Biology subjects

Ohata, S.

Publications and source records attributed to Ohata, S..

8 recordsLinked to original sources

bioRxiv2025

N-acetylchitohexaose induces immune priming against Pseudomonas aeruginosa infection in the silkworm, Bombyx mori

Lysine 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 B. 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 [A], 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 against P. aeruginosa infection, with median effective doses (ED) values of 0.62 and 0.48 {micro}g/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 structural similarity to known LysM domains. Moreover, GN6-induced antimicrobial peptide expression and enhanced infection resistance demonstrate immune priming in this model, supporting an evolutionarily conserved glycan-sensing pathway in invertebrates.

immunology↗

bioRxiv2025

Synergistic effects of Cyp51 isozyme-specific azole antifungal agents on fungi with multiple cyp51 isozyme genes

Pathogenic fungi pose significant societal challenges. The limited availability of therapeutic targets due to the eukaryotic nature of fungi emphasizes the importance of available drug targets such as Cyp51, a crucial enzyme in ergosterol biosynthesis, inhibited by azole antifungals. This study explored the susceptibility patterns of azole antifungals against Cyp51 isozyme deletion strains ({Delta}cyp51A and {Delta}cyp51B) in Trichophyton rubrum, the predominant dermatophyte species. Distinct susceptible patterns were observed among azole antifungals for {Delta}cyp51A and {Delta}cyp51B. Although most azole antifungal agents exhibited increased antifungal activity against {Delta}cyp51A, select agents demonstrated increased antifungal activity against {Delta}cyp51B. Remarkably, fluconazole, sulconazole, and imazalil exhibited relatively increased activity against {Delta}cyp51A, whereas prochloraz demonstrated increased activity against {Delta}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. Hence, the two Cyp51 isozymes, Cyp51A and Cyp51B, may be inhibited by distinct azole antifungals, exerting a synergistic effect with the dual azole antifungal combination. 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. This study proposes a novel therapeutic approach for addressing fungal infections through the combination of antifungal drugs that inhibit the same enzymatic activity but exhibit different isozyme selectivity. It also emphasizes the potential for developing drugs targeting specific isozymes, a previously underutilized approach in the realm of antifungal drug development.

microbiology↗

bioRxiv2024

Inhibition of microtubule polymerization and dynein impairs the nuclear localization of the ependymoma-associated ZFTA-RELA fusion protein and NF-κB activation

Ependymomas are rare and chemotherapy-resistant gliomas. One subclass of supratentorial ependymomas, ST-EPN-ZFTA, expresses a fusion protein consisting of a nuclear protein, zinc finger translocation associated (ZFTA), and v-rel reticuloendotheliosis viral oncogene homolog A (RELA), an effector transcription factor of the nuclear factor-{kappa}B (NF-{kappa}B) pathway (ZFTA-RELA). Constitutive localization of ZFTA-RELA to the nucleus hyperactivates the oncogenic NF-{kappa}B signaling pathway, thereby contributing to the pathogenesis of ST-EPN-ZFTA. To identify compounds that inhibit NF-{kappa}B activity induced by ZFTA-RELA, we established a high-throughput screening system using the NF-{kappa}B-responsive luciferase reporter cell line 6E8, which expresses ZFTA-RELA in a doxycycline-dependent manner. A chemical library of 9600 compounds selected for their structural diversity was screened, and a colchicine derivative was identified. Among colchicine and six of its derivatives, the IC50 on ZFTA-RELA-dependent NF-{kappa}B-responsive luciferase activity in 6E8 cells was found to be the lowest for colchicine at 90 nM. Interestingly, microtubule polymerization inhibitors (colchicine and vinblastine) and dynein inhibitors (ciliobrevin D and dynarrestin) impaired ZFTA-RELAs nuclear localization and NF-{kappa}B activity in 6E8 cells. These findings indicate that microtubule polymerization and dynein play pivotal roles in activating the NF-{kappa}B pathway in ST-EPN-ZFTA by promoting the nuclear localization of ZFTA-RELA. Consequently, inhibition of microtubule polymerization may be a therapeutic strategy for ST-EPN-ZFTA.

cell biology↗

bioRxiv2024

Structural insight into the nuclear transportation mechanism of PPARg by Transportin-1

The spatial and temporal control of protein is essential for normal cellular function. Proteins working in the nucleus have nuclear localization signal (NLS) sequences and are escorted into the nucleus by cognate nuclear transport receptors. A wealth of experimental data about NLS has been accumulated, and nuclear transportation mechanisms are established at the biochemical and structural levels. The peroxisome proliferator-activated receptors (PPARs) are ligand-dependent transcription factors that control various biological responses. We recently reported that the transportation of PPAR{gamma} is mediated by Transportin-1, but PPAR{gamma} lacks a typical NLS sequence recognized by Transportin-1. Moreover, the recognition mechanism remains largely unknown. In this study, we determined the Cryo-EM structure of PPAR{gamma} in complex with Transportin-1 and revealed that Transportin-1 gripped the folded DNA binding domain and the Hinge region of PPAR{gamma}, indicating that PPAR{gamma} recognizes a folded domain with an extended region as a nuclear localization signal, not a canonical unstructured signal sequence, confirmed by the mutation analyses in vitro and in cultured cells. Our study is the first snapshot structure working in nuclear transportation, not in transcription, of PPAR{gamma}.

molecular biology↗

bioRxiv2024

An efficient gene targeting system using Δku80 and functional analysis of Cyp51A in Trichophyton rubrum.

