Identification of Novel Mutations Contributing to Azole Tolerance of Aspergillus fumigatus through In Vitro Exposure to Tebuconazole

Abstract

Azole resistance of Aspergillus fumigatus is a global problem. The major resistance mechanism is through cytochrome P₄₅₀ 14-α sterol demethylase Cyp51A alterations such as a mutation(s) in the gene and the acquisition of a tandem repeat in the promoter. Although other azole tolerance and resistance mechanisms, such as the hmg1 (a 3-hydroxy-3-methylglutaryl coenzyme-A reductase gene) mutation, are known, few reports have described studies elucidating non-Cyp51A resistance mechanisms. This study explored genes contributing to azole tolerance in A. fumigatus by in vitro mutant selection with tebuconazole, an azole fungicide. After three rounds of selection, we obtained four isolates with low susceptibility to tebuconazole. These isolates also showed low susceptibility to itraconazole and voriconazole. Comparison of the genome sequences of the isolates obtained and the parental strain revealed a nonsynonymous mutation in MfsD, a major facilitator superfamily protein (Afu1g11820; R337L mutation [a change of R to L at position 337]), in all isolates. Furthermore, nonsynonymous mutations in AgcA, a mitochondrial inner membrane aspartate/glutamate transporter (Afu7g05220; E535Stop mutation), UbcD, a ubiquitin-conjugating enzyme E2 (Afu3g06030; T98K mutation), AbcJ, an ABC transporter (Afu3g12220; G297E mutation), and RttA, a putative protein responsible for tebuconazole tolerance (Afu7g04740; A83T mutation), were found in at least one isolate. Disruption of the agcA gene led to decreased susceptibility to azoles. Reconstruction of the A83T point mutation in RttA led to decreased susceptibility to azoles. Reversion of the T98K mutation in UbcD to the wild type led to decreased susceptibility to azoles. These results suggest that these mutations contribute to lowered susceptibility to medical azoles and agricultural azole fungicides.

Keywords: Aspergillus fumigatus; azoles; drug resistance.

FIG 1

The appearance and diameters of colonies of ΔpyrG, TBCZ31, TBCZ34, and TBCZ37 strains. Representative macroscopic images and colony diameters after culturing on MM with 10 mM uracil at 35°C for 96 h are shown. Bars and error bars represent the mean values ± standard deviations (SD) from three independent colonies. The asterisk denotes a significant difference (P < 0.01).

FIG 2

The appearance and diameters of colonies of TBCZ15 and the parental strain (ΔpyrG) cultured on MM with 10 mM uracil at 35°C for 96 h. Macroscopic images of TBCZ15 and the parental strain and colony diameters are shown. Bars and error bars represent the mean values ± SD from three independent colonies. The asterisk denotes a significant difference (P < 0.05).

FIG 3

The appearance and diameters of colonies of mfsD disruption strains. Macroscopic images and diameters of two independent mfsD disruption strains, ΔmfsD-1 and ΔmfsD-2, and their parental strain AfS35, cultured on MM at 35°C for 72 h, are shown. Bars represent the mean values ± SD from three independent colonies.

FIG 4

The appearance and diameters of colonies of TBCZ34to34 and TBCZ34to24. Representative macroscopic images and colony diameters after culturing on MM with 10 mM uracil at 35°C for 96 h are shown. Bars represent the mean values ± SD from three independent colonies. The asterisk denotes a significant difference (P < 0.01).

FIG 5

Putative adaptation routes for TBCZ in this study.