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


OBJECTIVE
We investigated the possibility of G418 resistance as a secondary selectable marker for genetic transformation in dermatophytes.


METHODS
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.


RESULTS
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.


CONCLUSIONS
This study has provided a new selectable marker for genetic transformation in dermatophytes.
Teikyo University Institute of Medical Mycology, Teikyo University, Hachioji, Tokyo 192-0395, Japan. tsyamada@main.teikyo-u.ac.jp
Tsuyoshi
T
Yamada
Koichi
K
Makimura
Tatsuya
T
Hisajima
Maki
M
Ito
Yoshiko
Y
Umeda
Shigeru
S
Abe
D000563:Amebicides / Q000494:pharmacology; D003470:Culture Media; D021983:Cyclophilins / Q000235:genetics; D025141:Drug Resistance, Fungal / Q000235:genetics*; D005819:Genetic Markers; D005839:Gentamicins / Q000494:pharmacology; D049452:Green Fluorescent Proteins / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011092:Polyethylene Glycols; D011523:Protoplasts / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000235:genetics; D014170:Transformation, Genetic; D014249:Trichophyton / Q000187:drug effects / Q000235:genetics*
D000563:Amebicides; D003470:Culture Media; D005819:Genetic Markers; D005839:Gentamicins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D011092:Polyethylene Glycols; C010680:antibiotic G 418; D021983:Cyclophilins