Ishii Masaki

Abstract Terbinafine (TBF) is a first-line allylamine antifungal used to treat dermatophytosis caused by Trichophyton species, targeting squalene epoxidase (SQLE). The recent emergence of TBF-resistant Trichophyton rubrum (T. rubrum) strains represents a significant global clinical challenge. Although the SQLE Leu393Phe substitution is a commonly reported resistance mechanism in Japan, direct genetic evidence demonstrating its effect when introduced into a T. rubrum recipient strain has been lacking. In this study, we established the causal relationship between the SQLE Leu393Phe substitution and TBF resistance via targeted gene replacement in T. rubrum. A homologous recombination plasmid containing SQLE from the TBF-resistant T. rubrum strain BGUTR13, which harbours the 1177TTA→TTC mutation encoding Leu393Phe, together with the neomycin phosphotransferase II (nptII) selection marker, was constructed and introduced into the TBF-susceptible strain T. rubrum CBS118892 Δku80. The resulting mutant transformants exhibited a >8,000-fold increase in TBF minimum inhibitory concentration compared with the parental strain, whereas control transformants carrying nptII but lacking the SQLE Leu393Phe substitution remained susceptible. Structural modelling indicated that the Leu393Phe substitution causes substantial steric hindrance within the SQLE binding pocket, displacing TBF’s aliphatic chain and disrupting key hydrophobic interactions. Molecular docking simulations predicted reduced TBF binding affinity (ΔG changed from −6.3 to −0.8 kcal/mol) following the mutation. These findings provide the first robust genetic evidence that the single SQLE Leu393Phe substitution is sufficient to confer high-level TBF resistance in T. rubrum. These findings highlight the importance of monitoring SQLE mutations in clinical isolates and provide a foundation for developing strategies to address antifungal resistance in dermatophytes.

ABSTRACT The resistance of Trichophyton indotineae to azoles is mainly due to the overexpression of TinCYP51B, resulting from additional copies of this gene in two types of strains (type I and type II). Due to its large size and the significant number of duplicated blocks, whole-genome sequencing has been unable to cover the entire TinCYP51B locus. Through optical genome mapping (OGM), we have successfully determined the copy number of the TinCYP51B gene in the genomes of resistant strains. The TinCYP51B copy number was lower in the type I strains than in the type II strains, while the TinCYP51B expression level was higher in the type I strains. To explain this paradox, we have revealed that polycistronic transcription of multiple TinCYP51B open reading frames (ORFs) alongside monocistronic transcription occurs in type I azole-resistant strains. In contrast, type II strains generated only the transcripts encoding one CYP51B polypeptide. OGM has also revealed that a 970 kb region on chromosome 3 is inverted in type I strains and the azole-susceptible strain TIMM20115, as compared to type II strains and the azole-susceptible strain TIMM20114. This has led to the hypothesis that under azole stress, type I resistant strains originate from susceptible strains such as TIMM20115, which possesses a single TinCYP51B gene. Conversely, it is believed that type II azole-resistant strains evolve from susceptible strains such as TIMM20114, which also has only one TinCYP51B gene. In conclusion, strains of Trichophyton indotineae can be divided into two groups in which a distinct type of resistance has developed.