ヒト初乳由来分泌型IgAはボツリヌスB型16S毒素と結合し，毒素のT84細胞への結合を抑制する

Botulinum neurotoxin produced by Clostridium botulinum type B is in the form of a complex of 12S and 16S toxins. Food-borne botulism is caused by these complex toxins which are ingested orally and absorbed from the digestive tract. Here, we show that the human milk SIgA binds to the type B16S toxin. The binding of SIgA to 16S toxin and HA was inhibited by carbohydrates such as galactose, suggesting that the interaction of carbohydrate side chain of the SIgA with the HA of the 16S toxin is important for SIgA-16S complex formation. We also demonstrate that SIgA inhibits the attachment of 16S toxin to intestinal epithelial cells. These data suggest that the interaction of antigen nonspecific SIgA with 16S toxin has a large influence on the absorption of 16S toxin from the intestinal epithelium, and that SIgA may provide insight into developing a therapeutic agent for type B food-borne botulism

Multivalency effects of hemagglutinin component of type B botulinum neurotoxin complex on epithelial barrier disruption

金沢大学医薬保健研究域医学系Hemagglutinin (HA) is one of the components of botulinum neurotoxin (BoNT) complexes and it promotes the absorption of BoNT through the intestinal epithelium by at least two specific mechanisms: cell surface attachment by carbohydrate binding, and epithelial barrier disruption by E-cadherin binding. It is known that HA forms a three-arm structure, in which each of three protomers has three carbohydrate-binding sites and one E-cadherin-binding site. A three-arm form of HA is considered to bind to these ligands simultaneously. In the present study, we investigated how the multivalency effect of HA influences its barrier-disrupting activity. We prepared type B full-length HA (three-arm form) and mini-HA, which is a deletion mutant lacking the trimer-forming domain. Size-exclusion chromatography analysis showed that mini-HA exists as dimers (two-arm form) and monomers (one-arm form), which are then separated. We examined the multivalency effect of HA on the barrier-disrupting activity, the E-cadherin-binding activity, and the attachment activity to the basolateral cell surface. Our results showed that HA initially attaches to the basal surface of Caco-2 cells by carbohydrate binding and then moves to the lateral cell surface, where the HA acts to disrupt the epithelial barrier. Our results showed that the multivalency effect of HA enhances the barrier-disrupting activity in Caco-2 cells. We found that basal cell surface attachment and binding ability to immobilized E-cadherin were enhanced by the multivalency effect of HA. These results suggest that at least these two factors induced by the multivalency effect of HA cause the enhancement of the barrier-disrupting activity. © 2017 The Societies and John Wiley & Sons Australia, LtdEmbargo Period 12 month

Botulinum hemagglutinin disrupts the intercellular epithelial barrier by directly binding E-cadherin

Botulinum neurotoxin's nontoxic HA protein binds E-cadherin to disrupt cell–cell adhesion in a species-specific manner

Collagen adhesion gene is associated with blood stream infections caused by methicillin-resistant Staphylococcus aureus

Objectives: Methicillin-resistant Staphylococcus aureus (MRSA) causes hospital- and community-acquired infections. It is not clear whether genetic characteristics of the bacteria contribute to disease pathogenesis in MRSA infection. We hypothesized that whole genome analysis of MRSA strains could reveal the key gene loci and/or the gene mutations that affect clinical manifestations of MRSA infection. Methods: Whole genome sequences (WGS) of MRSA of 154 strains were analyzed with respect to clinical manifestations and data. Further, we evaluated the association between clinical manifestations in MRSA infection and genomic information. Results: WGS revealed gene mutations that correlated with clinical manifestations of MRSA infection. Moreover, 12 mutations were selected as important mutations by Random Forest analysis. Cluster analysis revealed strains associated with a high frequency of bloodstream infection (BSI). Twenty seven out of 34 strains in this cluster caused BSI. These strains were all positive for collagen adhesion gene (cna) and have mutations in the locus, those were selected by Random Forest analysis. Univariate and multivariate analysis revealed that these gene mutations were the predictor for the incidence of BSI. Interestingly, mutant CNA protein showed lower attachment ability to collagen, suggesting that the mutant protein might contribute to the dissemination of bacteria. Conclusions: These findings suggest that the bacterial genotype affects the clinical characteristics of MRSA infection. (c) 2019 The Author(s). Published by Elsevier Ltd on behalf of International Society for Infectious Diseases

