Difficulties of molecular dissociation of Clostridium botulinum type G progenitor toxin.

Clostridium botulinum type G progenitor toxin was chromatographed on DEAE-Sephadex and Q-Sepharose equilibrated with 0.05 M Tris-HCl buffer, pH 8.0, containing 0.2 M urea. The toxin was eluted in a single protein peak from DEAE-Sephadex, but it was eluted in four protein peaks from Q-Sepharose; the third peak was toxic and the others were nontoxic. The third peak, appearing to be the toxic component, had a molecular mass of 150,000. In SDS-polyacrylamide gel electrophoresis, purified type G progenitor toxin migrated in six bands, with molecular masses of 150,000, 140,000, 58,000, 10,800, 10,600, and 10,400. Type G progenitor toxin may be composed of a toxin component with a molecular mass of 150,000 and a nontoxic component in a manner similar to progenitor toxins of other types. Type G toxic component, whether it was reduced or not, migrated in a single band to the same relative positions in SDS-PAGE; type A toxic component reduced with 2-mercaptoethanol migrated in two bands.

Binding of Clostridium botulinum type B toxin to rat brain synaptosome.

Purified toxin and its subunits from Clostridium botulinum type B were labeled with 125iodine and binding of them to rat brain synaptosomes was studied. Labeled toxin and heavy chain were shown to bind to synaptosomes and there was no significant difference in the molar quantity of bound toxin and heavy chain at several concentrations of synaptosomes, whereas labeled light chain did not bind to synaptosomes. The binding of labeled heavy chain to synaptosomes was inhibited by unlabeled toxin and heavy chain to a similar degree as that of labeled toxin. The binding of labeled toxin and heavy chain to synaptosomes were inhibited by a monoclonal antibody which is specific for the heavy chain.

Immunochemical identification of the ADP-ribosyltransferase in botulinum C1 neurotoxin as C3 exoenzyme-like molecule.

Botulinum C1 neurotoxin and C3 exoenzyme were purified to apparent homogeneity from the culture filtrate of Clostridium botulinum type C strain 003-9. Both preparations catalyzed ADP-ribosylation of the same substrate, the Mr 22,000 rho gene product (Gb). When the light and heavy chains of C1 toxin were separated, ADP-ribosyltransferase activity in the toxin was quantitatively recovered in the light chain fraction. Anti-C1 toxin antiserum precipitated the ADP-ribosyltransferase activity and the neurotoxicity of C1 toxin in parallel, whereas it had no effect on C3 exoenzyme. On the other hand, anti-C3 exoenzyme antiserum precipitated the ADP-ribosyltransferase activities of both C3 exoenzyme and C1 toxin. This antibody, however, did not precipitate the neurotoxicity of C1 toxin. The ADP-ribosyltransferase in C1 toxin was quantitatively adsorbed onto the anti-C3 antibody column and separated from the majority of C1 toxin protein. The enzyme was then eluted with acidic urea and Western blotting analysis of this eluate revealed the appearance of a protein band positively stained with anti-C3 antibody at a position similar to that of C3 exoenzyme. Quantitative determination by enzyme-linked immunosorbent assay showed that the C3-like immunoreactivity is present in the C1 toxin molecules at the molecular ratio of 1 to 1,000. These results suggest that the ADP-ribosyltransferase activity in C1 toxin is expressed by a C3-like molecule which is present in a small amount in the toxin preparation and appears to bind to the toxin component(s). The above results also indicate that the ADP-ribosyltransferase in C1 toxin is not related to its neurotoxin action.

Association between the cell-wall peptidoglycan and the progenitor toxin of Clostridium botulinum type C.

Clostridium botulinum type C progenitor toxin with a molecular weight of 500 k daltons (C1 L toxin), purified from the bacterial cells, bound at pH 2 to the cell-wall peptidoglycan derived from certain strains. The carbohydrate moiety of the peptidoglycan contained arabinose and galactose at a certain ratio, both of which may directly be associated with the binding. The binding, being dependent on the quality and quantity of the sugars, enhanced the oral toxicity of the toxin to the chicken as well as the mouse.

Clostridium botulinum type D neurotoxin: purification and detection.

A method is reported for the purification of type D botulinum toxin using a combination of low and high pressure ion exchange chromatography. The procedure produced homogeneous toxin in its free form in 3 days, with a specific toxicity in mice of 5.4 x 10(7) LD50/mg protein. Polyclonal antibodies against the pure toxin were raised in rabbits and detected the toxin in both ELISA and western blotting. The antibodies also detected type C1 botulinum toxin using these techniques, confirming the presence of cross-reacting antigenic determinants in these two proteins.

