石井 雅樹

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 PPARg is mediated by Transportin-1, but PPARg 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 PPARg in complex with Transportin-1 and revealed that Transportin-1 gripped the folded DNA binding domain and the Hinge region of PPARg, indicating that PPARg 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 PPARg.

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 knockout 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 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. IMPORTANCE Superficial fungal infections, such as athlete's foot, affect more than 10% of the world's population and have a significant impact on quality of life. Despite the fact that treatment-resistant fungi are a concern, there are just a few antifungal drug targets accessible, as opposed to the wide range of therapeutic targets found in bacterial infections. As a result, additional alternatives are sought. In this study, we generated a PAK TrCla4 deletion strain (∆Trcla4) of Trichophyton rubrum. The ∆Trcla4 strain exhibited deficiencies in mycelial growth, hyphal morphology, and polarized actin localization at the hyphal tip. IPA-3 and FRAX486, small chemical inhibitors of mammalian PAK, were discovered to limit fungal mycelial proliferation. According to our findings, fungal PAKs are interesting therapeutic targets for the development of new antifungal medicines.

The fusion protein of uncharacterised zinc finger translocation associated (ZFTA) and effector transcription factor of tumorigenic NF-κB signalling, RELA (ZFTA-RELA), is expressed in more than two-thirds of supratentorial ependymoma (ST-EPN-RELA), but ZFTA's expression profile and functional analysis in multiciliated ependymal (E1) cells have not been examined. Here, we showed the mRNA expression of mouse Zfta peaks on embryonic day (E) 17.5 in the wholemount of the lateral walls of the lateral ventricle. Zfta was expressed in the nuclei of FoxJ1-positive immature E1 (pre-E1) cells in E18.5 mouse embryonic brain. Interestingly, the transcription factors promoting ciliogenesis (ciliary TFs) (e.g., multicilin) and ZFTA-RELA upregulated luciferase activity using a 5' upstream sequence of ZFTA in cultured cells. Zftatm1/tm1 knock-in mice did not show developmental defects or abnormal fertility. In the Zftatm1/tm1 E1 cells, morphology, gene expression, ciliary beating frequency and ependymal flow were unaffected. These results suggest that Zfta is expressed in pre-E1 cells, possibly under the control of ciliary TFs, but is not essential for ependymal development or flow. This study sheds light on the mechanism of the ZFTA-RELA expression in the pathogenesis of ST-EPN-RELA: Ciliary TFs initiate ZFTA-RELA expression in pre-E1 cells, and ZFTA-RELA enhances its own expression using positive feedback.

Recognition of (1→3)-β-d-glucans (BGs) by invertebrate β-1,3-d-glucan recognition protein (BGRP) plays a significant role in the activation of Toll pathway and prophenoloxidase systems in insect host defense against fungal invasion. To examine the structure diversity of BGRPs for the recognition of physiochemically different BGs, the binding specificity of BGRPs cloned from four different insects to structure different BGs was characterized using ELISA. Recombinant BGRPs expressed as Fc-fusion proteins of human IgG1 bound to the solid phase of BGs. Based on the binding specificities, the BGRPs were categorized into two groups with different ultrastructures and binding characters; one group specifically binds BGs with triple-helical conformation, while the other group recognizes BGs with disordered conformations like single-helical or partially opened triple helix. The BGRPs from the silkworm and the Indian meal moth bound to the BGs with a triple-helical structure, whereas BGRPs from the red flour beetle and yellow mealworm beetle showed no binding to triple-helical BGs, but bound to alkaline-treated BGs that have a partially opened triple-helical conformation. This evidence suggests that the insect BGRPs can distinguish between different conformations of BGs and are equipped for determining the diversity of BG structures.

Hyperglycemia caused by excessive intake of sucrose leads to lifestyle-related diseases such as diabetes. Administration of a lactic acid bacterial strain to mice suppresses sucrose-induced hyperglycemia, but evidence for a similar effect in humans is lacking. Here we show that Enterococcus faecalis YM0831, identified using an in vivo screening system with silkworms, suppressed sucrose-induced hyperglycemia in humans. E. faecalis YM0831 also suppressed glucose-induced hyperglycemia in silkworms. E. faecalis YM0831 inhibited glucose uptake by the human intestinal epithelial cell line Caco-2. A transposon insertion mutant of E. faecalis YM0831, which showed decreased inhibitory activity against glucose uptake by Caco-2 cells, also exhibited decreased inhibitory activity against both sucrose-induced and glucose-induced hyperglycemia in silkworms. In human clinical trials, oral ingestion of E. faecalis YM0831 suppressed the increase in blood glucose in a sucrose tolerance test. These findings suggest that E. faecalis YM0831 inhibits intestinal glucose transport and suppresses sucrose-induced hyperglycemia in humans.