Age, gender, will, and use of home-visit nursing care are critical factors in home care for malignant diseases; a retrospective study involving 346 patients in Japan

<p>Abstract</p> <p>Background</p> <p>We aimed to clarify the factors affecting outcomes of home care for patients with malignant diseases.</p> <p>Methods</p> <p>Of 607 patients who were treated in 10 clinics specialized in home care between January and December 2007 at Chiba, Fukuoka, Iwate, Kagoshima, Tochigi and Tokyo prefectures across Japan, 346 (57%; 145 men and 201 women) had malignant diseases. We collected information on medical and social backgrounds, details of home care, and its outcomes based on their medical records.</p> <p>Results</p> <p>Median age of the patients was 77 years (range, 11-102), and 335 patients were economically self-sufficient. Their general condition was poor; advanced cancer (n = 308), performance status of 3-4 (n = 261), and dementia (n = 121). At the beginning of home care, 143 patients and 174 family members expressed their wish to die at home. All the patients received supportive treatments including fluid replacement and oxygenation. Median duration of home care was 47 days (range, 0-2,712). 224 patients died at home. For the remaining 122, home care was terminated due to complications (n = 109), change of attending physicians (n = 8), and others (n = 5). The factors which inhibited the continuity of home care were the non-use of home-visit nursing care (hazard ratio [HR] = 1.78, 95% confidence interval [CI]: 1.05-3.00, <it>p </it>= 0.03), the fact that the patients themselves do not wish to die at home (HR = 1.83, CI: 1.09-3.07, <it>p </it>= 0.02), women (HR = 1.81, CI: 1.11-2.94, <it>p </it>= 0.02), and age (HR = 0.98, CI: 0.97-1.00, <it>p </it>= 0.02).</p> <p>Conclusions</p> <p>Continuation of home care is influenced by patients' age, gender, will, and use of home-visit nursing.</p

ボツリヌス毒素の体内侵入機構を利用した粘膜ワクチンの開発

金沢大学医薬保健研究域医学系我々はこれまでに血清型A型ボツリヌス毒素が生体防御を担う細胞である腸管M細胞の機能を利用することにより体内に侵入し、中毒を引き起こすことを明らかにした。本研究では、他の血清型(B型)毒素との比較解析から腸管吸収に寄与する因子を同定し、各血清型の吸収機構を利用した新しい薬物送達システム・粘膜ワクチンの開発を目指す。B型毒素複合体はマウスにおいてA型と比較して数十倍経口毒性が高く腸管内局在が異なることから、腸管吸収機構が異なることが示唆される。我々はこの吸収機構の違いに関与する候補因子としてムチン層に着目し、毒素複合体中のHAとムチンとの相互作用の違いが毒素の吸収に影響していることを明らかにした。We previously reported that type A BoNT complex (BoNT and non-toxic component) exploits intestinal microfold (M) cells to breach the intestinal epithelial barrier. In this study, we analyzed the intestinal absorption mechanism of other serotype BoNT (type B) complexes, and aimed to develop a novel delivery system for vaccines and drugs using the invasion mechanism of BoNT. Type B BoNT complex showed potent oral toxicity and different intestinal localization in mice compared with type A, suggesting that type B BoNT has a different intestinal absorption mechanism. We focused on the mucin layer as a possible factor leading to the different intestinal absorption mechanism, and analyzed the interactions of type A and type B BoNT complexes with mucin. As the result, it was found that a difference in the interaction of hemagglutinin (HA) in the toxin complex with mucin affects the intestinal absorption of BoNT.研究課題/領域番号:18K07107, 研究期間(年度):2018-04-01 – 2021-03-31出典：「ボツリヌス毒素の体内侵入機構を利用した粘膜ワクチンの開発」研究成果報告書　課題番号18K07107（KAKEN：科学研究費助成事業データベース（国立情報学研究所）） （https://kaken.nii.ac.jp/report/KAKENHI-PROJECT-18K07107/18K07107seika/）を加工して作