Evaluation of a monoclonal antibody-based immunoassay for detecting type B Clostridium botulinum toxin produced in pure culture and an inoculated model cured meat system.

A monoclonal antibody-based amplified ELISA method for detecting Clostridium botulinum type B toxin was evaluated for its ability to detect the toxin in the supernatant fluid of pure cultures and after growth from Cl. botulinum spores inoculated into pork slurries. Slurries containing NaCl (1.5-4.5% w/v) and polyphosphate (0.3% w/v) were either unheated or heated 80 degrees C/5 min followed by 70 degrees C/2 h before incubation at 15 degrees, 20 degrees or 27 degrees C. Presence of specific toxin was confirmed by mouse bioassay and results were compared with those of the amplified ELISA method. A total of 48 strains, consisting of 38 Cl. botulinum and 10 Cl. sporogenes (putrefactive anaerobes), and 140 slurry samples were tested. Cultures of eight out of nine strains of type B Cl botulinum and 73 of 101 slurry samples containing type B toxin were positive by ELISA; the remaining 28 slurry samples contained type B toxin at levels below or close to the detection limit (20 LD50/ml) of the type B ELISA. No false-positive reactions occurred with Cl. botulinum types A, C, D, E or F, or with the 10 strains of Cl. sporogenes. Toxin produced by one strain of Cl. botulinum type B (NCTC 3807) was not detected by this single monoclonal antibody-based amplified ELISA. With a mixture of two monoclonal antibodies, however, the toxin from NCTC 3807 could be detected without reducing the sensitivity of the ELISA.

Comparison of toxins of Clostridium butyricum and Clostridium botulinum type E.

The toxin of Clostridium butyricum strains isolated from two infants with botulism is neutralized by antitoxin for type E botulinum toxin. This toxin and that of a C. botulinum type E strain were purified by the same protocol. Both toxins were Mr 145,000 proteins which, when activated with trypsin, were composed of an H subunit of Mr 105,000 and an L subunit of Mr 50,000. The activated specific toxicity of purified butyricum toxin based on an intravenous assay was 2 X 10(8) mouse 50% lethal doses (LD50s)/mg of protein, but that based on an intraperitoneal assay was 7 X 10(7) LD50s/mg, compared with 6 X 10(7) LD50s/mg for type E toxin as determined by both methods. Immunodiffusion tests with antitoxin raised with type E toxin indicated that the two toxins were serologically very similar except for a spur formed by type E toxin. The close similarities of the two toxins suggest that toxigenic C. butyricum could arise when a wild-type strain, which is normally nontoxigenic, acquires the toxin gene of a C. botulinum type E strain.

Distribution of Clostridium botulinum in Japan and in Shinkiang district of China.

Soil specimens obtained from several areas of Japan, which are closely located to or facing the Continental land of China, were examined for the distribution of Clostridium botulinum, especially pertaining to types A and B. A total of 266 specimens of Japan, when cultured, showed no type A or B toxicity, although 30 (11.3%), 4 (1.5%), and 10 (3.8%) of the specimens showed C1, C2, and type E toxicities, respectively. On the contrary, types A and/or B toxicities were shown, by the same method, in 14 of 20 specimens of Shinkiang district, China. The highest number of C. botulinum cells found in one gram of soil specimen was 25 for type A and 10 for type B.

Activation of Clostridium botulinum type E toxin purified by two different procedures.

Clostridium botulinum type E toxin was purified from culture supernates and from cell extracts by two methods. The specific activity [2 X 10(4) mouse LD50 (mg protein)-1] of the toxin purified from cell extract under slightly acidic conditions was lower than that [3 X 10(5) LD50 (mg protein)-1] of the toxin purified from culture supernate under slightly alkaline conditions. Both toxin preparations were activated by trypsin treatment, but to different extents, the degree of activation of the toxin from cell extract being about 30-fold higher than that of the toxin from culture supernate. The two toxin preparations had the same electrophoretic mobility on SDS-polyacrylamide gels and antigenic specificity as revealed by agar gel double-immunodiffusion tests. The antigenic specificity of the two toxin preparations was unaltered by trypsin treatment. In SDS-polyacrylamide gel electrophoresis, a single band of Mr 144,000 was demonstrated before trypsin treatment and two bands of Mr 100,000 and 55,000 appeared after trypsin treatment. The two toxin preparations were labelled with 125I and chymotryptic peptide maps were obtained before and after trypsin treatment. The two toxin preparations without trypsin treatment demonstrated many differences in their peptide maps, but the preparations after trypsin activation had similar peptide maps. These results indicate that the toxin obtained from culture fluid was a partially activated form, and that its molecular conformation was different from that of the toxin from cell extract. Differences in specific activity and activation ratio by trypsin treatment may be due to differences in the conformation of the toxin molecules.

