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.     

Homogeneity and Molecular Weight of Toxin of Clostridium botulinum Type B

Gerwing et al. described the isolation and purification from culture filtrates of the toxin of Clostridium botulinum type B and characterized it as a homogeneous protein of less than 10,000 molecular weight. Analysis by various methods of samples of this toxin obtained from Gerwing et al., and preparations produced by their methods in our laboratories, furnished convincing evidence that neither her preparation nor ours was homogeneous. The molecular weight of the toxic component isolated from either of the preparations was 100,000 or greater and resembled, in a number of respects, the alpha component isolated by us from the crystalline toxin of C. botulinum type A.     

Differentiation of Clostridium difficile Toxin from Clostridium botulinum Toxin by the Mouse Lethality Test

The mouse lethality test is the most sensitive method for confirming the diagnosis of infant botulism. Both Clostridium difficile and Clostridium botulinum produce heat-labile toxins which are lethal for mice and can be found in the feces of infants. These two toxins can be distinguished from one another in this assay when both are present in the same fecal specimen because they appear to be immunologically distinct toxins.     

Radioimmunoassay for Type A Toxin of Clostridium botulinum

A preparation of pure type A toxin of Clostridium botulinum was labeled with (131)I in the presence of chloramine-T and carrier-free isotope. The radioactive toxin ((131)I Tox) was used in a radioimmunoassay procedure similar to that of Berson and Yalow. Dilutions of antibody to the toxin, capable of binding 50% of the added (131)I Tox, were mixed with a sample of the labeled toxin and various concentrations of unlabeled toxin ('Cold' Tox). When concentration of Cold Tox were plotted against the ratio (131)I bound/(131)I not bound, a standard curve was established that could be used to estimate the concentration of Cold Tox in a test mixture. This assay was sensitive to as little as 100 mouse minimum lethal dose and was highly specific for the serological type of the toxin used.     

Toxin production by Clostridium botulinum in grass.

Investigations on farms where botulism has occurred in cows showed that proteolytic Clostridium botulinum type B was present in newly made grass silages. Experiments were undertaken to study growth and toxin production of C. botulinum in grass. Of the strains tested only proteolytic strains of C. botulinum types A and B were able to produce toxin with grass as a substrate. Proteolytic strains of type B produced both medium (12S) and large (16S) toxin forms. The minimal water activity (aw) for toxin production at pH 6.5 and 5.8 was 0.94. At pH 5.3, toxin was produced at an aw of 0.985. These results indicate that proteolytic strains of C. botulinum (if present) may multiply and produce toxin in wilted grass silages.     

Toxin production by Clostridium botulinum in grass.

Investigations on farms where botulism has occurred in cows showed that proteolytic Clostridium botulinum type B was present in newly made grass silages. Experiments were undertaken to study growth and toxin production of C. botulinum in grass. Of the strains tested only proteolytic strains of C. botulinum types A and B were able to produce toxin with grass as a substrate. Proteolytic strains of type B produced both medium (12S) and large (16S) toxin forms. The minimal water activity (aw) for toxin production at pH 6.5 and 5.8 was 0.94. At pH 5.3, toxin was produced at an aw of 0.985. These results indicate that proteolytic strains of C. botulinum (if present) may multiply and produce toxin in wilted grass silages.     

Purification and Properties of Clostridium botulinum Type F Toxin

Clostridium botulinum type F toxin of proteolytic Langeland strain was purified. Toxin in whole cultures was precipitated with (NH₄)₂SO₄. Extract of the precipitate was successively chromatographed on diethylaminoethyl-cellulose at pH 6.0, O-(carboxymethyl) cellulose at pH 4.9, Sephadex G-200 at pH 8.1, quaternary aminoethyl-Sephadex at pH 4.9, and finally diethylaminoethyl-cellulose at pH 8.1. The procedure recovered 14% of the toxin assayed in the starting culture. The toxin was homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, double gel diffusion serology, and isoelectric focusing. Purified toxin had a molecular weight of 150,000 by gel filtration and 155,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Specific toxicity was 9.6 × 10⁶ mean lethal doses per absorbancy (278 nm) unit. Sub-units of 105,000 and 56,000 molecular weight are found when purified toxin is treated with a disulfide reducing agent and electrophoresed on sodium dodecyl sulfate-polyacrylamide gels. Reciprocal cross neutralizations were demonstrated when purified type F and E toxins were reacted with antitoxins which were obtained with immunizing toxoids prepared with purified toxins.     