ボツリヌス神経毒素複合体と腸管免疫系細胞との相互作用の解析

金沢大学医薬保健研究域医学系ボツリヌス食中毒はボツリヌス神経毒素複合体を経口的に摂取することにより発症する。我々はこれまでに、中毒発症に必須な過程であるにも関わらず不明であった血清型A1型神経毒素複合体の腸管吸収機構を明らかにした。本研究において、我々は血清型B1型神経毒素複合体がA1型とは異なる機構で腸管から宿主内へ侵入することを発見した。さらにA1型、B1型いずれの神経毒素複合体も腸管上皮直下のsub-epithelial dome (SED)において免疫細胞と共局在していた。これらの結果から、腸管免疫細胞はA1型およびB1型神経毒素の血中への移行に影響しており、ボツリヌス食中毒の発症に関与していることが示唆された。Food-borne botulism is caused by ingestion of botulinum neurotoxin (BoNT) complexes which composed of BoNT and one or more neurotoxin-associated proteins (NAPs). We have uncovered the site(s) and mechanism of intestinal absorption of type A1 BoNT complex, heretofore largely unknown, which is essential for the onset of food-borne botulism.In this study, we found that type B1 BoNT complex invades into the host from intestine by different mechanisms from type A1. Furthermore, we observed that both type A and type B BoNT complex interact with immune cells which are located in sub-epithelial dome (SED). These results suggest that intestinal immune cells influence the entry of type A1 or type B1 BoNT into the blood stream and are involved in the onset of food-borne botulism.研究課題/領域番号:16K19123, 研究期間(年度):2016-04-01 - 2018-03-3

腸管上皮バリアを破壊するボツリヌスHAの基質分子の探索及び作用機構の解析

本研究では、ボツリヌス神経毒素複合体中の無毒成分の一つであるHemagglutinin(HA)の持つ腸管上皮細胞間バリア破壊作用について解析した。HAのバリア破壊作用を引き起こす基質分子の探索において、Recombinant HAを用いた解析により、ヒト腸管上皮細胞からHAと特異的に結合する標的分子が得られた。次にHAの作用メカニズムを解析する目的で、生体内における動態を解析した結果、HAは特異的な細胞から取り込まれ、その細胞からバリア破壊を引き起こすことが明らかとなった。In this study, we analyzed the mechanism of disruption of epithelial paracellular barrier function caused by botulinum HA. Using recombinant HA, we purified HA-binding proteins from human intestinal epithelial cell lines. Next, we analyzed the localization of botulinum toxins in vivo . The HA bound and transcytosed to the basolateral side and disrupted the epithelial barrier at the specific cells

Functional dissection of the Clostridium botulinum type B hemagglutinin complex: identification of the carbohydrate and E-cadherin binding sites.

Botulinum neurotoxin (BoNT) inhibits neurotransmitter release in motor nerve endings, causing botulism, a condition often resulting from ingestion of the toxin or toxin-producing bacteria. BoNTs are always produced as large protein complexes by associating with a non-toxic protein, non-toxic non-hemagglutinin (NTNH), and some toxin complexes contain another non-toxic protein, hemagglutinin (HA), in addition to NTNH. These accessory proteins are known to increase the oral toxicity of the toxin dramatically. NTNH has a protective role against the harsh conditions in the digestive tract, while HA is considered to facilitate intestinal absorption of the toxin by intestinal binding and disruption of the epithelial barrier. Two specific activities of HA, carbohydrate and E-cadherin binding, appear to be involved in these processes; however, the exact roles of these activities in the pathogenesis of botulism remain unclear. The toxin is conventionally divided into seven serotypes, designated A through G. In this study, we identified the amino acid residues critical for carbohydrate and E-cadherin binding in serotype B HA. We constructed mutants defective in each of these two activities and examined the relationship of these activities using an in vitro intestinal cell culture model. Our results show that the carbohydrate and E-cadherin binding activities are functionally and structurally independent. Carbohydrate binding potentiates the epithelial barrier-disrupting activity by enhancing cell surface binding, while E-cadherin binding is essential for the barrier disruption