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The catabolic pathway for flavodoxin has yet to be clarified for any bacterial species. In this study, we found that the flavin mononucleotide in the flavodoxin of Helicobacter pylori is degraded to riboflavin via the phosphomonoesterase activity of class C acid phosphatase. The result is a conversion of holoflavodoxin to apoflavodoxin.

Two proteins representing the heavy-chain subunits of botulinum neurotoxin types C and D were expressed in Escherichia coli , and their vaccine potential was evaluated. Mice were vaccinated with doses ranging from 0.5 to 10 μg and were challenged with 10 to 10 5 50% lethal doses of toxin. For the type C subunit protein, C50, two doses of 2 μg were required for full protection, while, for type D subunit protein, D50, two 1-μg doses were required. A bivalent vaccine consisting of a mixture of these two proteins also provided protection against both botulinum neurotoxin type C and type D challenge. Antibody levels in serum were determined by both enzyme-linked immunosorbent assays and serum neutralization assays

Type E botulinum toxin (BoNT/E)-producing Clostridium butyricum strains isolated from botulism cases or soil specimens in Italy and China were analyzed by using nucleotide sequencing of the bont/E gene, random amplified polymorphic DNA (RAPD) assay, pulsed-field gel electrophoresis (PFGE), and Southern blot hybridization for the bont/E gene. Nucleotide sequences of the bont/E genes of 11 Chinese isolates and of the Italian strain BL 6340 were determined. The nucleotide sequences of the bont/E genes of 11 C. butyricum isolates from China were identical. The deduced amino acid sequence of BoNT/E from the Chinese isolates showed 95.0 and 96.9% identity with those of BoNT/E from C. butyricum BL 6340 and Clostridium botulinum type E, respectively. The BoNT/E-producing C. butyricum strains were divided into the following three clusters based on the results of RAPD assay, PFGE profiles of genomic DNA digested with Sma Io rXhoI, and Southern blot hybridization: strains associated with infant botulism in Italy, strains associated with food-borne botulism in China, and isolates from soil specimens of the Weishan lake area in China. A DNA probe for the bont/E gene hybridized with the nondigested chromosomal DNA of all toxigenic strains tested, indicating chromosomal localization of the bont/E gene in C. butyricum. The present results suggest that BoNT/E-producing C. butyricum is clonally distributed over a vast area. Type E botulinum toxin (BoNT/E)-producing Clostridium butyricum was first isolated from two cases of infant botulism in Italy in 1984 (1, 8). In 1997, we isolated BoNT/E-producing C. butyricum from the food implicated in food-borne botulism in China (10). Because our results indicated that type E food-borne botulism can be caused by BoNT/E-producing C. butyricum, we reexamined the cultural and biochemical properties of BoNT/E-producing organisms that had previously been isolated from type E food-borne botulism cases and found that two isolates were identifiable as C. butyricum (9). In addition, we isolated several strains of BoNT/E-producing C. butyricum from soil specimens of China (9). In 1998, an outbreak of food-borne botulism was reported in India and was strongly suggested to be caused by BoNT/E-producing C. butyricum (2). These studies indicate that soil is the principal habitat of BoNT/E-producing C. butyricum and that this organism may be widely distributed throughout the world (9). For improved surveillance of BoNT/E-producing C. butyricum, biochemical and genetic analysis of this organism is required. In this study, we performed molecular analysis of the strains isolated in Italy and China, by using nucleotide sequencing of the bont/E gene, random amplified polymorphic DNA (RAPD) assay, pulsed-field gel electrophoresis (PFGE), and Southern blot hybridization for the bont/E gene.

Infection with Helicobacter pylori induces humoral immune responses against various antigens of the bacterium. Heat shock proteins (hsps) are immunodominant antigens in various diseases including H. pylori infection. In the present study, we measured the anti-hsp antibody titers in 42 patients with H. pylori -infected peptic ulcers during a bacterial eradication study. The patients were treated with a proton pump inhibitor and antimicrobial agents to eradicate the organism. Their sera were obtained at pretreatment and at 1 month and 6 months after the eradication therapy. The titers of immunoglobulin G antibodies to the H. pylori hsp, whole-cell lysate, and urease (30-kDa subunit) antigens in serum were measured by a capture enzyme-linked immunosorbent assay. The levels of H. pylori hsp60 antibodies in sera collected 1 month after treatment had declined significantly, even when changes in the titers of antibodies to whole-cell and urease antigens were not apparent. These results suggest that measurement of antibodies to H. pylori hsp60 in serum is useful for the early monitoring of the effectiveness of eradication therapy.

