Establishment of a gene recombination method for a major human skin commensal fungus, Malassezia restricta, using Agrobacterium tumefaciens-mediated gene transfer system

Abstract

Malassezia restricta is the most predominant fungus in the microbiome of human skin. This microorganism can cause or exacerbate Malassezia-associated skin dermatitis, seborrheic dermatitis, atopic dermatitis, and pityriasis versicolor. The virulence factors of M. restricta have not been analyzed because a gene recombination system has not been developed. In this study, we established an Agrobacterium tumefaciens-mediated gene transfer (ATMT) system, optimized for generating gene-deficient mutants of M. restricta. A mutant of FKB1 gene, which encodes the FKBP12 protein that binds to the calcineurin inhibitor tacrolimus, was generated using the ATMT system. Subsequently, the FKB1 gene was reintroduced into the FKB1 gene-deficient mutant for obtaining a gene-complemented strain. The wild-type strain of M. restricta was sensitive to tacrolimus, whereas the FKB1 gene-deficient mutant was resistant to tacrolimus; the phenotypic drug susceptibility in the mutant was restored by reintroducing the FKB1 gene. Contrastingly, the FKB1 gene-deficient mutant was not resistant to cyclosporine A, which also inhibits calcineurin by binding to cyclophilin A. The gene recombination system for M. restricta will facilitate in elucidating the molecular mechanisms causing Malassezia-associated dermatitis.

Keywords: Agrobacterium tumefaciens-mediated gene transfer system; FKB1; Malassezia restricta; gene recombination; skin microbiome.