Shinya Ohata

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. Keywords Trichophyton rubrum, squalene epoxidase, terbinafine, transformation, docking simulation, dermatophytosis, tinea, antifungal resistance, gene replacement, homologous recombination, Leu393Phe SQLE mutation, allylamine resistance

ABSTRACT Lysin motif (LysM) domain-containing receptors are evolutionarily conserved pattern recognition receptors (PRRs) that serve as key mediators of glycan sensing and innate immune activation in plants and mammals. In invertebrates, however, their role in activating innate immunity remains poorly understood, although some evidence for immunosuppressive functions exists. In this study, we performed in silico structural analyses and identified a putative Bombyx mori LYSMD3 homolog ( XP_004933441.1 ). This protein exhibits high structural similarity in the LysM domain to human LYSMD3, with a root-mean-square deviation (RMSD) of 0.559 Å, indicating close structural alignment. RNA-seq analysis of hemocytes isolated from silkworm larvae injected with N -acetylchitohexaose (GN6), a chitin-derived oligosaccharide and known ligand of human LYSMD3, revealed transcriptional activation of innate immune effectors, including antimicrobial peptide (AMP) genes such as cecropins . GN6 also induced cecropin transcription in isolated hemocytes in vitro , and Western blotting of hemolymph confirmed elevated cecropin B protein levels. Furthermore, GN6 and chitin significantly improved survival outcomes against P. aeruginosa infection, with median effective doses (ED₅₀) values of 0.62 and 0.48  µg/larva, respectively. In contrast, N -acetylglucosamine (GlcNAc) and shorter oligosaccharides (GN2–GN5) were ineffective. These findings provide the first molecular-level evidence of a putative glycan receptor in silkworms based on the structural similarity to known LysM domains. Moreover, GN6-induced antimicrobial peptide expression and enhanced infection resistance demonstrate immune activation in this model, supporting an evolutionarily conserved glycan-sensing pathway in invertebrates.

ABSTRACT Pathogenic fungi pose significant societal challenges due to limited therapeutic targets resulting from the eukaryotic nature of fungi. This limitation emphasizes the importance of enhancing susceptibility to inhibitors of Cyp51, a crucial enzyme in ergosterol biosynthesis targeted by azole antifungals. In Cyp51 isozyme deletion strains (Δ cyp51A and Δ cyp51B ) of Trichophyton rubrum , the predominant dermatophyte species, we found that Cyp51B is essential for basal mycelial growth, while Cyp51A functions as an inducible isozyme associated with azole tolerance. Based on these differential functions, we hypothesized that each isozyme would show distinct susceptibility to azole antifungals. Our study demonstrated that most azoles exhibited increased antifungal activity against Δ cyp51A , while select agents demonstrated increased antifungal activity against Δ cyp51B . Remarkably, fluconazole, sulconazole, and imazalil exhibited relatively increased activity against Δ cyp51A , whereas prochloraz demonstrated increased activity against Δ cyp51B . Combining these isozyme-selective agents exerted synergistic effects against the wild-type strain and the parent ku80 -knockout strain but not against individual Cyp51 knockout mutants. Our data revealed that the two Cyp51 isozymes can be selectively inhibited by different azole antifungals, resulting in a synergistic effect when combined. This synergistic effect was also observed on another fungal species, Aspergillus welwitschiae , which also has two Cyp51 isozymes. These data demonstrate that combining azole antifungals with different Cyp51 isozyme selectivities exerts synergistic effects against fungi possessing multiple Cyp51 isozymes. These findings advance antifungal therapeutic strategies by demonstrating that the combination of antifungals with different Cyp51 isozyme selectivities offers a promising approach for treating fungal infections, opening new avenues for isozyme-specific drug development.