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Clostridium argentinense produces botulinum neurotoxin type G (BoNT/G). We sequenced and analyzed the plasmid harboring the bont/G gene, designated pCAG, in C. argentinense strain 2740. The pCAG consisted of 140,070 bp containing the bont/G gene cluster. Although this gene cluster showed high similarities in its DNA sequence and ORF arrangement to those of other bont gene clusters, the other regions of the plasmid did not. A phylogenetic study suggested that pCAG had a unique evolutionary history compared with other clostridial bont-harboring plasmids. This suggests that pCAG is possibly a novel type of plasmid expressing the bont/G gene in C. argentinense.

In Clostridium botulinum, the characteristics of type C and D strains are quite different from other types, and they are classified as group III. They produce C2 binary toxin and C3 exoenzyme in addition to type C and D neurotoxins. Two different phages and many plasmids are identified in the organisms. The genes of neurotoxin and C3 exoenzyme are converted from toxigenic strains to non-toxigenic strains by the specific bacteriophages (phages), whereas, the C2 toxin gene is carried by large or small plasmids. Classification of type C and D strains has been in confusion because 1) antigenicity of type C and D neurotoxins is complex, 2) the cells produce two types of toxins, neurotoxin and C2 toxin, and 3) some non-toxigenic strains can be converted to produce C or D neurotoxin by the infection with phages. Until now, entire nucleotide sequences of cell chromosomes, phages, and plasmids have been determined. Since both genetic and protein-chemical analyses have been clarifying the above confusions, these data are reviewed historically.

This study aims to evaluate the effect of hyperimmune immunoglobulin Y (IgY) against human rotavirus (HRV) among pediatric patients receiving standard supportive treatment for rotavirus-associated diarrhea mostly with an enteric non-cholera co-pathogen in a hospital setting. Two natural HRV reassortant clinical strains ATCC VR 2273 and ATCC VR 2274 were used as mixed immunizing antigens in poultry hens to generate anti-HRV IgY (Rotamix IgY). The Rotamix IgY was used in laboratory and clinical studies against control or placebo IgY. The control or placebo IgY was prepared using tissue culture medium from mock-infected MA104 cell line as antigen for poultry immunization. In vitro, Rotamix IgY exhibited multi-serotypic cross neutralization activities along with synergistic effects against major global serotypes G1, G2, G3, G4 and other human or animal rotavirus strains when compared with mono-specific IgY. Suckling mice (ICR strain) pre-treated orally once with Rotamix IgY and then challenged with rotavirus 3h later showed a significant dose-dependent reduction in frequency (p0.05) and duration (p0.05) of diarrhea compared to placebo IgY-treated mice. Out of 114 children aged between 3 and 14 months and with diarrhea upon admission in a Myanmar hospital, 54 dehydrated and rotavirus-positive children were randomized into Rotamix IgY group and placebo IgY group. Of these, only 52 children had complete data with n=26 children per study group. Ninety-two percent of patients in each of these groups were positive for co-infecting enteric non-cholera pathogen and all patients received standard supportive therapy for diarrhea. The patients were monitored for volume and duration of oral rehydration fluid (ORF) and intravenous fluid (IVF) intake, daily stool frequency and overall duration of diarrhea, and frequency and duration of rotavirus shedding. Compared to placebo IgY group, the Rotamix IgY group had statistically significant reduction in mean ORF intake (p=0.004), mean duration of intravenous fluid administration (p=0.03), mean duration of diarrhea from day of admission (p0.01) and mean duration of rotavirus clearance from stool from day of admission (p=0.05). Overall, our novel approach using oral Rotamix IgY for rotavirus-infected children mostly with non-cholera enteric pathogen co-infection appears to be a promising, safe and effective adjunct to management of acute diarrhea in pediatric patients.

