Amino acid substitution in Trichophyton rubrum squalene epoxidase associated with resistance to terbinafine

Abstract

There has only been one clinically confirmed case of terbinafine resistance in dermatophytes, where six sequential Trichophyton rubrum isolates from the same patient were found to be resistant to terbinafine and cross-resistant to other squalene epoxidase (SE) inhibitors. Microsomal SE activity from these resistant isolates was insensitive to terbinafine, suggesting a target-based mechanism of resistance (B. Favre, M. Ghannoum, and N. S. Ryder, Med. Mycol. 42:525-529, 2004). In this study, we have characterized at the molecular level the cause of the resistant phenotype of these clinical isolates. Cloning and sequencing of the SE gene and cDNA from T. rubrum revealed the presence of an intron in the gene and an open reading frame encoding a protein of 489 residues, with an equivalent similarity (57%) to both yeast and mammalian SEs. The nucleotide sequences of SE from two terbinafine-susceptible strains were identical whereas those of terbinafine-resistant strains, serially isolated from the same patient, each contained the same single missense introducing the amino acid substitution L393F. Introduction of the corresponding substitution in the Candida albicans SE gene (L398F) and expression of this gene in Saccharomyces cerevisiae conferred a resistant phenotype to the transformants when compared to those expressing the wild-type sequence. Terbinafine resistance in these T. rubrum clinical isolates appears to be due to a single amino acid substitution in SE.

FIG. 1.

Alignment of the SE sequences from S. cerevisiae, C. albicans, Schizosaccharomyces pombe, Neurospora crassa, and humans. The three consensus sequences considered as the hallmark of flavoprotein hydroxylases have been framed. The position of the amino acid substitution L393F in the terbinafine-resistant T. rubrum is shown in boldface.

FIG. 2.

Expression of C. albicans SE in S. cerevisiae. Western blot analysis of whole-cell trichloroacetic acid extracts from S. cerevisiae transformed with the vector pYES2 (lane 1) or the pYES2 constructs consisting of CaSE-S172L (lane 2), CaSE (Ser¹⁷²) (lane 3), and CaSE-L398F (lane 4). Transformants (2 × 10⁴ CFU/ml) were cultured in synthetic medium containing 2% galactose for 2 days at 30°C. Proteins were size fractionated on a sodium dodecyl sulfate-8% polyacrylamide gel, transferred onto an Immobilon-P membrane, and analyzed with rabbit polyclonal anti-SE peptide antibody (6). The positions of the size markers (kDa) are indicated on the left. The faint band seen in lane 1 probably corresponds to endogenous SE.