Using a silkworm evaluation system, we previously evaluated various substances that suppress postprandial hyperglycemia. Enterococcus faecalis YM0831, a lactic acid bacterium that inhibits glucose uptake by the human intestinal Caco-2 cell line, exhibited hyperglycemia-suppressing effects in the silkworm system. In the present study, we found that Kothala himbutu (Salacia reticulata) extract, a traditional medicine containing α-glucosidase inhibitors, suppressed sucrose-induced hyperglycemia in the silkworm system. Moreover, combined oral administration of lactic acid bacteria YM0831 with Kothala himbutu extract had stronger suppressive effects on sucrose-induced hyperglycemia than single administration of either component. These findings suggest that the silkworm system provides a simple way to evaluate the effects of supplements on the suppression of blood glucose level induced by sucrose ingestion.

Diabetes and obesity result from sucrose-induced hyperglycemia. Prevention of hyperglycemia contributes to inhibit the onset of these life-related diseases. Here we show that polysaccharides obtained from soil bacteria inhibit sucrose-induced hyperglycemia in an in vivo silkworm evaluation system. Ethanol precipitates of extracellular polysaccharides were prepared from viscous bacterial colonies. Among 24 samples obtained from different bacterial species, oral administration of 6 samples from Rhizobium altiplani, Cupriavidus sp., Paenibacillus polymyxa, Pantoea eucalypti, Variovorax boronicumulans, and Xanthomonas cynarae suppressed sucrose-induced hyperglycemia in silkworm insect larvae. The R. altiplani fraction treated further with DNase I, RNase A, and proteinase K, followed by phenol extraction also exhibited suppressive activity. Our results suggest that silkworms provide an efficient screening system of bacterial polysaccharides that inhibit sucrose-induced hyperglycemia.

Cyclodextrins (CyDs) are cyclic oligosaccharides consisting of six to eight glucose residues. Administration of α-CyD (six glucose residues) inhibits sucrose-induced hyperglycemia in humans. Here we show that oral administration of α-CyD and dimethyl α-CyD suppresses sucrose-induced hyperglycemia in an in vivo evaluation system using silkworms. On the other hand, β-CyD (seven glucose residues), γ-CyD (eight glucose residues), and their derivatives did not show the suppressive effect. These findings suggest that dimethyl α-CyD is a new inhibitor against sucrose-induced hyperglycemia and the silkworm system is useful for evaluation of suppressive activities of α-CyD derivatives against postprandial hyperglycemia.

ショ糖は、様々な食品に添加される代表的な甘味料である。しかし、ショ糖の過剰な摂取は血糖値の上昇を導き、肥満や糖尿病などの生活習慣病の発症や悪化を引き起こす。よって、ショ糖摂取による血糖値の上昇を抑制する物質の発見は、生活習慣病の予防法や治療法の開発に有用である。血糖値は、ショ糖の腸管内における分解、吸収のあと、全身の様々な臓器の働きにより調節される。そのため血糖値の上昇を阻害する物質の活性を評価するためには動物実験が必要となる。我々は、マウスなどの哺乳動物よりも費用や倫理的な問題が小さいカイコを動物モデルとした食後高血糖モデルを確立した。カイコにショ糖を加えた餌を与えると、体液中のグルコース濃度が速やかに上昇した。カイコの餌にα-グリコシダーゼ阻害剤であるアカルボースやボグリボースを添加するとショ糖を加えた餌の摂食による体液中のグルコース濃度の上昇は抑えられた。よって、カイコを用いて、ショ糖摂取後の血糖値の上昇を抑制する化合物が評価できることが明らかとなった。カイコは、ショ糖の摂食による高血糖を抑制する化合物や食品を探索するために有用な実験動物である。(著者抄録)

The silkworm infection model has the potential to replace conventional animal models for evaluation of the efficacy and toxicity of investigational antifungal agents. Silkworms are relatively inexpensive, can be simply grown in large numbers and can be easily infected with pathogenic fungi, including mutant strains. Antifungal agents can then be injected into the silkworm either via the hemolymph to mimic intravenous administration or directly into the gut for oral administration, and their antifungal effect can be evaluated. Common features regarding the mechanisms of pharmacokinetics between the silkworm and mammals result in consistent therapeutic effectiveness of antifungal agents. ASP2397, a promising new antifungal agent, was discovered using the silkworm model. The conclusion is that silkworms can be a more ethical and less expensive alternative to standard animal models, particularly for the identification and testing of new antifungal agents.