Heterogeneities of two components of C2 toxin produced by Clostridium botulinum types C and D.

Botulinum C2 toxin (C2T) is composed of two dissimilar protein components, designated components I and II, which are linked with neither covalent nor noncovalent bonds. The heterogeneity of these two components of C2T produced by Clostridium botulinum type C and D strains was examined. Of 21 strains examined, 19 strains produced the two components, while the others produced neither component I nor component II. The 19 producers of C2T could be divided into three groups based on the differences in antigenicity, molecular weight and biological activity of components I and II. The results provide evidence of heterogeneity in the molecular structure of the two components of C2T, which is possibly a cause of the differences in the biological activity of the toxin observed in different strains.

Solubilization and partial properties of receptor substance for bacteriophage alpha 2 induced from Clostridium botulinum type A 190L.

Bacteriophage alpha 2, one of the two inducible phages from Clostridium botulinum type A 190L, had a latent period of 55 min and an average burst size of 75 in C. botulinum type A Hall used as the host bacterium. The phage particles were adsorbed on the cell walls extracted with hot trichloroacetic acid (TCA-walls). The receptor substance for the phage was solubilized from the TCA-walls with Achromopeptidase and fractionated by gel filtration on Sephadex G-150. The fraction having the highest level of receptor activity for the phage contained large amounts of muramic acid and glucosamine. Both authentic muramic acid and glucosamine significantly inactivated the phage, whereas glucose, galactose, L-and D-alanine, diaminopimeric acid, or D-glutamic acid did not exhibit similar activity. There results strongly suggest that the receptor site for phage alpha 2 is closely associated with glycan moieties of the cell wall peptidoglycan.

Use of monoclonal antibodies in enzyme linked immunosorbent assay (ELISA) for detection of botulinum type B toxins.

Use of polyclonal antibodies failed to correlate mouse assay with enzyme linked immunosorbent assay (ELISA) in titration of culture fluid of different strains of Clostridium botulinum type B. If ELISA is performed with such a monoclonal antibody that is capable of neutralizing the toxin, however, the lethal toxicity can be determined quantitatively.

Purification and amino acid composition of type A botulinum neurotoxin.

A method to purify type A botulinum neurotoxin from a 64 liter bacterial culture is reported. The procedure includes cation exchange chromatography at pH 7.0. The final product, essentially homogeneous (according to polyacrylamide gel-sodium dodecylsulfate electrophoresis), is a mixture of two forms of the neurotoxin (mol. wt 145,000); the dichain or nicked form (over 95%) and its precursor the single chain or unnicked form. Two batches of the neurotoxin purified by the method described here and one batch purified according to the method of Sugii and Sakaguchi were similar in purity and amino acid composition. The best estimate of the number of amino acid residues per neurotoxin molecule (mol. wt 145,000) is: Asp200Thr75Ser79Glu114Pro44Gly64Ala53Val70CyS10Met22Ile111Leu104Tyr71 Phe68Lys100His14Arg43Trp17.

Taxonomic relationships among Clostridium novyi Types A and B, Clostridium haemolyticum and Clostridium botulinum type C.

The present study was undertaken to examine the genetic relationships among the closely related species, Clostridium novyi types A and B, C. haemolyticum and C. botulinum type C. These species were tested for DNA-DNA homology and thermostability of DNA duplexes and sorted into three genetically related groups: I, C. novyi type A; II, C. novyi type B, C. haemolyticum and one C. botulinum type C strain (Stockholm); III, the remaining C. botulinum type C strains. A few biochemical criteria corresponding to the genetic differences were recommended to differentiate each group. These studies imply that C. haemolyticum might be considered as C. novyi type D and that there are two genetically different groups in C. botulinum type C.

[Preparation of neurotoxin and hemagglutinin from Clostridium botulinum A and characterization of its neurotoxin]. / Poluchenie neirotoksina i gemaggliutinina Clostridium botulinum tipa A i kharakteristika neirotoksina.

