Alliinase from Ensifer adhaerens and Its Use for Generation of Fungicidal Activity

A bacterium Ensifer adhaerens FERM P-19486 with the ability of alliinase production was isolated from a soil sample. The enzyme was purified for characterization of its general properties and evaluation of its application in on-site production of allicin-dependent fungicidal activity. The bacterial alliinase was purified 300-fold from a cell-free extract, giving rise to a homogenous protein band on polyacrylamide gel electrophoresis. The bacterial alliinase (96 kDa) consisted of two identical subunits (48 kDa), and was most active at 60°C and at pH 8.0. The enzyme stoichiometrically converted (-)-alliin ((-)-S-allyl-L-cysteine sulfoxide) to form allicin, pyruvic acid, and ammonia more selectively than (+)-alliin, a naturally occurring substrate for plant alliinase ever known. The C-S lyase activity was also detected with this bacterial enzyme when S-alkyl-L-cysteine was used as a substrate, though such a lyase activity is absolutely absent in alliinase of plant origin. The enzyme generated a fungicidal activity against Saccharomyces cerevisiae in a time- and a dose-dependent fashion using alliin as a stable precursor. Alliinase of Ensifer adhaerens FERM P-19486 is the enzyme with a novel type of substrate specificity, and thus considered to be beneficial when used in combination with garlic enzyme with respect to absolute conversion of (±)-alliin to allicin

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

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

宿主消化管内におけるボツリヌス毒素の動態解明

ボツリヌス菌が産生するボツリヌス神経毒素(BoNT)は、重篤な食中毒である食餌性ボツリヌス症を引き起こす。BoNTは無毒タンパク質(NAPs）と複合体を形成し、NAPの機能によって宿主腸管から効率よく吸収される。本複合体はpH 7.0以上の緩衝液中でBoNTとNAPsに解離することから、弱アルカリ性である宿主腸管でも解離すると考えられてきた。本研究では、宿主腸管に存在する宿主由来因子が、弱アルカリ性環境下で本複合体の解離を阻害することを明らかにした。このことは、宿主腸管内でも本毒素複合体が解離しないことを示唆しており、本研究によってボツリヌス食中毒の発症機構における重要な知見が得られた。研究課題/領域番号:26860287, 研究期間(年度):2014-04-01 - 2017-03-

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

HA/B complex binds to mucins through carbohydrate recognition by HA1/B and HA3/B.

<p>(A) Mucin binding of GST-HA1/B. Mucin binding of GST, GST-HA1/B, and GST-HA1/B harboring Asn286 to Ala mutation (N286A) was assessed as described in Materials and Methods. Binding of GST-HA1/B was measured in the presence or absence of 100 mM glucose (Glc) or galactose (Gal). HA1/B preferentially bound to PGM in a galactose-binding-dependent manner. (B) Mucin binding of GST, GST-HA3/B, and GST-HA3/B harboring Arg528 to Ala mutation (R528A). Binding of GST-HA3/B was assessed with the coated mucin pretreated with (+Neu) or without 5 mU/ml neuraminidase. HA3/B preferentially bound to BSM in a sialic-acid-dependent manner. (C) Mucin binding of HA/B complex (WT) and those harboring either HA1/B N286A or HA3/B R528A, or both (HA/B harboring HA1 N286A mutation, HA1 m; HA/B harboring HA3 R528A mutation, HA3 m; HA/B harboring HA1N286A and HA3 R528A mutations, HA1 m/HA3 m). The results show that carbohydrate binding activities of HA1 and HA3 are fully functional in the HA/B complex. Values are means ± S.E.M. from three independent experiments. *<i>p</i><0.05, unpaired <i>t</i> test.</p

Crystal structure and the carbohydrate and E-cadherin binding sites of the HA/B complex.

<p>The galactose binding sites of HA1 (Asn286) are colored blue and marked with dotted blue circles. The sialic acid-binding sites of HA3 (Arg528) are colored magenta and marked with dotted magenta circles. The E-cadherin binding sites of HA3 (Leu473, M508, and Lys607) are colored red and marked with dotted red circles. These sites are structurally independent. Each of the three HA monomers is colored pale cyan, pale yellow, or pink.</p

Carbohydrate binding of HA/B is not required for E-cadherin binding.

<p>(A) Caco-2 cells were grown on Transwell chambers, and treated with 10 nM HA/B (WT) or those harboring carbohydrate-binding-defective mutations (HA/B harboring HA1 N286A mutation, HA1 m; HA/B harboring HA3 R528A mutation, HA3 m; HA/B harboring HA1 N286A and HA3 R528A mutations, HA1 m/HA3 m) from the lower side of the chambers. HA1 mutation affected the barrier-disrupting activity, whereas HA3 mutation largely did not. (B) Caco-2 cell monolayers were treated with 10 nM HA/B or the indicated concentrations of HA/B carbohydrate-binding-defective mutant (HA1m/HA3m). These HAs were treated with the lower side of the chamber. Values are means ± S.E.M. of triplicate wells. The mutant showed sufficient activity to reduce the TER value completely when applied at higher concentrations (50 nM and 100 nM). (C) Caco-2 cells were treated the same as in A. After 1 h of treatment, cells were fixed and the basolateral surface of the cell was stained with the anti-B16S antibody. E-cadherin staining is also indicated. Binding to the basolateral surface of the cells was reduced in the carbohydrate-binding-defective mutant. Scale bar: 50 µm. (D) Varying concentrations of the recombinant E-cadherin ectodomain protein (300, 100, and 30 nM) was pulled-down with resin-bound HA/B complex or its carbohydrate-binding-defective mutant. The mutations did not affect E-cadherin binding.</p

Fully Human Monoclonal Antibodies Effectively Neutralizing Botulinum Neurotoxin Serotype B

Botulinum neurotoxin (BoNT) is the most potent natural toxin known. Of the seven BoNT serotypes (A to G), types A, B, E, and F cause human botulism. Treatment of human botulism requires the development of effective toxin-neutralizing antibodies without side effects such as serum sickness and anaphylaxis. In this study, we generated fully human monoclonal antibodies (HuMAbs) against serotype B BoNT (BoNT/B1) using a murine&ndash;human chimera fusion partner cell line named SPYMEG. Of these HuMAbs, M2, which specifically binds to the light chain of BoNT/B1, showed neutralization activity in a mouse bioassay (approximately 10 i.p. LD50/100 &micro;g of antibody), and M4, which binds to the C-terminal of heavy chain, showed partial protection. The combination of two HuMAbs, M2 (1.25 &micro;g) and M4 (1.25 &micro;g), was able to completely neutralize BoNT/B1 (80 i.p. LD50) with a potency greater than 80 i.p. LD50/2.5 &micro;g of antibodies, and was effective both prophylactically and therapeutically in the mouse model of botulism. Moreover, this combination showed broad neutralization activity against three type B subtypes, namely BoNT/B1, BoNT/B2, and BoNT/B6. These data demonstrate that the combination of M2 and M4 is promising in terms of a foundation for new human therapeutics for BoNT/B intoxication