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.     

Rapid Detection of Clostridium botulinum Toxin by Capillary Tube Diffusion

A micro capillary agar-gel diffusion system for the detection of botulinal toxin in foods and cultures was developed and evaluated. Toxins types A, B, and E, produced in culture broth with and without added trypsin, and type E toxin, produced in inoculated canned clams, were tested with this system and with the mouse bioassay procedure. With nontrypsinized toxin, the capillary diffusion system detected as little as 100 minimal lethal doses (MLD) per ml but was effective only at higher levels, 10(6) to 1.5 x 10(7) MLD/ml, when used with trypsinized toxin. The inability to detect lower levels of trypsinized toxin was due to thioglycolate present in the medium used to produce toxin. Evidently, trypsinization of toxin produces polypeptides still held together by disulfide bonds. Cleavage of these bonds by reduction with thioglycolate reduces the sensitivity of the capillary method. Trypsinized toxin produced in broth without thioglycolate was detected as readily as nontrypsinized toxin. Toxin was detected in canned clams containing as low as 100 MLD/ml. No cross-reactions were observed with type E toxin and types A and B antitoxins. Extensive studies using the capillary method for detecting types A and B toxins were not performed; however, a suspected sample of commercially canned mushrooms gave a positive type B reaction but not a type A reaction. This typing was confirmed later by the mouse bioassay. Toxin was present at a level of 100 MLD/ml. The procedure developed may prove useful as a rapid screening method for the detection of botulinal toxin in foods, with final identification made by using the mouse bioassay.     

Significance of 12S Toxin of Clostridium botulinum Type E

The pathogenesis of type E botulism is discussed as an aspect of the physicochemical and biological properties of 12S toxins (prototoxin and trypsin-activated 12S toxin) and the Ealpha and Ebeta components of each 12S toxin. A molecular weight of 350,000 was determined for each 12S toxin and 150,000 for Ealpha and Ebeta. Owing to the structure comprising the subunits Ealpha and Ebeta, 12S toxins are much more stable than Ealpha at low pH values and high temperatures. Such was also the case with type A 19S toxin and its alpha component. The Ealpha component alone accounts for the total toxicity of type E toxin. The toxic substance detected in the blood of the animals administered 12S toxins orally or parenterally was identified as Ealpha from the molecular size and the chromatographic pattern. Prototoxin escaping from detoxification in the stomach owing to the subunit structure may undergo dissociation in the intestine to release the Ealpha component. After absorption, the activated Ealpha appeared in the circulating blood without any further signs of dissociation or enzymatic digestion.     

Toxin Production in Clostridium botulinum as Demonstrated by Electron Microscopy

Spheroplasts of Clostridium botulinum 62A were prepared with the use of lysozyme. These spheroplasts were then exposed to ferritin-labeled type A antitoxin. Ultrathin sections of these specimens revealed the ferritin-labeled antibody symmetrically arranged around the outer spore coats but not within the spore cortex. The ferritin-labeled antibody was also observed in the bacterial cytoplasm. Here it was arranged in aggregates and strands, although it was not associated with any identifiable cell structure. Controls included sections of C. botulinum spheroplasts treated with a 1.5% solution of ferritin as well as spheroplasts of C. roseum and Bacillus subtilis treated with conjugated type A antitoxin or a 1.5% solution of ferritin. No intracellular or extracellular ferritin was demonstrable in these specimens.     

