Enhanced gene replacements inKu80disruption mutants of the dermatophyte,Trichophyton mentagrophytes

Abstract

The frequency of targeted gene disruption via homologous recombination is low in the clinically important dermatophyte, Trichophyton mentagrophytes. The Ku genes, Ku70 and Ku80, encode key components of the nonhomologous end-joining pathway involved in DNA double-strand break repair. Their deletion increases the homologous recombination frequency, facilitating targeted gene disruption. To improve the homologous recombination frequency in T. mentagrophytes, the Ku80 ortholog was inactivated. The nucleotide sequence of the Ku80 locus containing a 2788-bp ORF encoding a predicted product of 728 amino acids was identified, and designated as TmKu80. The predicted TmKu80 product showed a high degree of amino acid sequence similarity to known fungal Ku80 proteins. Ku80 disruption mutant strains of T. mentagrophytes were constructed by Agrobacterium tumefaciens-mediated genetic transformation. The average homologous recombination frequency was 73.3 ± 25.2% for the areA/nit-2-like nitrogen regulatory gene (tnr) in Ku80⁻ mutants, about 33-fold higher than that in wild-type controls. A high frequency (c. 67%) was also obtained for the Tri m4 gene encoding a putative serine protease. Ku80⁻ mutant strains will be useful for large-scale reverse genetics studies of dermatophytes, including T. mentagrophytes, providing valuable information on the basic mechanisms of host invasion.