Helicobacter pylori is a major etiologic agent in gastroduodenal disorders. In this study, immunoglobulin A (IgA) antibodies to H. pylori antigens were evaluated in serum and gastric juice specimens obtained from patients with gastritis or peptic ulcers by utilizing antibody capture enzyme-linked immunosorbent assays (ACELISAs). Urease α subunit (UA), urease β subunit (UB), the 66-kDa heat shock protein (HSP), and the 25-kDa protein (25K) were used as antigens for the ACELISAs. The antibody titers of the ACELISAs reflect the ratio of H. pylori -specific IgA to total IgA. The ratio is stable, although the antibody concentration fluctuates in gastric juice. By using ACELISAs it was possible to evaluate quantitatively not only serum IgA antibodies but also gastric juice secretory IgA (S-IgA) antibodies. In both serum IgA and gastric juice S-IgA ACELISAs, the titers of antibody to HSP and 25K were remarkably correlated with the histologic grade of gastritis, whereas those to UA and UB were not strongly correlated with histologic grade. Thus, it is useful for estimating the histologic grade of gastritis to quantify serum IgA and gastric juice S-IgA antibodies to HSP and 25K.

Helicobacter pylori is a major etiological agent in gastroduodenal disorders. The adhesion of H. pylori to human gastric epithelial cells is the initial step of H. pylori infection. Inhibition of H. pylori adhesion is thus a therapeutic target in the prevention of H. pylori infection. Experiments were performed to evaluate the effect of rebamipide, a novel antiulcer agent, on H. pylori adhesion to gastric epithelial cells. MKN-28 and MKN-45 cells, derived from human gastric carcinomas, were used as target cells. Ten H. pylori strains isolated from patients with chronic gastritis and gastric ulcer were used in the study. We evaluated the effect of rebamipide on H. pylori adhesion to MKN-28 and MKN-45 cells quantitatively using our previously established enzyme-linked immunosorbent assay. The adhesion of H. pylori to MKN-28 and MKN-45 cells was significantly inhibited by pretreatment of these cells with 100 μg of rebamipide per ml. However, the adhesion was not affected by the pretreatment of H. pylori with rebamipide. On the other hand, the viabilities of H. pylori , MKN-28 cells, and MKN-45 cells were not affected by rebamipide. Our studies suggest that rebamipide inhibits the adhesion of H. pylori to gastric epithelial cells.

The DNA fragment common to the genes encoding botulinum neurotoxin types C1 (BN/C1) and D (BN/D) was amplified by PCR from the culture supernatant of Clostridium botulinum type C strain 6813 (C6813) that was treated with either DNase I or proteinase K but not from the supernatant that was treated with both DNase I and proteinase K, suggesting the neurotoxin gene is located on a certain bacteriophage DNA. Thus, to isolate the neurotoxin gene, we performed PCR with the culture supernatant of C6813 and seven primer pairs designed from the genes encoding BN/C1 and BN/D. The coding region in the connected sequence encodes a neurotoxin composed of 1,280 amino acids with a molecular weight of 147,817. The neurotoxin from C6813 has 95% amino acid identity to BN/C1, except for its C-terminal one-third, which is quite similar to the C-terminal one-third of BN/D (95% identity). When we performed PCRs with four primer pairs designed from the 5'-terminal two-thirds of the BN/C1 gene and two primers from the 3'-terminal one-third of the BN/D gene, DNA fragments of the expected sizes (0.5 to 1.3 kbp) could be amplified from C. botulinum type C strains 6812 and 6814. These results suggest that some strains of C. botulinum type C contain the gene encoding the mosaic neurotoxin composed of parts of BN/C1 and BN/D.

We examined production of ADP-ribosyltransferase C3 in 11 strains of Clostridium botulinum type C and D and their nontoxigenic derivatives. Antisera to C3 proteins of type C organisms divided C3 proteins roughly into at least two groups, bearing no relation to their bacterial types. The C3 gene of type D strain South African was isolated from a toxigenic phage library, and the complete sequence of the C3 gene was determined. The C3 protein of type D strain South African had 98% homology to the C3 protein of type C strain 003-9 and 66% homology to that of type D strain 1873. These results indicate that there are two types of C3 protein in type D organisms, as there are in type C organisms.

The partial amino acid sequence of the light-chain (Lc) component of Clostridium botulinum type C1 toxin was determined. The sequence was quite similar to those of the other types of botulinum and tetanus toxins. Nine monoclonal antibodies against botulinum type E toxin were established by immunizing BALB/c mice with type E toxoid or its Lc component. Six antibodies reacted with the heavy-chain component and three reacted with the Lc component of the toxin. One of the latter three antibodies reacted with botulinum type B, C1, and D toxins and tetanus toxin, as well as botulinum type E toxin. This antibody recognized the Lc components of these toxins, indicating that there exists one common antigenic determinant on the Lc regions of these toxins.