Molecular Composition of the 16S Toxin Produced by a Clostridium botulinum Type D Strain, 1873

The 16S toxin was purified from a Clostridium botulinum type D strain 1873 (D-1873). Furthermore, the entire nucleotide sequences of the genes coding for the 16S toxin were determined. It became clear that the purified D-1873 16S toxin consists of neurotoxin, nontoxic nonhemagglutinin (NTNH), and hemagglutinin (HA), and that HA consists of four subcomponents, HA1, HA2, HA3a, and HA3b, the same as type D strain CB16 (D-CB16) 16S toxin. The nucleotide sequences of the nontoxic components of these two strains were also found to be identical except for several bases. However, the culture supernatant and the purified 16S toxin of D-1873 showed little HA activity, unlike D-CB16, though the fractions successively eluted after the D-1873 16S toxin peak from an SP-Toyopearl 650S column showed a low level of HA activity. The main difference between D-1873 and D-CB16 HA molecules was the mobility of the HA1 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Therefore it was presumed that the loss of HA activity of D-1873 16S toxin might be caused by the differences of processing HA after the translation.     

Kinetics of the Thermal Inactivation of Type E Clostridium botulinum Toxin

Rate of inactivation curves for the "free" toxin, prototoxin, or activated toxin in crude filtrates of Clostridium botulinum type E were nonlinear, consisting of a fast inactivating rate followed by a slow inactivating rate. Thermodynamic parameters were calculated over a temperature range of 125 to 145 F (51.7 to 62.8 C) for the two different inactivation rates. Energy of activation was low at the lower temperature and high at the higher temperature. The thermal requirement for inactivating similar concentrations of the "free" toxin, prototoxin, or activated toxin was considered to be the same.     

Storage Stability of Clostridium botulinum Toxin and Spores in Processed Cheese

Growth initiated from detoxified spores of Clostridium botulinum 62A resulted in toxin production of 50 to 10,000 mouse lethal doses (MLD) per gram of processed soft surface-ripened cheese. Regular assays during subsequent storage of toxic samples at 2 to 4 C revealed a characteristic two- to fivefold increase in toxin titer during the initial 1 week to 12 months of storage. Thereafter, the toxin titer remained constant for 2 to 4 years, after which the toxicity declined rapidly. At the end of 6 years of storage at 2 to 4 C, the samples still contained 20 to 5,000 MLD of toxin per gram, with the usual toxin level at 200 to 500 MLD. Toxic culture filtrates of C. botulinum incorporated into cheese and stored at 30 C for 60 days showed no decline in toxin in processed type I cheese, but toxin decreased slightly in processed type II and type III cheese. The surface flora of these cheeses did not attack but, on the contrary, protected C. botulinum toxin during storage at 30 C. Initial difficulties in recovering C. botulinum organisms from type I cheese were traced to growth inhibitory activity which could be removed by washing with distilled water in a centrifuge. Viable spores or vegetative cells could be recovered from all samples after 4 to 5 years of storage at 2 to 4 C. After 6 years, organisms were recovered from all except three samples of type I cheese. Two other samples showed a large decrease in viable organisms. In type III cheese, spores remained remarkably stable for 6 years at the level of the initial inoculum, i.e., approximately 10(5) spores per gram.     

Activation of Clostridium botulinum Type B Toxin by an Endogenous Enzyme

It was previously postulated, based on indirect evidence, that Clostridium botulinum type B produces neurotoxin which is initially of low toxicity but which then becomes activated to highly toxic form by the action of an endogenous enzyme(s). The first direct in vitro experimental evidence in support of this hypothesis is presented here. The mildly active toxin (progenitor toxin) produced by C. botulinum type B (Lamanna) was isolated from the filtrate of a 24-hr culture and partially purified chromatographically. An enzyme that activates the progenitor toxin was also isolated from the filtrate of a 96-hr culture and purified 200-fold. The enzyme hydrolyzes synthetic substrates of trypsin but not of chymotrypsin.     