A critical role in internalizing the Clostridium botulinum neurotoxin into gastrointestinal cells is played by nontoxic components complexed with the toxin. One of the components, a β-trefoil lectin has been known as HA33 or HA1. The HA33 from C. botulinum type A (HA33/A) has been predicted to have a single sugar-binding site, while type C HA33 (HA33/C) has two sites. Here we constructed HA33/C mutants and evaluated the binding capacities of the individual sites through mucin-assay and isothermal titration calorimetry. The mutant W176A (site I knockout) had a K d value of 31.5 mM for galactose (Gal) and 61.3 mM for N -acetylgalactosamine (GalNAc), while the K d value for N -acetylneuraminic acid (Neu5Ac) was too high to be determined. In contrast, the double mutant N278A/Q279A (site II knockout) had a K d value of 11.8 mM for Neu5Ac. We also determined the crystal structures of wild-type and the F179I mutant in complex with GalNAc at site II. The results suggest that site I of HA33/C is quite unique in that it mainly recognizes Neu5Ac, and site II seems less important for the lectin specificity. The architectures and the properties of the sugar-binding sites of HA33/C and HA33/A were shown to be drastically different.

Helicobacter pylori infection causes atrophic gastritis, peptic ulcer, and gastric cancer. The host immune response plays an important role in the pathogenesis of H. pylori-related diseases. Heat shock proteins are antigens involved in various diseases. This study evaluated seropositivity for antibodies to H. pylori heat shock protein 60 in patients with gastric cancer. Serum samples were obtained from 57 patients with gastric cancer (25 patients with diffuse-type gastric cancer and 32 with intestinal-type gastric cancer), 45 H. pylori-positive patients, and 49 H. pylori-negative patients without gastric cancer. Antibodies to heat shock protein 60 and H. pylori were assessed by enzyme-linked immunosorbent assay. The positivity rate for antibodies to hsp60 was significantly higher in H. pylori-positive patients than in H. pylori-negative patients (73.3% vs. 24.5%, p

Objectives To investigate the efficacy of a newly purified neurotoxin (NTX) in male rat prostates. Several reports have suggested that intraprostatic injection of botulinum toxin type A has demonstrated efficacy against symptomatic benign prostatic hyperplasia. NTXs associate with nontoxic components and form large complexes designated "progenitor toxins." In general, progenitor toxins are used, because they are easily obtained and are more stable than NTXs. However, they have side effects for some patients in whom anti-progenitor toxins, including anti-NTX antibodies, are produced after several injections. We purified NTXs without their nontoxic components using a simple procedure. Methods Adult male Sprague-Dawley rats were injected with 5 U of NTX or saline into their prostates, which were harvested and weighed after 1 or 4 weeks. The effects of the NTX on the prostate were histologically and immunohistochemically studied using hematoxylin-eosin, synaptophysin, and terminal deoxynucleotidyl-mediated deoxyuridine triphosphate nick end labeling staining. Results In the NTX-treated rats, the prostate weight was reduced and atrophy and diffuse apoptosis were observed. Moreover, synaptophysin-positive cells in the epithelium were decreased after NTX injection. Conclusions Intraprostatic NTX injection induces prostate apoptosis and atrophic change in the rat prostate. These results suggest that NTX injection might be a promising material for treatment of patients with benign prostatic hyperplasia.

The Clostridium botulinum type C 16S progenitor toxin contains a neurotoxin and several nontoxic components, designated nontoxic nonhemagglutinin (HA), HA1 (HA-33), HA2 (HA-17), HA3a (HA-22–23), and HA3b (HA-53). The HA3b subcomponent seems to play an important role cooperatively with HA1 in the internalization of the toxin by gastrointestinal epithelial cells via binding of these subcomponents to specific oligosaccharides. In this study, we investigated the sugar-binding specificity of the HA3b subcomponent using recombinant protein fused to glutathione S -transferase and determined the three-dimensional structure of the HA3a–HA3b complex based on X-ray crystallography. The crystal structure was determined at a resolution of 2.6 A. HA3b contains three domains, domains I to III, and the structure of domain I resembles HA3a. In crystal packing, three HA3a–HA3b molecules are assembled to form a three-leaved propeller-like structure. The three HA3b domain I and three HA3a alternate, forming a trimer of dimers. In a database search, no proteins with high structural homology to any of the domains (Z score > 10) were found. Especially, HA3a and HA3b domain I, mainly composed of β-sheets, reveal a unique fold. In binding assays, HA3b bound sialic acid with high affinity, but did not bind galactose, N -acetylgalactosamine, or N -acetylglucosamine. The electron density of liganded N -acetylneuraminic acid was determined by crystal soaking. In the sugar-complex structure, the N -acetylneuraminic acid-binding site was located in the cleft formed between domains II and III of HA3b. This report provides the first determination of the three-dimensional structure of the HA3a–HA3b complex and its sialic acid binding site. Our results will provide useful information for elucidating the mechanism of assembly of the C16S toxin and for understanding the interactions with oligosaccharides on epithelial cells and internalization of the botulinum toxin complex.