Leuconostoc carnosum #7-2, L. gelidum #4-2, and L. mesenteroides 8/11-3, which were isolated from fermented plant foods, are lactic acid bacteria. We previously reported that these bacteria are functional lactic acid bacteria whose innate immunity-stimulating activities are high based on a silkworm muscle contraction assay. The concentrations of these three lactic acid bacteria increased to more than 1 × 106 colony forming units (cfu)/mL in various vegetable and fruit juices when the pH values were appropriately adjusted. As the bacteria grew in the vegetable and fruit juices, the pH decreased and the concentrations of total sugars and glucose also decreased. These findings suggest that these functional lactic acid bacteria can be used to produce vegetable and fruit juices with reduced sugar levels, which is expected to be beneficial for human health.

We screened lactic acid bacteria that exhibited high innate immunity-stimulating activity by monitoring muscle contraction activity in silkworms. Heat-treated fractions of lactic acid bacteria, Leuconostoc carnosum #7-2, Leuconostoc gelidum #4-2, and Leuconostoc mesenteroides 8/11-3, had high (250-460 units/mg) innate immunity-stimulating activity. These lactic acid bacteria proliferated in milk to concentrations of 1 × 106 colony forming unit/mL. The present findings suggest that the silkworm muscle contraction assay is useful for screening lactic acid bacteria with high innate immunity-stimulating activity, and that the assay can be used for the production of fermented foods made from milk.

The development of effective drugs against fungal diseases involves performing infection experiments in animals to evaluate candidate therapeutic compounds. Cryptococcus neoformans is a pathogenic fungus that causes deep mycosis, resulting in respiratory illness and meningitis. Here we describe a silkworm system established to evaluate the safety and efficacy of therapeutic drugs against infection by Cryptococcus neoformans and the advantages of this system over other animal models. The silkworm assay system has two major advantages: 1) silkworms are less expensive to rear and their use is less problematic than that of mammals in terms of animal welfare, and 2) in vivo screenings for identifying candidate drugs can be easily performed using a large number of silkworms. The pharmacokinetics of compounds are consistent between silkworms and mammals. Moreover, the ED50 values of antibiotics are concordant between mammalian and silkworm infection models. Furthermore, the body size of silkworms makes them easy to handle in experimental procedures compared with other invertebrate infectious experimental systems, and accurate amounts of pathogens and chemicals can be injected fairly easily. These advantages of silkworms as a host animal make them useful for screening candidate drugs for cryptococcosis.

We propose Cryptococcus neoformans infection model using silkworm for understanding cryptococcosis and screening of therapeutically effective antibiotics. Silkworm is an insect whose rearing methods were established through a long history of the sericulture industry. Silkworm facilitates experiments using a large number of individuals because of low cost for rearing and few ethical problems caused by killing animals. Silkworm can be reared at 37˚C to perform infection experiments at same temperature to human body. Injection of accurate amounts of samples into hemolymph of silkworm by usual syringes is easy to be done since silkworm has an appropriate size to handle. Moreover two injection methods, injection into hemolymph and intestine, are distinguishable for silkworms. The former is correspondent to intravenous injection, and the latter is to oral administration in humans. Taking these advantages of silkworms as host animals, it is possible to evaluate the virulence factors in C. neoformans and the therapeutic efficacy of antifungal agents.

Candida albicans, Candida tropicalis, Candida grabrata, and Cryptococcus neoformans are causative pathogens of opportunistic diseases in immunocompromised human patients. Silkworms are killed by injection of these pathogenic fungi into their hemolymph. In this paper, we describe recent results by our laboratory and other researchers using gene-deficient strains of these pathogenic fungi. The silkworm is considered to be a useful model animal for understanding the pathogenicity of these fungi. Silkworms are also beneficial for evaluating therapeutically active anti-fungal reagents.

