Aspergillus fumigatus C-5 sterol desaturases Erg3A and Erg3B: role in sterol biosynthesis and antifungal drug susceptibility

Abstract

Two erg3 genes encoding C-5 sterol desaturase enzymes (Erg3A and Erg3B) in Aspergillus fumigatus were characterized with respect to their nucleotide sequences and null mutant phenotypes. Targeted disruption of the erg3A and erg3B genes and a double gene knockout, erg3A- erg3B-, showed that they are not essential for A. fumigatus viability. Mutant phenotypes clearly showed that Erg3B is a C-5 sterol desaturase, but no apparent role for Erg3A in A. fumigatus ergosterol biosynthesis was found. Susceptibility to amphotericin B, itraconazole, fluconazole, voriconazole, and ketoconazole was not altered in isolates in which erg3A and erg3B were knocked out alone and in combination.

FIG. 1.

Ergosterol biosynthesis pathway in S. cerevisiae (14, 17).

FIG. 2.

Diagrams of plasmid constructs used for creating the A. fumigatus erg3A-deficient mutant strain (pUM208) (I), the erg3B⁻ strain (pUM232) (II), and the double erg3A⁻ erg3B⁻ mutant (pUM235) (III). Arrows represent the full coding sequences, gray bars represent the genomic sequence, unfilled boxes indicate the hygromycin resistance cassette (hph) (I and II) or phleomycin resistance cassette (phle) (III), and black bars indicate PCR fragments used as probes. The restriction enzymes used were ApaI (A), BamHI (B), EcoRV (E), KpnI (K), NotI (N), and SacI (S). On the right, Southern hybridization analysis of erg3A⁻ single mutant strain CM-A80 (I), erg3B⁻ strain CM-B866 (II), and the double erg3A⁻ erg3B⁻ strain (III) is shown. Genomic DNAs were digested with BamHI and hybridized using the erg3A gene probe for CM-A80 (I). CM-B866 (II) and CM-AB118 (III) were detected by digesting the genomic DNAs with EcoRV and hybridizing with the erg3B gene probe.