An efficient gene targeting system using Δku80and functional analysis of Cyp51A inTrichophyton rubrum.

Trichophyton rubrumis one of the most frequently isolated fungi in patients with dermatophytosis. Despite its clinical significance, the molecular mechanisms of drug resistance and pathogenicity ofT. rubrumremain to be elucidated because of the lack of genetic tools, such as efficient gene targeting systems. In this study, we generated aT. rubrumstrain that lacks the nonhomologous end-joining-related geneku80(Δku80) and then developed a highly efficient genetic recombination system with gene targeting efficiency that was 46 times higher than that using the wild-type strain. Cyp51A and Cyp51B are 14-α-lanosterol demethylase isozymes inT. rubrumthat promote ergosterol biosynthesis and are the targets of azole antifungal drugs. The expression ofcyp51AmRNA was induced by the addition of the azole antifungal drug efinaconazole, whereas no such induction was detected for cyp51B, suggesting that Cyp51A functions as an azole-responsive Cyp51 isozyme. To explore the contribution of Cyp51A to susceptibility to azole drugs, the neomycin phosphotransferase (nptII) gene cassette was inserted into thecyp51A3′-UTR region of Δku80to destabilize the mRNA ofcyp51A. In this mutant, although the expression level ofcyp51AmRNA was comparable to that of the parent strain, the induction ofcyp51AmRNA expression by efinaconazole was diminished. The minimum inhibitory concentration for several azole drugs of this strain was reduced, suggesting that dermatophyte Cyp51A contributes to the tolerance for azole drugs. These findings suggest that an efficient gene targeting system using Δku80inT. rubrumis applicable for analyzing genes encoding drug targets.