Development of an efficient gene-targeting system for elucidating infection mechanisms of the fungal pathogen Trichosporon asahii

Abstract

Trichosporon asahii is a pathogenic fungus that causes severe, deep-seated fungal infections in neutropenic patients. Elucidating the infection mechanisms of T. asahii based on genetic studies requires a specific gene-targeting system. Here, we established an efficient gene-targeting system in a highly pathogenic T. asahii strain identified using the silkworm infection model. By comparing the pathogenicity of T. asahii clinical isolates in a silkworm infection model, T. asahii MPU129 was identified as a highly pathogenic strain. Using an Agrobacterium tumefaciens-mediated gene transfer system, we obtained a T. asahii MPU129 mutant lacking the ku70 gene, which encodes the Ku70 protein involved in the non-homologous end-joining repair of DNA double-strand breaks. The ku70 gene-deficient mutant showed higher gene-targeting efficiency than the wild-type strain for constructing a mutant lacking the cnb1 gene, which encodes the beta-subunit of calcineurin. The cnb1 gene-deficient mutant showed reduced pathogenicity against silkworms compared with the parental strain. These results suggest that an efficient gene-targeting system in a highly pathogenic T. asahii strain is a useful tool for elucidating the molecular mechanisms of T. asahii infection.

A critical role of calcineurin in stress responses, hyphal formation, and virulence of the pathogenic fungus Trichosporon asahii

“…The ku70 gene-deficient strain of the T. asahii MPU129 strain has high homologous recombination efficiency, making it useful as a parent strain 28 . Using this strain, we generated the cna1 gene-deficient mutant.…”

“…The T. asahii strain (MPU129 ku70 gene-deficient mutant) used in this study was generated as previously reported 28 . The T. asahii MPU129 ku70 gene-deficient mutant was grown on Sabouraud dextrose agar (SDA) (1% hipolypepton [Nihon Pharmaceutical Co., Ltd., Tokyo, Japan], 4% dextrose, and 1.5% agar [both from FUJIFILM Wako Pure Chemical Industries, Osaka, Japan]) containing G418 (50 μg/ml) and incubated at 27 °C for 2 days.…”

“…Competent cells for electroporation were prepared according to a previous report 28 . T. asahii was spread on an SDA plate and cultured at 27 °C for 1 day.…”

“…In the present study, we used a recently developed technique 28 to generate calcineurin gene-deficient T. asahii mutants and characterized the phenotypes related to stress resistance and virulence in the silkworm infection model. The cna1 and cnb1 gene-deficient mutants exhibited sensitivity to chemicals known to cause membrane damage and ER stress, as well as attenuated hyphal formation and virulence against silkworms.…”

A joint PCR-based gene-targeting method using electroporation in the pathogenic fungus Trichosporon asahii

“…Phenotypic analyses using gene-deficient mutants are important for clarifying the molecular mechanisms of infection and drug resistance in T. asahii. We established a method for generating a gene-deficient mutant of T. asahii using an Agrobacterium tumefaciens-mediated gene transfer system (Matsumoto et al 2021). The efficiency of genetic recombination in T. asahii was increased by deleting the ku70 gene that encodes the Ku70 protein, a subunit of the non-homologous end-joining repair enzyme (Matsumoto et al 2021).…”