Immune responses against heat shock protein 60 (HSP60) of pathogen-origin are thought to be defensive events which, due to molecular mimicry, misdirect to a human counterpart. Therefore, atherosclerosis may be serologically predicted by anti-HSP60 antibodies (Abs). In the present study, we analyzed the clinical prevalence of the serum IgG Abs against Helicobacter pylori (Hp)-derived HSP60 (Hp-HSP60) or its peptide fragments in patients with cardiovascular disease (CVD; n = 250), as compared to those in age- and gender-matched non-CVD patients ( n = 293). Anti-Hp cell lysate Abs frequently appeared in Hp-infected patients who were not associated with CVD. In contrast, Abs against the particular amino acid sequence Hp-HSP60 II3 (II3 region, Glu 141 –Leu 160 , in Hp-HSP60) predominantly appeared in CVD patients, as well as IgG anti-human HSP60 (Hu-HSP60 w ). Furthermore, neither titer of anti-Hp-HSP60 II3 nor anti-Hu-HSP60 w Abs was correlated with the levels of high sensitivity C-reactive protein (hsCRP). This data strongly suggested that IgG anti-Hp-HSP60 II3 Abs cross-reacted with Hu-HSP60 w were independent diagnostic markers relevant to CVD. Further, the 20 amino acid residues (Glu 141 –Leu 160 ) might be predominant CVD-associated epitopes that induce anti-Hu-HSP60 auto-Abs, whose location was predicted in the tertiary structure of Hu-HSP60.

It has been reported that Clostridium botulinum type C 16S progenitor toxin (C16S toxin) first binds to the sialic acid on the cell surface of mucin before invading cells [A. Nishikawa, N. Uotsu, H. Arimitsu, J.C. Lee, Y. Miura, Y. Fujinaga, H. Nakada, T. Watanabe, T. Ohyama, Y. Sakano, K. Oguma, The receptor and transporter for internalization of Clostridium botulinum type C progenitor toxin into HT-29 cells, Biochem. Biophys. Res. Commun. 319 (2004) 327–333]. In this study we investigated the binding properties of the C16S toxin to glycoproteins. Although the toxin bound to membrane blotted mucin derived from the bovine submaxillary gland (BSM), which contains a lot of sialyl oligosaccharides, it did not bind to neuraminidase-treated BSM. The binding of the toxin to BSM was inhibited by N -acetylneuraminic acid, N -glycolylneuraminic acid, and sialyl oligosaccharides strongly, but was not inhibited by neutral oligosaccharides. Both sialyl α2–3 lactose and sialyl α2–6 lactose prevented binding similarly. On the other hand, the toxin also bound well to porcine gastric mucin. In this case, neutral oligosaccharides might play an important role as ligand, since galactose and lactose inhibited binding. These results suggest that the toxin is capable of recognizing a wide variety of oligosaccharide structures.

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.

A mutant of Escherichia coli enterotoxin induces specific Thl-type T cells to varicella-zoster vaccine. The mutant increased IL-12p40, TNFalpha and nitric oxide production by IFNgamma-stimulated bone marrow macrophages but cholera toxin did not. Anti-TNFalpha antibodies blocked its stimulation of IL-12p40 production but iNOS inhibitor did not. IL-12p40 and IL-12p35 production was stimulated at the level of mRNA formation by the mutant. Cholera toxin suppressed IL-12beta1 expression by spleen T cells stimulated with anti-CD3 antibodies but the mutant did not. These findings indicate that the mutant may induce Thl-type response to the vaccine through its IL-12 and TNFalpha induction by macrophages.