In drug development, the toxicity of candidate chemicals must be carefully examined in an animal model. Here we developed a live imaging technique using silkworms for a noninvasive toxicity test applicable for drug screening. Injection of carbon tetrachloride, a tissue-injuring chemical, into transgenic silkworms expressing green fluorescent protein (GFP) induced leakage of GFP from the tissues into the hemolymph. The leakage of GFP was suppressed by pre-administration of either cimetidine, a cytochrome P450 inhibitor, or N-acetyl cysteine, a free-radical scavenger. The transgenic silkworm was made transparent by feeding a diet containing chemicals that inhibit uric acid deposition in the epithelial cells. In the transparent silkworms, GFP fluorescence in the fat body could be observed from outside the body. Injection of salicylic acid or iron sulfate, tissue-injuring chemicals, into the transparent silkworms decreased the fluorescence intensity of the GFP in the fat body. These findings suggest that the transparent GFP-expressing silkworm model is useful for evaluating the toxicity of chemicals that induce tissue injury.

We previously reported that sugar levels in the silkworm hemolymph, i.e., blood, increase immediately (within 1 h) after intake of a high-glucose diet, and that the administration of human insulin decreases elevated hemolymph sugar levels in silkworms. In this hyperglycemic silkworm model, however, administration of pioglitazone or metformin, drugs used clinically for the treatment of type II diabetes, have no effect. Therefore, here we established a silkworm model of type II diabetes for the evaluation of anti-diabetic drugs such as pioglitazone and metformin. Silkworms fed a high-glucose diet over a long time-period (18 h) exhibited a hyperlipidemic phenotype. In these hyperlipidemic silkworms, phosphorylation of JNK, a stress-responsive protein kinase, was enhanced in the fat body, an organ that functionally resembles the mammalian liver and adipose tissue. Fat bodies isolated from hyperlipidemic silkworms exhibited decreased sensitivity to human insulin. The hyperlipidemic silkworms have impaired glucose tolerance, characterized by high fasting hemolymph sugar levels and higher hemolymph sugar levels in a glucose tolerance test. Administration of pioglitazone or metformin improved the glucose tolerance of the hyperlipidemic silkworms. These findings suggest that the hyperlipidemic silkworms are useful for evaluating the hypoglycemic activities of candidate drugs against type II diabetes.

Staphylococcus aureus Newman strain and several methicillin-resistant S. aureus (MRSA) clinical isolates were grown on agar plates prepared with conventional lots of tryptic soy broth (TSB). Cell growth of these strains was inhibited on agar plates containing TSB of a particular product lot (lot A), whereas the cell growth of S. aureus RN4220 strain and several other MRSA clinical isolates was not inhibited. The cell growth of a strain of S. epidermidis was also inhibited on agar plates containing TSB of lot A, whereas the cell growth of Bacillus subtilis, Lactococcus lactis, Klebsiella pneumonia, Salmonella enterica, Serratia marcescens, Pseudomonas aeruginosa, and Escherichia coli was not inhibited. Although cell growth of the Newman strain was inhibited on agar plates containing TSB of lot A that was autoclaved in stainless steel or glass containers, cell growth inhibition was not observed when the medium was autoclaved in polypropylene containers. Compounds that inhibited the cell growth of the Newman strain were extracted from a polypropylene tube that was preincubated with liquid medium prepared from TSB of lot A. These findings suggest that polypropylene-binding compounds in TSB of lot A inhibited the cell growth of S. aureus Newman strain, some MRSA clinical isolates, and S. epidermidis.

We established a transgenic silkworm strain expressing the human insulin receptor (hIR) using the GAL4/UAS system. Administration of human insulin to transgenic silkworms expressing hIR decreased hemolymph sugar levels and facilitated Akt phosphorylation in the fat body. The decrease in hemolymph sugar levels induced by injection of human insulin in the transgenic silkworms expressing hIR was blocked by co-injection of wortmannin, a phosphoinositide 3-kinase inhibitor. Administration of bovine insulin, an hIR ligand, also effectively decreased sugar levels in the transgenic silkworms. These findings indicate that functional hIRs that respond to human insulin were successfully induced in the transgenic silkworms. We propose that the humanized silkworm expressing hIR is useful for in vivo evaluation of the therapeutic activities of insulin receptor agonists.