Shinya Ohata

ABSTRACT The increasing prevalence of dermatophyte resistance to terbinafine, a key drug in the treatment of dermatophytosis, represents a significant obstacle to treatment. Trichophyton rubrum is the most commonly isolated fungus in dermatophytosis. In T. rubrum , we identified TERG_07844, a gene encoding a previously uncharacterized putative protein kinase, as an ortholog of budding yeast Saccharomyces cerevisiae polyamine transport kinase 2 (Ptk2), and found that T. rubrum Ptk2 (TrPtk2) is involved in terbinafine tolerance. In both T. rubrum and S. cerevisiae , Ptk2 knockout strains were more sensitive to terbinafine compared with the wild types, suggesting that promotion of terbinafine tolerance is a conserved function of fungal Ptk2. Pma1 is activated through phosphorylation by Ptk2 in S. cerevisiae . Overexpression of T. rubrum Pma1 (TrPma1) in T. rubrum Ptk2 knockout strain (ΔTrPtk2) suppressed terbinafine sensitivity, suggesting that the induction of terbinafine tolerance by TrPtk2 is mediated by TrPma1. Furthermore, omeprazole, an inhibitor of plasma membrane proton pump Pma1, increased the terbinafine sensitivity of clinically isolated terbinafine-resistant strains. These findings suggest that, in dermatophytes, the TrPtk2-TrPma1 pathway plays a key role in promoting intrinsic terbinafine tolerance and may serve as a potential target for combinational antifungal therapy against terbinafine-resistant dermatophytes.

Dermatophytes invade and colonize host superficial tissues via hyphal growth. Although cytoskeletal reorganization and its regulation are essential for hyphal growth, the molecular mechanisms in dermatophytes and their applicability as antifungal drug targets remain poorly understood. The p21-activated kinase (PAK) is a downstream effector of the small GTPases Rac and CDC42, also known as p21, and is involved in various molecular and cellular functions, including actin polymerization and cell morphogenesis. In this study, we investigated the contribution of the PAK protein TrCla4 to morphogenesis and mycelial growth in Trichophyton rubrum, the most frequently isolated fungus in dermatophytosis (athlete’s foot). The actin polymerization inhibitor, cytochalasin A inhibited actin accumulation at the hyphal tip and mycelial growth of T. rubrum, suggesting the involvement of the actin cytoskeleton in mycelial growth. In the Trcla4 knock-out strain (ΔTrcla4), we observed defects in mycelial growth, hyphal branching, and the accumulation of polymerized actin at the hyphal tip. Chemical inhibitors of TrRac-dependent TrCla4 kinase activity, FRAX486 and IPA-3, also inhibited spore germination and mycelial growth. Interestingly, ΔTrcla4 showed no additional inhibition of mycelial growth when treated with these inhibitors, indicating that their inhibitory effects are primarily mediated through TrCla4. In an invertebrate dermatophyte infection model, animals infected with ΔTrcla4 had higher survival rates than those infected with the wild-type, and IPA-3 and FRAX486 treatments both slightly but significantly improved animal survival rates. These results suggest that the dermatophyte PAK promotes mycelial growth by facilitating actin polymerization at the hyphal tip, making it a potential therapeutic target for dermatophytosis.

Nuclear factor-kappa B (NF-κB) signaling is an intracellular signaling pathway involved in inflammatory responses and the pathogenesis of various cancers, including ependymoma, which is a rare and chemotherapy-resistant glioma. Several isoforms of fusion proteins that consist of a nuclear protein, zinc finger translocation associated (ZFTA), and RELA (ZFTA-RELA), an NF-κB-signaling effector transcription factor, cause excessive activation of the NF-κB signaling pathway and result in supratentorial ependymomas (ST-EPN-RELA). As inhibitors of NF-κB activity induced by ZFTA-RELA are expected to be therapeutic agents for ST-EPN-RELA, we established an NF-κB responsive luciferase reporter cell line that expresses the most common isoform of ZFTA-RELA in a doxycycline-dependent manner. Using this reporter cell line, we screened fungus extracts for compounds that inhibit the NF-κB activity induced by ZFTA-RELA expression and identified aszonalenin, an alkaloid from Aspergillus novofumigatus. We also purified analogs of aszonalenin, namely acetylaszonalenin and epi-aszonalenin B and C. In a luciferase assay using cells constitutively expressing luciferase (counter assay), acetylaszonalenin and epi-aszonalenin C showed non-specific inhibition of the luciferase activity. Aszonalenin and epi-aszonalenin B inhibited the NF-κB responsive luciferase activity by expressing ZFTA-RELA more strongly than the luciferase activity in the counter assay. The upregulation of endogenous NF-κB responsive genes, such as CCND1, ICAM1, and L1CAM, by ZFTA-RELA expression was inhibited by epi-aszonalenin B, but not by aszonalenin. This study suggests that epi-aszonalenin B may be a lead compound for the therapeutic development of ST-EPN-RELA.