The bacterium Clostridium botulinum type C produces a progenitor toxin (C16S toxin) that binds to O-linked sugar chains terminating with sialic acid on the surface of HT-29 cells prior to internalization [A. Nishikawa, N. Uotsu, H. Arimitsu, J.C. Lee, Y. Miura, Y. Fujinaga, H. Nakada, T. Watanabe, T. Ohyama, Y. Sakano, K. Oguma, Biochem. Biophys. Res. Commun. 319 (2004) 327–333] [21]. Based on this, it was hypothesized that the C16S toxin is internalized via clathrin-coated pits. To examine this possibility, the internalized toxin was observed with a fluorescent antibody using confocal laser-scanning microscopy. The confocal images clearly indicated that the C16S toxin was internalized mainly via clathrin-coated pits and localized in early endosomes. The toxin was colocalized with caveolin-1 which is one of the components of caveolae, however, implying the toxin was also internalized via caveolae. The confocal images also showed that the neurotoxin transported to the endosome was transferred to the Golgi apparatus. However, the non-toxic components were not merged with the Golgi marker protein, TGN38, implying the neurotoxin was dissociated from progenitor toxin in endosomes. These results suggested that the C16S toxin was separated to the neurotoxin and other proteins in endosome and the neurotoxin was further transferred to the Golgi apparatus which is the center for protein sorting.

Orally ingested botulinum toxin enters the circulatory system and eventually reaches the peripheral nerves, where it elicits a response of neurological dysfunction. In this study, we report the important findings concerning the mechanism of Clostridium botulinum type C progenitor toxin (C16S) endocytic mechanism. C16S toxin bound to high molecular weight proteins on the surface of human colon carcinoma HT-29 cells and was internalized, but not if the cells were pretreated with neuraminidase. Benzyl-GalNAc which inhibited O-glycosylation of glycoproteins also interfered in the toxin's ability to bind the cell surface. On the other hand, the toxin was internalized in spite of pretreatment of the cells with PPMP, an inhibitor of ganglioside synthesis. These results suggest that the glycoproteins, like mucin, fulfill the important roles of receptor and transporter of C16S toxin.

The nontoxic–nonhemagglutinin (NTNHA) component, in both isolated form and the neurotoxin (NT)/NTNHA complexed form, was prepared protease-free from toxin complexes produced by Clostridium botulinum type D strain 4947. NTNHA in both preparations was found to be spontaneously converted to the nicked NTNHA form leading to 15- and 115-kDa fragments with the excision of several amino acid residues at specific sites on SDS–PAGE during long-term incubation, while that of the NT/NTNHA/hemagglutinin complexed form remained unnicked single-chain polypeptides under the same conditions. Considering that the NTNHA preparation contained small amounts of the nicked form of NTNHA and the addition of trypsin accelerated the cleavage, it is speculated that a nicked form of NTNHA remaining after the purification and/or NTNHA itself catalyzes the cleavage of intact NTNHA.

Using SDS-PAGE, we found that one subcomponent, hemagglutinin (HA-33), from the Clostridium botulinum progenitor toxin of type D strain 1873 and type C strain Yoichi had slightly smaller molecular sizes than those of type C and D reference strains, but other components did not. Based on N- and C-terminal sequence analyses of HA-33, a deletion of 31 amino acid residues from the C-terminus at a specific site was observed in the HA-33 proteins of both strains. The progenitor toxins from both strains showed poor hemagglutination activities, titers of 2(1) or less, which were much lower than titers from the reference strains (2(6)), and did not bind to erythrocytes. These results suggest strongly that the short C-terminal region of the HA-33 plays an essential role in the hemagglutination activity of the botulinum progenitor toxin. Additionally, a sequence motif search predicted that the C-terminal region of HA-33 has a carbohydrate-recognition subdomain.

The ability of an anti-TNF-alpha antibody to confer protection against enterohaemorrhagic Escherichia coli (EHEC) O157 was investigated in germfree IQI mice. The use of an antibiotic levofloxacin (LVFX) alone or with the antibody was also studied. Protection included an increase in survival rate. Treatment with the anti-TNF-alpha antibody inhibited the histological signs associated with EHEC infection but did not prevent the colonization of EHEC or production of Shiga toxin (Stx). No clinical signs were observed and EHEC was completely eliminated in the mouse model receiving both anti-TNF-alpha antibody and LVFX. Anti-TNF-alpha antibody suppressed inflammatory cytokine response in the mouse kidney and brain by EHEC infection.