Molecular Analysis of an Extrachromosomal Element Containing the C2 Toxin Gene Discovered in Clostridium botulinum Type C

Clostridium botulinum cultures are classified into seven types, types A to G, based on the antigenicity of the neurotoxins produced. Of these seven types, only types C and D produce C2 toxin in addition to the neurotoxin. The C2 toxin consists of two components designated C2I and C2II. The genes encoding the C2 toxin components have been cloned, and it has been stated that they might be on the cell chromosome. The present study confirmed by using pulsed-field gel electrophoresis and subsequent Southern hybridization that these genes are on a large plasmid. The complete nucleotide sequence of this plasmid was determined by using a combination of inverse PCR and primer walking. The sequence was 106,981 bp long and contained 123 potential open reading frames, including the c2I and c2II genes. The 57 products of these open reading frames had sequences similar to those of well-known proteins. It was speculated that 9 these 57 gene products were related to DNA replication, 2 were responsible for the two-component regulatory system, and 3 were σ factors. In addition, a total of 20 genes encoding proteins related to diverse processes in purine catabolism were found in two regions. In these regions, there were 9 and 11 genes rarely found in plasmids, indicating that this plasmid plays an important role in purine catabolism, as well as in C2 toxin production.Clostridium botulinum is a gram-positive, spore-forming, anaerobic bacterium. Cultures of this species produce poisonous botulinum neurotoxins (BoNTXs) that are lethal to humans and animals and are classified into seven types, types A to G, based on the antigenicity of the BoNTXs produced (actually, the cultures producing type G toxin were recently classified in a new species, Clostridium argentinens [40]).C. botulinum type C and D cultures produce a binary C2 toxin in addition to C (or C1) and D BoNTXs; this additional toxin consists of two nonlinked proteins, C2I and C2II (28), that occur independently in the culture supernatant and are not chemically joined to each other. The C. botulinum C2 toxin used here is a representative of the family of binary actin-ADP-ribosylating toxins, which includes, in addition to C2 toxin, the Clostridium perfringens iota toxin, Clostridium difficile toxin, Clostridium spiroforme toxin, and the vegetative insecticidal proteins from Bacillus cereus (4).The enzyme component of C2 toxin (C2I) ADP ribosylates G-actin at arginine 177 (1). This leads to depolymerization of actin filaments and finally to cell rounding. The proteolytically activated binding-translocation component (C2IIa) forms heptamers, which assemble with C2I and bind to the cellular receptor (5). Following receptor-mediated endocytosis, C2IIa forms pores in the membrane of acidic endosomes. Subsequently, C2I translocates across the membrane into the cytosol through these C2IIa pores.The production of C1 and D BoNTXs is governed by bacteriophages (12, 13, 18, 19, 26), and both toxin genes have been cloned from the corresponding phage DNAs (16, 21). Recently, we determined the whole-genome sequence of a type C toxin-converting phage (c-st) genome (31). Eklund et al. reported that C2 toxin toxigenicity (mouse lethality) became clear when strains were cultured in fortified egg-meat medium and the culture supernatants were treated with trypsin (12). They also reported that C2 toxin production was not related to the BoNTX-converting phages; some non-BoNTX-producing cells produced C2 toxin. Fujii et al. (15) and Kimura et al. (22) determined the whole nucleotide sequences of the c2I and c2II genes and speculated that these genes might be located on the bacterial chromosome (15, 22).In this study, we determined that these genes are present not on the cell chromosome but on a large plasmid; we first speculated that this was this was the case based on the results of both pulsed-field gel electrophoresis (PFGE) and Southern hybridization analysis and then confirmed it by determining the complete nucleotide sequence of the plasmid. Since the plasmid was extremely unstable, we could not purify the complete plasmid DNA; therefore, we determined the whole-genome sequence by using the inverse PCR method, which enables rapid determination of the flanking regions of unknown sequences and determination of unidentified sequences in the genome, and the primer-walking method. This is the first case in which an entire DNA sequence of a large plasmid was determined using only these two procedures. The process used to determine the whole-plasmid DNA sequence and several interesting features of the plasmid are described below.