relevant factors and genes. However, this is not possible in species for which dominant selectable markers for genetic studies are unavailable. To overcome the limitation in selectable markers for the dermatophyte Arthroderma vanbreuseghemii (anamorph: Trichophyton mentagrophytes), we adapted the flippase (FLP) recombinase-recombination target (FRT) site-specific recombination system from the yeast Saccharomyces cerevisiae as a selectable marker recycling system for this fungus. Taking into account practical applicability, we designed FLP/FRT modules carrying two FRT sequences as well as the flp gene adapted to the pathogenic yeast Candida albicans (caflp) or a synthetic codon-optimized flp (avflp) gene with neomycin resistance (nptII) cassette for one-step marker excision. Both flp genes were under control of the Trichophyton rubrum copper-repressible promoter (PCTR4). Molecular analyses of resultant transformants showed that only the avflp-harbouring module was functional in A. vanbreuseghemii. Applying this system, we successfully produced the Ku80 recessive mutant strain devoid of any selectable markers. This strain was subsequently used as the recipient for sequential multiple disruptions of secreted metalloprotease (fungalysin) (MEP) or serine protease (SUB) genes, producing mutant strains with double MEP or triple SUB gene deletions. These results confirmed the feasibility of this system for broad-scale genetic manipulation of dermatophytes, advancing our understanding of functions and networks of individual genes in these fungi.
authors.affiliation: Department of Chemical and Biological Sciences, Faculty of Science, Japan Women's University, 2-8-1, Mejirodai, Bunkyo, Tokyo 112-8681, Japan.
authors.forename: Yohko
authors.initials: Y
authors.lastname: Yamada
authors.affiliation: Teikyo University Institute of Medical Mycology, 359 Otsuka, Hachioji, Tokyo 192-0395, Japan.
authors.forename: Mari
authors.initials: M
authors.lastname: Maeda
authors.affiliation: Laboratory of Space and Environmental Medicine, Graduate School of Medicine, Teikyo University, 2-11-1, Kaga, Itabashi, Tokyo 173-8605, Japan.
authors.forename: Mohamed Mahdi
authors.initials: MM
authors.lastname: Alshahni
authors.affiliation: Department of Dermatology, Centre Hospitalier Universitaire Vaudois, Av. de Beaumont 29, 1011 Lausanne, Switzerland.
authors.forename: Michel
authors.initials: M
authors.lastname: Monod
authors.affiliation: Research and Development, Kneipp GmbH, Winterhäuser Str. 85, 97084 Würzburg, Germany.
authors.forename: Peter
authors.initials: P
authors.lastname: Staib
authors.affiliation: Teikyo University Institute of Medical Mycology, 359 Otsuka, Hachioji, Tokyo 192-0395, Japan.
authors.forename: Tsuyoshi
authors.initials: T
authors.lastname: Yamada
mesh_terms: D003883:Arthrodermataceae / Q000235:genetics*; D004254:DNA Nucleotidyltransferases / Q000378:metabolism*; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D017353:Gene Deletion; D015870:Gene Expression; D018390:Gene Targeting / Q000379:methods*; D005819:Genetic Markers; D005827:Genetics, Microbial / Q000379:methods*; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D017422:Sequence Analysis, DNA
chemical_list: D004271:DNA, Fungal; D005819:Genetic Markers; D004254:DNA Nucleotidyltransferases; C045119:FLP recombinase