The genetic structure of neurotoxin genes in a Clostridium botulinum strain producing both type A and B neurotoxins (type AB) was investigated. Analyses by polymerase chain reaction (PCR) using type-specific primers corresponding to the coding regions for N-terminals of light-chains and C-terminals of heavy-chains of type A and type B neurotoxins, and Southern hybridization of total DNA showed that the type AB strain I.P.7212 carries at least one copy each of type A and B neurotoxin genes. Partial nucleotide sequences obtained by direct sequencing of the PCR products indicate that the type A and B genes carried by this strain are not classical type A and non-proteolytic type B genes, but are similar to the type A gene present in a strain which had caused infant botulism in Kyoto and the type B gene present in a proteolytic type B strain.

Botulinum type D neurotoxin inhibited Ca(2+)-evoked norepinephrine secretion in digitonin permeabilized PC12 cells. Inhibition by the toxin required prior incubation with dithiothreitol (DTT). The inhibition was dependent on both concentration and incubation times of the toxin, and was affected by Ca2+ concentration. With less than 0.7 microM Ca2+ almost complete inhibition was observed; however, above 0.7 microM, Ca2+ stimulated additional norepinephrine release in a dose-dependent manner.

Helicobacter pylori has recently been implicated as an etiologic agent of gastroduodenal disorders. Comparing the antibody to H. pylori in the sera of patients with that of normal controls by Western blot analysis, a unique antibody was detected in the sera of patients, which reacted with the 25-kilodalton antigen of H. pylori. On the other hand, monoclonal antibody CP3 prepared in the authors' laboratory also recognized the 25-kilodalton antigen of H. pylori. Whether the serum antibody of the patient recognized the CP3 antigen purified by monoclonal antibody CP3 was then examined. Western blot analysis showed that the patient's serum reacted strongly with the affinity-purified CP3 antigen. Using monoclonal antibody CP3, an enzyme-linked immunosorbent assay to detect CP3 antibody in sera was established. In patients with chronic gastritis and gastric ulcers, the titer of CP3 antibody was significantly higher than in normal controls and correlated with the histological grade of antral gastritis. The detection of CP3 antibody in sera is useful in the diagnosis of chronic gastritis and gastric ulcer associated with H. pylori infection and also in evaluation of the grade of gastritis.

Uncommon serotypes were tested for their ability to induce aggregation in platelet-rich plasma. Both uncommon and common serotypes induced platelet aggregation in rabbit platelet-rich plasma, but serotonin release was higher with the uncommon serotypes. Aggregation at ATP release varied between uncommon and common serotypes. With human platelet-rich plasma, only uncommon serotypes showed aggregation. Differences in serotype selectivity and the rate of aggregation were noted among platelet donors. About half of the patients with Behçet's disease and 30% of health controls showed platelet aggregation. Plasma from non-responder patients with Behçet's disease inhibited aggregation of healthy responder platelets within 20 min. Thus selective binding of uncommon serotypes of Strep. sanguis to platelets might cause the vasculitis in Behçet's disease.

Clostridium botulinum125I-labeled C1 neurotoxin bound to NG108 hybridoma cell line. Unlabeled type C1 neurotoxin inhibited the binding of the labeled C1 toxin but neither types D nor E toxin. 125I-labeled type D neurotoxin bound to rat brain synaptosomes but did not bind to NG108 cells. It is suggested that receptors for types C and D or E toxins on neuronal cell membranes are different.

Germination of spores of Clostridium botulinum type G under various conditions of heat activation, incubation temperature and minimum nutrition was studied. The spores were optimally germinated in a mixture of L-cysteine + L-lactacte + bicarbonate. The rate and extent of germination were greatly increased when unheated spores were heat-activated at 75°C for 10 min before exposure to the germination medium. Maximum germination was obtained at incubation temperatures of 37 to 45°C within 1 h. However, germination occurred substantially even at 4°C if the incubation time was extended to 48 h.