Trichophyton rubrum is one of the most frequently isolated fungi in patients with dermatophytosis. Despite its clinical significance, the molecular mechanisms of drug resistance and pathogenicity of T. rubrum remain to be elucidated because of the lack of genetic tools, such as efficient gene targeting systems. In this study, we generated a T. rubrum strain that lacks the nonhomologous end-joining-related gene ku80 ({Delta}ku80) and then developed a highly efficient genetic recombination system with gene targeting efficiency that was 46 times higher than that using the wild-type strain. Cyp51A and Cyp51B are 14--lanosterol demethylase isozymes in T. rubrum that promote ergosterol biosynthesis and are the targets of azole antifungal drugs. The expression of cyp51A mRNA was induced by the addition of the azole antifungal drug efinaconazole, whereas no such induction was detected for cyp51B, suggesting that Cyp51A functions as an azole-responsive Cyp51 isozyme. To explore the contribution of Cyp51A to susceptibility to azole drugs, the neomycin phosphotransferase (nptII) gene cassette was inserted into the cyp51A 3'-UTR region of {Delta}ku80 to destabilize the mRNA of cyp51A. In this mutant, although the expression level of cyp51A mRNA was comparable to that of the parent strain, the induction of cyp51A mRNA expression by efinaconazole was diminished. The minimum inhibitory concentration for several azole drugs of this strain was reduced, suggesting that dermatophyte Cyp51A contributes to the tolerance for azole drugs. These findings suggest that an efficient gene targeting system using {Delta}ku80 in T. rubrum is applicable for analyzing genes encoding drug targets. Key PointsO_LIA novel gene targeting system using {Delta}ku80 strain was established in T. rubrum C_LIO_LICyp51A in T. rubrum responds to the azole antifungal drug efinaconazole C_LIO_LICyp51A contributes to azole drug tolerance in T. rubrum C_LI

microbiology↗

bioRxiv2024

Targeting dermatophyte Cdc42 and Rac GTPase signaling to hinder hyphal elongation and virulence

The identification of novel molecular targets for antifungal drugs is critical due to limited treatment options and drug-resistance threats. We screened inhibitors of small GTPases, molecular switches in signal transduction, in Trichophyton rubrum, the primary cause of dermatophytosis. Our study found that chemical and genetic inhibition of Cdc42 and Rac GTPases, which are involved in cellular morphological changes, significantly impair hyphal formation, and are crucial for pathogenic fungal growth and virulence. Genetic repression of Cdc24, a guanine nucleotide exchange factor of Cdc42 and Rac, led to hyphal growth defects, abnormal cell morphology, and cell death. Chemical screening identified EHop-016 as an inhibitor of Cdc24 activity, which improved outcomes in in vitro nail infection and invertible infection models of T. rubrum. Our results suggest the Cdc24-Cdc42/Rac pathway as a promising therapeutic target for antifungal agent development, with EHop-016 as a potential lead compound.

microbiology↗

bioRxiv2023

The Ptk2-Pma1 pathway enhances tolerance to terbinafine in Trichophyton rubrum

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 to the wild types, suggesting that promotion of terbinafine tolerance is a conserved function of fungal Ptk2. The T. rubrum Ptk2 knockout strain ({Delta}TrPtk2) was sensitive to omeprazole, an inhibitor of plasma membrane proton pump Pma1, which is activated through phosphorylation by Ptk2 in S. cerevisiae. Overexpression of T. rubrum Pma1 (TrPma1) in {Delta}TrPtk2 suppressed terbinafine sensitivity, suggesting that the induction of terbinafine tolerance by TrPtk2 is mediated by TrPma1. Furthermore, omeprazole 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.

microbiology↗

bioRxiv2019

Matrix mechanotransduction mediated by thrombospondin-1/integrin/YAP signaling pathway in the remodeling of blood vessels

RationaleThe extracellular matrix (ECM) initiates mechanical cues and transduces intracellular signaling through matrix-cell interactions. In the blood vessels, additional mechanical cues derived from the pulsatile blood flow and pressure play a pivotal role in homeostasis and disease development. Currently, the nature of the cues from ECM and how they coordinate with a mechanical microenvironment in large blood vessels to maintain the integrity of the vessel wall are not fully understood.\n\nObjectiveThe aim of this study was to elucidate the crucial mediator(s) and molecular signaling pathway(s) involved in matrix mechanotransduction during remodeling of the vessel wall.\n\nMethods and ResultsWe performed secretome analysis using rat vascular smooth muscle cells (SMCs) under cyclic stretch and examined matrix-cell interactions and cell behavior. We found that the matricellular protein thrombospondin-1 (Thbs1) was secreted upon cyclic stretch and bound to integrin v{beta}1, thereby recruiting vinculin and establishing focal adhesions. RNA-sequence (RNA-seq) analysis revealed that deletion of Thbs1 in vitro markedly affected the target gene expression of Yes-associated protein (YAP). Consistently, we found that Thbs1 promotes nuclear shuttling of YAP in response to cyclic stretch, which depends on the small GTPase Rap2 and Hippo pathway, and is not influenced by alteration of actin fibers. Deletion of Thbs1 in mice inhibited Thbs1/integrin/YAP signaling, leading to maladaptive remodeling of the aorta in response to pressure overload by transverse aortic constriction (TAC), whereas it suppressed neointima formation upon carotid artery ligation, exerting context-dependent effects on the vessel wall.\n\nConclusionsThbs1 serves as a mechanical stress-triggered extracellular mediator of mechanotransduction that acts via integrin v{beta}1 to establish focal adhesions and promotes nuclear shuttling of YAP. We thus propose a novel mechanism of matrix mechanotransduction centered on Thbs1, connecting mechanical stimuli to YAP signaling during vascular remodeling in vivo.\n\nSubject codesO_LIVascular Disease\nC_LIO_LIGenetically Altered and Transgenic Models\nC_LIO_LIVascular Biology\nC_LIO_LICell Signaling/Signal Transduction\nC_LI

molecular biology↗