Identification of Amino Acid Substitution(s) in the Squalene Epoxidase Sequence from Trichophyton rubrum Isolates Resistant to Terbinafine.



Background Normally dermatophytes are highly susceptible to terbinafine, which has squalene epoxidase (SE) as the target. Recently, however, a few Trichophyton rubrum clinical isolates have appeared exhibiting primary resistance to terbinafine. As a means to identifying the mechanism of resistance in these strains, SE from these isolates, along with strains normally susceptible to terbinafine, have been cloned and sequenced. Methods A central fragment of SE gene from T. rubrum standard strain NFI 1895 was PCR amplified from genomic DNA using degenerate primers. The 5'- and 3'- sequences were obtained by rapid amplification of cDNA ends from total RNA. The full length squalene epoxidase gene or cDNA from four T. rubrum strains, two susceptible and two resistant to terbinafine, was then amplified with specific primers. Results T. rubrum squalene epoxidase gene codes for a protein of 489 residues. It contains one intron of 62 nucleotides interrupting the open reading frame. Comparison of T. rubrum SE protein sequence with those of Saccharomyces cerevisiae, Candida albicans and human showed it to be 57%, 56% and 58% homologous (43%, 40% and 41% identical) respectively. As with the mammalian sequence, T. rubrum SE does not contain a specific stretch of 34 residues found in yeast SEs (C. albicans and S. cerevisiae). SE protein sequence from two strains susceptible to terbinafine was identical whereas the sequence from each of the two terbinafine-resistant strains contained a single amino acid substitution, either L393F or F397L. Conclusion Our results suggest that amino acid substitution(s) in SE might be responsible for the resistant phenotype of these T. rubrum clinical isolates. Such genetic variations, which are dependent on random mutations in the genome, would explain the rarity of terbinafine-resistant dermatophytes.

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42nd Interscience Conference on Antimicrobial Agents and Chemotherapy
    • ICAAC 42nd