A procedure for preparation of electrophoretically and serologically homogeneous neurotoxin and a highly purified hemagglutinin from the culture fluid of Cl. botulinum A, strain 501 is described. The yield of neurotoxin with specific activity of 80-100 X 10(6) DLM/mg of protein is 5-20%. Neurotoxin has a molecular weight of 150,000, sedimentation coefficient of 7.1S, pI of 6.2-6.3; the maximum of its fluorescence corresponds to 332 nm. The toxin molecule contains 4 SH-groups. Neurotoxin consists of two subunits with molecular weights of 98,000 and 56,000. The storage of neurotoxin at -20 degrees C causes inactivation and electrophoretical heterogeneity of the protein. The inactivation leads to an alteration of the toxin molecule charge, a shift of the lambda max of fluorescence and a loss of the SH-group reactivity without affecting the molecular weight or serological properties of the protein. The data obtained suggest that the conformational state of toxin molecules is essential for its biological activity.

Proposed mechanism for sensitization by hypochlorite treatment of Clostridium botulinum spores.

Hypochlorite-treated Clostridium botulinum 12885A spores, but not buffer-treated spores, could be germinated with lysozyme, indicating that their coats are made permeable to lysozyme by hypochlorite treatment so that the cortex is accessible. Hypochlorite-treated spores and spores extracted with 8 M urea-2-mercaptoethanol (pH 3.0) were sensitive to certain components of recovery media, but spores sensitized to lysozyme by other treatments were not. These data indicate that hypochlorite does more than increase coat permeability to lysozyme. Scanning electron microscopy revealed a more open-appearing surface of hypochlorite-treated spores, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that a greater amount of protein was removed from hypochlorite-treated and other lysozyme-sensitized spores than from buffer-treated spores. The data suggest that spore coat proteins may be removed by hypochlorite treatment, and this may be responsible for the sensitivity of spores and for their observed ability to germinate in lysozyme.

Purification and amino acid composition of type E botulinum neurotoxin.

The procedure we have adopted to purify type E botulinum neurotoxin (mol. wt. approximately 147,000) from bacterial cultures consistently yields a pure protein (a single band in polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate). Our procedure is a modification of one of the five published procedures. Other procedures have failed to yield pure neurotoxin. To develop reliable data on the amino acid composition, three batches of the neurotoxin were analyzed, each batch isolated from a separate neurotoxin producing culture. The best estimate of the number of amino acid residues per neurotoxin molecule was: Asp240 Thr75 Ser98 Glu118 Pro45 Gly58 Ala40 Val62 CyS7 Met17 Ile123 Leu107 Tyr70 Phe62 Lys97 His15 Arg34 Trp16.

Amino acid composition of Clostridium botulinum type F neurotoxin.

To develop reliable data on the amino acid composition of type F botulinum neurotoxin, three batches of the neurotoxin were analyzed. Each batch was isolated from a separate neurotoxin producing bacterial culture. Two batches had inoculum from one source and the other batch one from a different source. Two batches of the neurotoxin were purified by the same method and one was purified by a different method. The neurotoxin preparations were found comparable in purity and similar in amino acid composition. The best estimate of number of amino acid residues per neurotoxin molecule (mol. wt. 155,000) was: Asp218 Thr80 Ser105 Glu128 Pro47 Gly69 Ala47 Val72 CyS9 Met14 Ile128 Leu104 Tyr86 Phe60 Lys90 His13 Arg51 Trp23.

Separation and characterization of heavy and light chains from Clostridium botulinum type C toxin and their reconstitution.

Clostridium botulinum type C toxin consists of a heavy and a light chain with molecular weights of 98,000 and 53,000, respectively, which are linked by one disulfide bond. The two components were separated from each other by quaternary aminoethyl Sephadex A-50 column chromatography by stepwise elution with NaCl in 27.5 mM borax-45 mM sodium dihydrogen phosphate buffer, pH 8.0, containing 5% 2-mercaptoethanol at 0 degrees C. The purified components had different amino acid compositions and antigenicities, and the toxicity of the toxin was neutralized completely by either anti-heavy chain Fab or anti-light chain Fab. the two components could be reconstituted to form an active molecule with recovered toxicity which varied according to the method used. Maximum recovery was obtained in a system in which the intersubunit S--S bond was first formed in the presence of high concentration of neutral salts, after which the concentration of salt was gradually decreased. The reconstituted preparation was highly toxic and had the same properties as the parental toxin on chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and immunodiffusion. By the use of three perturbants, the fractions of exposed tryptophans and tyrosines of the preparation were found to be almost the same as that of the parental toxin.