Genetic transformation of the dermatophyte, Trichophyton mentagrophytes, based on the use of G418 resistance as a dominant selectable marker

Abstract

Dermatophytes are closely related keratinophilic fungal pathogens and are the causative agents of a superficial cutaneous infection called dermatophytosis (ringworm). A lack of gene manipulation techniques has prevented detailed analyses of the mechanisms of host invasion by dermatophytes. We have introduced the tetracycline-regulatable (TR) gene expression system into dermatophytes to facilitate functional analyses of genes essential for growth and virulence. As the TR gene expression system consists of two plasmid vector components, two dominant selectable markers are required for genetic transformation. In dermatophytes, only the hygromycin B phosphotransferase gene (hph) is available as a selectable marker. We investigated the possibility of G418 resistance as a secondary selectable marker for genetic transformation in dermatophytes. A series of plasmid vectors carrying the neomycin phosphotransferase gene (nptII) were introduced into the protoplasts of Trichophyton mentagrophytes, one of the most clinically important dermatophyte species, by polyethylene glycol (PEG)-mediated transformation. Transformants were selected on selective medium containing G418 at 300-500 microg/ml. Molecular biological analyses indicated that colonies appearing on the selective medium harbored nptII in their chromosomes. Colonies produced from protoplasts transformed with the enhanced green fluorescent protein (eGFP) gene-T. mentagrophytes cyclophilin cDNA (TmcypB) fusion vector also exhibited GFP fluorescence throughout their mycelia, but accumulation of the GFP-TmCYPB fusion protein in specific intracellular compartments was not observed. This study has provided a new selectable marker for genetic transformation in dermatophytes.