Purification and characterization of Clostridium botulinum type B oxin

Gerwing, Julia (The University of British Columbia, Vancouver, B.C., Canada), Claude E. Dolman, David V. Kason, and Jack H. Tremaine. Purification and characterization of Clostridium botulinum type B toxin. J. Bacteriol. 91:484-487. 1966.-A toxic component of low molecular weight has been isolated from a type B strain of Clostridium botulinum by methods involving ammonium sulfate precipitation and elution through diethylaminoethyl cellulose at pH 5.6. The material thus isolated was shown to be monophoretic and monodisperse in the ultracentrifuge. End-group analysis indicated the presence of a single N-terminal amino acid residue, which was identified as arginine. On the basis of biophysical studies and amino acid analyses, a molecular weight between 9,000 and 10,000 was calculated.     

Isolation and immunochemical study of the toxic complex of Cl. botulinum type F]

The toxic comples of Cl. botulinum, type F, was separated into the toxic and nontoxic protein fractions by the methods of ion exchange chromatography and gel filtration in accordance with a specially devised purification scheme. Highly purified, electrophoretically and serologically homogeneous toxin with a molecular weight of 150,000 and potency equal to 10 X 10(6) DLM per 1 mg of protein was isolated from the toxic fraction. The nontoxic protein component had faintly pronounced hemagglutinating properties and was essentially different from type A and B hemagglutinins. The toxic complex of Cl. botulinum, type F, was shown to contain a proteolytically active fraction.     

Isolation and properties of highly purified C1. botulinum toxin type E]

A new method of isolation of highly purified Cl. botulinum toxin of E type from the cultural fluid of strain 188 centrifugates was developed. The method allows to isolate the toxin both in a precursor and in activated forms with a yield of 10--15%. The method includes fractionation by ammonium sulfate, ultrafiltration and subsequent column chromatography on DEAE-cellulose, Sephadex G-200 and DEAE-Sephadex A-50. The preparations were found homogeneous during polyacrylamide gel electrophoresis and immunoprecipitation in agar with antitoxic horse serum. The potential specific toxicity of the preparations is 1--1,2.10(7) DLM/mg of protein. The molecular weight of the toxin is about 160 000; the molar extinction coefficient is equal to 278 nm. The isoelectric point lies around pH 6.0. The highly purified Cl. botulinum toxin of E type was found stable upon storage.     

Purification of Clostridium botulinum Type F Progenitor Toxin

Clostridium botulinum type F progenitor toxin was purified to a homogeneous state as judged by gel filtration on Sephadex G-200, ultracentrifugation, and disc electrophoresis. The sedimentation constant, corrected to water at 20 C, of type F progenitor toxin was determined to be 10.3 and the molecular weight to be 235,000 by ultracentrifugation at pH 6.0. The purified toxin contained a toxicity of 1.2 x 10(8) 50% lethal doses/mg of N. In agar gel double diffusion, it formed two precipitin lines at pH 6.0. The progenitor toxin of type F differs from that of type A in that it contains no hemagglutinin and from that of type E in that it is not activable.     

Clostridium botulinum Type E Toxin: Effect of pH and Method of Purification on Molecular Weight

The toxin of Clostridium botulinum type E was isolated from intact cells and from toxic culture filtrates by column chromatography at three pH values, 4.5, 5.3, and 6.0. At pH 6.0 and 5.3, the isolated toxin was in a form with a molecular weight (MW) of 86,000. This toxin was homogeneous on polyacrylamide gel electrophoresis and gel filtration and had an optical density ratio, 280 nm/260 nm, greater than 2.0. It did not dissociate at higher pH levels, but was dissociated into nontoxic components of approximately 12,000 MW when reduced and alkylated in the presence of 6 M guanidine hydrochloride. At pH 4.5, smaller amounts of an impure toxic moiety with a MW of 12,000 were found. After storage for 6 months, the 86,000-MW moiety had lost 60% of its lethality. Gel filtration revealed that the bulk of the toxicity was associated with a component having a MW of 150,000. Toxic components with MW of 12,000 and over 200,000 were also found. The toxin appears to polymerize or aggregate when in a pure form, so that most, if not all, of the MW previously reported for the toxin may belong to different polymers of a monomer with a MW of 12,000 or less. Treatment of the 86,000-MW toxin with trypsin resulted in an 18- to 128-fold increase in lethality, but no detectable change in MW.     