Monoclonal antibodies against type C1 toxin produced by Clostridium botulinum type C strain Stockholm (C-ST) were prepared by fusion of BALB/c myeloma cells P3X63-Ag8, with spleen cells from the mice immunized by C-ST toxoid. About 5% of single-cell colonies in wells were found to produce antibodies against the toxin as determined by an enzyme-linked immunosorbent assay (ELISA). Four different hybridoma cell lines, no. 9, 12, 14, and 17, were established, cloned by limiting dilution, and intraperitoneally injected into mice to obtain the ascites fluids containing high-titered antibodies. The reactions of these antibodies to type C1 and D toxins of strains C-ST, D-1873, and D-South African (D-SA) were observed by both neutralization and ELISA tests. Three monoclonal antibodies, no. 9, 14, and 17, reacted with C-ST toxin, but only no. 17 highly neutralized the toxin. These antibodies did not react with type D toxins. On the contrary, no. 12 reacted with toxins of both C-ST and D-SA (but not of D-1873) and commonly neutralized these two toxins. This indicates that there is a common antigenic part between C-ST and D-SA toxin molecules which participates in the toxin-neutralizing reaction. The neutralization profiles of C-ST toxin by no. 12 and 17 antibodies were different in a time-to-death test of mice. The mechanisms of neutralization by no. 12 and 17 may be different.

Binding of type C neurotoxin (C1 toxin) from Clostridium botulinum (strain Stockholm) to neuroblastoma cell lines was studied by using biotinylated anti-toxin antibody and avidin-biotinylated peroxidase complex. The neurotoxin bound with high efficiency to mouse neuroblastoma (NS-20Y and NIE-115) cells and to hybridomas of rat glioblastoma and mouse neuroblastoma (NG108-C15) cells. The toxin bound little to human neuroblastoma, rat astrocytoma, and nonneural cell lines. Binding of the neurotoxin to NG108-C15 cells was inhibited by gangliosides (GT1b and GM1) and by monoclonal antibodies (CA-12 and C-9), although inhibition was not complete. Sequential preincubation of C1 toxin with GT1b and CA-12 caused complete inhibition. A Scatchard plot of binding of 125I-labeled C1 toxin to NG108-C15 cells showed a hyperbolic curve. Monoclonal antibody CA-12 but not C-9 neutralized the lethal activity of the toxin toward mice. Only C-9 clearly inhibited toxin binding to GT1b. These results suggest that NG108-C15 cells have at least two kinds of receptors for C1 toxin. From the results of binding tests with neuraminidase-, pronase-, and trypsin-treated NG108-C15 cells, the chemical nature of the high-affinity site was presumed to be a glycoprotein containing sialic acid. GT1b may have an important role in low-affinity sites.

Five Clostridium botulinum strains were used in the present work, two of type C, C-Stockholm (C-ST) and C-CB19, and three of type D, D-South African (D-SA), D-1873, and D-CB16. The toxins, except for those of C-CB19 and D-CB16, were purified, and antisera were prepared in rabbits. To clarify the antigenicity of the toxins, neutralization and agar gel double-diffusion tests were performed. Anti-C-ST toxin serum neutralized two kinds of type C 1 toxin to a similar extent. Antisera against D-SA and D-1873 toxins, however, showed different neutralizing activity toward three type D toxins. Precipitin lines formed between D-SA and D-1873 toxins, and their antisera spurred to each other. From anti-D-SA toxin serum, two neutralizing fractions, one which neutralized D-SA, D-1873, and D-CB16 and one which neutralized only D-SA, were obtained. These results indicate that the antigenicities of D-SA and D-1873 toxins are not identical. Anti-C-ST toxin serum produced cross-neutralization on type D toxins, although the neutralization titer differed depending on the kind of toxin used. A precipitin line was formed with D-SA toxin, but not with D-1873; the developed line spurred to the C-ST toxin precipitin line. Two anti-D toxin sera also caused cross-neutralization on type C 1 toxins. However, the neutralizing activity of each serum to the same type C 1 toxin was different, and only anti-D-SA toxin serum developed a precipitin line with C-ST toxin which spurred to the D-SA toxin precipitin line. From anti-D-SA toxin serum, two different fractions capable of neutralizing C 1 and D toxins were obtained; one neutralized C-ST, C-CB19, and D-SA toxins, but not D-1873 and D-CB16, and the other neutralized all five toxins. There may be at least two common parts among C-ST, C-CB16, and D-SA toxin molecules.