Shinya Ohata

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.