神经毒素原性酪酸梭菌与E型肉毒梭菌毒素的精制及特性比较

对首次自Ｅ型肉毒中毒食品中分离到的一株神经毒素原性酪酸梭菌（ＬＣＬ１５５）所产生的神经毒素,同Ｅ型肉毒梭菌（Ｅ１５３）所产生的神经毒素进行了精制及特性比较,发现（１）两菌神经毒素的分子量,Ｎａｔｉｖｅ－ＰＡＧＥ测试均为３２０ｋＤａ;ＳＤＳ－ＰＡＧＥ测试则均为１４７ｋＤａ,非毒性非血凝素部分均为１２８ｋＤａ;用胰蛋白酶激活神经毒素后发现两菌神经毒素均由分子量为１０３ｋＤａ的Ｈ链和４８ｋＤａ的Ｌ链组成。（２）两菌神经毒素柱层析图像基本一致,但在菌体毒素提取效果及精制效果诸方面,分离的酪酸梭菌却都较差。（３）胰蛋白酶激活试验表明：两菌神经毒素达到最大毒力所需激活时间不等。在相同温度下,分离的酪酸梭菌毒素只需５ｍｉｎ,而Ｅ型肉毒梭菌毒素却需３０ｍｉｎ,提示两菌神经毒素激活动力学上存在差异。（４）琼脂双扩散试验结果表明两菌神经毒素的抗原性是一致的,没有发现沉淀线呈交叉或部分交叉现象。     

ADP-ribosylation of a Mr 21,000 membrane protein by type D botulinum toxin

When crude membrane fraction from bovine adrenal gland was incubated with type D botulinum toxin in the presence of NAD, a membrane protein with a molecular weight of 21,000 was specifically ADP-ribosylated. This ADP-ribosylation occurred dependent on the dose of the toxin and was abolished by prior boiling ADP-ribose transfer to the membrane protein was significantly suppressed when agmatine and L-arginine methyl ester were included in the reaction mixture. Dithiothreitol stimulated this ADP-ribosylation about 3-fold. Incubation of membrane fractions from mouse brain and pancreas with this toxin also resulted in ADP-ribosylation of a protein of the same molecular weight. These results suggested that type D botulinum toxin catalyzed transfer of an ADP-ribose moiety of NAD to the specific membrane protein common to secretory cells.     

Characterization and reconstitution of functional hemagglutinin of the Clostridium botulinum type C progenitor toxin.

The purified progenitor toxin of Clostridium botulinum type C strain 6814 (C-6814) forms a large complex composed of 150-kDa neurotoxin (NT), 130-kDa nontoxic-nonhemagglutinin (NTNHA), and hemagglutinin (HA) components. The HA component consisted of a mixture of several subcomponents with molecular masses of 70, 55, 33, 26-21 and 17 kDa. We isolated the HA subcomponents from the progenitor toxin by chromatography in the presence of denaturants. The isolated HA subcomponents, designated as i-HA-33, i-HA-55, i-HA-70 and i-HA-33/17, were nearly homogeneous on SDS/PAGE, but the HA-17 and HA-26-21 components were not purified. Some HA subcomponents, designated as f-HA-33 and f-HA-33/17 complex, existed free of the progenitor toxin in the culture medium and they were separately purified. Every HA subcomponent so far isolated shows binding activity to erythrocytes. The hemagglutination activities of each HA subcomponent had a titer of 25 for the f-HA-33/17 complex, and below 23 for the other f- and i-HA subcomponents, while the parent progenitor L toxin was 28. The reconstitution of various combinations of f- and i-HA subcomponents was attempted via mixing and tested for hemagglutination activity. When the i-HA-33/17 complex and i-HA-55 were mixed, the hemagglutination activity was recovered to a titer of 29, which was slightly higher than that of the parent toxin. These data imply that a combination of at least HA-33, -17 and -55 subcomponents is required for full hemagglutination activity of the botulinum progenitor toxin, but each single HA subcomponent shows weak or no aggregation of erythrocytes.     

The existence of the ribonucleoprotein branching enzyme in rabbit skeletal muscle and ribozyme corresponding to it]

Amylose isomerase (AI) preparations were isolated from rabbit muscles after Petrova et al., as well as by the additional fractionation steps. Their homogeneity, enzymatic activity and RNA, isolated from those preparations, were characterized. AI preparations, as described by Petrova et al., proved to be heterogeneous in respect to the protein and RNA; by using additional fractionation methods RNA and protein have been separated from each other, which proves that a homogeneous stable ribonucleoprotein complex, exerting AI activity, does not exist. It was shown by three independent methods that AI preparations isolated after Petrova do not display branching, but have amylolytic activity. RNA, isolated along with the AI preparations, proved to be mainly total tRNA degraded to different degrees. No RNA corresponding to the previously sequenced 2.5S RNA could be detected in these preparations. RNA preparations do not manifest neither branching, nor amylolytic activity. Our data prove that there is no ribozyme, whose existence has been suggested previously.     

Isolation and molecular size of Clostridium botulinum type C toxin.

A procedure is described for the purification of hemagglutinin-free Clostridium botulinum type C toxin. The toxin was purified approximately 1,000-fold from the original culture supernatant in an overall yield of 60% to a final specific toxicity of 4.4 x 10(7) minimal lethal doses/mg of protein. The toxin had a molecular weight of 141,000 and consisted of a heavy and a light chain. The molecular weights of the subunits were approximately 98,000 and 53,000. When comparing the molecular size and composition of type C toxin to that of botulinum toxins of different types, some common features may be suggested; i.e., the toxin has a molecular weight between 141,000 to 160,000 and is comprised of a heavy and a light chain linked by disulfide bonds (or bond).     

Isolation and molecular size of Clostridium botulinum type C toxin.

A procedure is described for the purification of hemagglutinin-free Clostridium botulinum type C toxin. The toxin was purified approximately 1,000-fold from the original culture supernatant in an overall yield of 60% to a final specific toxicity of 4.4 x 10(7) minimal lethal doses/mg of protein. The toxin had a molecular weight of 141,000 and consisted of a heavy and a light chain. The molecular weights of the subunits were approximately 98,000 and 53,000. When comparing the molecular size and composition of type C toxin to that of botulinum toxins of different types, some common features may be suggested; i.e., the toxin has a molecular weight between 141,000 to 160,000 and is comprised of a heavy and a light chain linked by disulfide bonds (or bond).     

Cloning of a DNA fragment encoding the 5'-terminus of the botulinum type E toxin gene from Clostridium butyricum strain BL6340

Chromosomal DNA was extracted from toxigenic Clostridium butyricum strain BL6340 isolated from a case of infant botulism. After digestion by EcoRI, a DNA fragment of about 1 kbp was cloned into Escherichia coli using lambda gt11, and was subcloned into pUC118. The E. coli cells transformed with this cloned fragment produced a 33 kDa protein which reacted with monoclonal antibodies recognizing the light chain (Lc) component of botulinum type E toxin. The nucleotide sequence of the cloned fragment was determined. The sequence was similar to that from botulinum type E toxin gene fragments previously determined by our laboratory (strains Mashike, Otaru and Iwanai). Several highly homologous sequences among the botulinum type A, C, E, butyricum and tetanus toxin genes were found in both translated and untranslated regions. These results suggest that the toxin gene of C. butyricum may have evolved by transfer from C. botulinum.     

The isolation of an easily reversible postsynaptic toxin from the venom of a sea snake, Laticauda semifasciata

A weakly neurotoxic component (Ls-III) was isolated by CM-cellulose column chromatography from the venom of a sea snake Laticauda semifasciata. The content of component LsIII was about 10-20% of the venom as determined by u.v. absorption at 280nm. Component LsIII was homogeneous on rechromatography and disc electrophoresis, and its molecular weight was shown to be 7100 by ultracentrifugation and 7300 by sodium dodecyl sulphate-polyacrylamide-gel disc electrophoresis. The isoelectric point of component LsIII was pH7.2. Component LsIII consisted of 66 amino acid residues including 10 half-cystine residues. The LD(50) of component LsIII by intramuscular injection was 1.24mug/g body wt. for mice and 0.45mug/g for baby chicks, which is about eight to ten times less toxic than erabutoxins a, b and c, all of which are contained in the same venom. Experiments with three isolated muscle preparations from different species indicated that component LsIII was a post-synaptically acting toxin, the action of which was easily reversed by washing.     

Isolation of Clostridium botulinum type G from Swiss soil specimens by using sequential steps in an identification scheme

After Clostridium botulinum type G organisms and toxin were identified in necropsy specimens in cases of unexplained death in adults and infants (O. Sonnabend, W. Sonnabend, R. Heinzle, T. Sigrist, R Dirnhofer, and U. Krech, J. Infect. Dis. 143:22-27, 1981), extensive research to detect C. botulinum type G in soil samples from Switzerland was done. A total of 41 specimens from virgin soil and from cultivated land were examined for the presence of C. botulinum type G and other toxin types. Because of the lack of the lipase marker in type G, the detection of C. botulinum type G was based on the demonstration of type G organisms in enrichment cultures by a type G-specific enzyme-linked immunosorbent assay to detect both the type G toxin and antigen; enrichment cultures in which type G toxin or antigen was identified by enzyme-linked immunosorbent assay were then tested by a type G-specific gel immunodiffusion agar procedure. This method not only isolated strains of type G but also strains of Clostridium subterminale, a nontoxigenic variant of C. botulinum type G. As a consequence of the observed cross-reactions caused by strains of C. subterminale within this test system, all isolates of type G had to be definitively confirmed by mouse bioassay. The sequential steps of these methods seem to be very useful for detecting C. botulinum type G organisms. C. botulinum type G strains were isolated in five soil samples from different locations in close association with cultivated land.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation of Clostridium botulinum type G from Swiss soil specimens by using sequential steps in an identification scheme.

After Clostridium botulinum type G organisms and toxin were identified in necropsy specimens in cases of unexplained death in adults and infants (O. Sonnabend, W. Sonnabend, R. Heinzle, T. Sigrist, R Dirnhofer, and U. Krech, J. Infect. Dis. 143:22-27, 1981), extensive research to detect C. botulinum type G in soil samples from Switzerland was done. A total of 41 specimens from virgin soil and from cultivated land were examined for the presence of C. botulinum type G and other toxin types. Because of the lack of the lipase marker in type G, the detection of C. botulinum type G was based on the demonstration of type G organisms in enrichment cultures by a type G-specific enzyme-linked immunosorbent assay to detect both the type G toxin and antigen; enrichment cultures in which type G toxin or antigen was identified by enzyme-linked immunosorbent assay were then tested by a type G-specific gel immunodiffusion agar procedure. This method not only isolated strains of type G but also strains of Clostridium subterminale, a nontoxigenic variant of C. botulinum type G. As a consequence of the observed cross-reactions caused by strains of C. subterminale within this test system, all isolates of type G had to be definitively confirmed by mouse bioassay. The sequential steps of these methods seem to be very useful for detecting C. botulinum type G organisms. C. botulinum type G strains were isolated in five soil samples from different locations in close association with cultivated land.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

The nucleotide and deduced amino acid sequences of EcoRI fragment containing the 5'-terminal region of Clostridium botulinum type E toxin gene cloned from Mashike, Iwanai and Otaru strains

Chromosomal DNAs were extracted from toxigenic three Clostridium botulinum type E strains isolated from food-borne botulism. After digestion by EcoRI, the fragments were cloned into Escherichia coli by using bacteriophage lambda gt11 and screened with monoclonal antibody recognizing the light chain component of botulinum type E toxin. The fragments (about 1 kbp size) cloned from each strain were recloned into a plasmid vector pUC118. The E. coli cells transformed with the recombinant plasmids produced 33 kDa protein with or without IPTG (isopropyl-beta-D-thiogalactopyranoside) which reacted with the monoclonal antibody. The nucleotide sequences of the cloned EcoRI fragments from the three type E strains were identical and contain the 5'-terminal region of the type E toxin gene. It was also found that there exist several highly homologous nucleotide sequences among the botulinum types A, C and E, and tetanus toxin genes in both translated and untranslated regions.     

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.     

Production of a homogeneous Cl. botulinum type B neurotoxin

The method for obtaining the neurotoxin, or alpha-fraction of the toxin, of Cl. botulinum, type B, is described. In accordance with this method, the toxin was precipitated three times with hydrochloric acid in the isoelectric zone with subsequent extraction with phosphate (pH 6.8) and citrate-phosphate (pH 5.6) buffers, then fractionated in columns with DEAE cellulose (pH 5.6), DEAE Sephadex A-50 (pH 7.2) and Sephadex G-200 (pH 7.2). The homogeneous neurotoxin preparations with molecular weights ranging from 145,000 to 160,000 and having the isoelectric point at pH 5.5 and toxicity 5.0--10.0 x 10(7) Dlm per 1 mg protein were obtained.     

Intracellular Synthesis of Clostridium botulinum Type B Toxin I. Demonstration of Toxin Synthesis and Sedimentation Studies on Toxic Products

The synthesis and nature of the toxin of Clostridium botulinum type B were studied in growing cells. It was demonstrated that the toxin was synthesized in the latter stage of logarithmic growth and was released into the culture supernatant fluid during lysis of the cells. Studies were done on the sedimentation properties of intracellular toxin. Two components were demonstrated, one of low molecular weight and low specific activity and one of high molecular weight with specific activity similar to that isolated from culture lysates. The high molecular weight toxin was shown to be composed of an aggregate of small subunits, separable with either high ionic strength or sodium dodecyl sulfate.