Development of an operational synaptobrevin-based fluorescent substrate for tetanus neurotoxin quantification.

Tetanus neurotoxin (TTx), produced by Clostridium tetani, is a two-chain polypeptide with a heavy molecular chain (HC; 100 kDa) and a light molecular chain (LC; 50 kDa) linked by a disulphide bridge. The low-molecular-mass chain is classified as a zinc metalloprotease (EC 3.4.24.68) with specific hydrolysis on synaptobrevin. With the known enzymic activity for the LC of TTx, we developed a quantification method using a quenched fluorescence peptide substrate based on the synaptobrevin sequence (fragments 73-82), suitable for direct determination of the whole TTx (HC+LC) even in crude production batches, without the necessity of purification and reduction steps to isolate the LC of TTx. The rate of substrate hydrolysis was 200 nmol/min and it was totally inhibited by EDTA, anti-recombinant fragment C antibody, and the cleavage was in a single bond (Gln-Phe) with purified and crude TTx. Besides, ELISA applied to the anti-TTx serum produced at our Institute showed cross-reaction with every fraction of the crude TTx extract. Another aspect is that TTx activity depends on the storage time, reaching a maximum on day 10. The results obtained suggest that the use of the new fluorescent substrate, Abz-synaptobrevin(73-82)-EDDnp, enables easy and quick determination of TTx. It is a good alternative to some of the existing methods such as flocculation assay, and it can replace, under some conditions, the biological assays (minimal mortal dose).

Detection of type A, B, and E botulism neurotoxin genes in Clostridium botulinum and other Clostridium species by PCR: evidence of unexpressed type B toxin genes in type A toxigenic organisms.

We studied the effectiveness of the PCR in detecting the type A, B, and E botulism neurotoxin genes in 209 strains of Clostridium botulinum and 29 strains of other Clostridium spp. All 79 strains that produced type A toxin, 77 strains that produced type B toxin, and 51 organisms that produced type E toxin (46 C. botulinum and 5 C. butyricum) were PCR positive in reactions with primers targeting sequences specific for their respective toxin genes. The PCR for type A toxin was positive for one type B toxin-producing strain that produced a small amount of type A toxin in addition to a large amount of type B toxin. Surprisingly, the type B toxin gene was detected in addition to the type A toxin gene in 43 type A toxin-producing strains, only 1 of which could be shown by bioassay to produce biologically active type B toxin in culture. The type B gene was also detected in two strains of C. subterminale, which were determined to be nontoxigenic by bioassay. While the PCR was sensitive and specific in detecting the neurotoxin genes, the discovery of unexpressed toxin genes indicates that PCR results may not be adequate for establishing type B neurotoxigenicity.

Chelation of zinc antagonizes the neuromuscular blocking properties of the seven serotypes of botulinum neurotoxin as well as tetanus toxin.

Botulinum neurotoxin types A, B (unactivated and activated), C, D, E, F and G, as well as tetanus toxin, paralyzed transmission in mouse phrenic nerve-hemidiaphragm preparations. Toxin-induced blockade of transmission was antagonized by chelators [e.g., ethylenediamine tetraacetic acid, tetrakis(2-pyridylmethyl)ethylenediamine or diethylene-triaminepentaacetic anhydride], but this effect was dependent on incubation conditions. Pretreatment of toxin with chelators failed to produce antagonism, but pretreatment of tissues did produce antagonism. Of the various chelators tested, tetrakis(2-pyridylmethyl)ethylenediamine produced the greatest effect. Antagonism of toxin-induced neuromuscular blockade could be partially reversed by washing chelators from tissues and could be fully reversed by adding an excess of zinc. The ability of chelators to antagonize clostridial neurotoxins was specific and did not extend to phospholipase A2 neurotoxins. Ligand-binding studies with radioiodinated toxin and brain membrane preparations showed that chelators did not antagonize toxicity by inhibiting toxin association with receptors. Similarly, pharmacological experiments with unlabeled toxin- and type-specific antibodies demonstrated that chelators did not act by blocking receptor-mediated internalization of toxin. The chelators appeared to exert their effects by antagonizing the intracellular actions of clostridial neurotoxins. Electrophysiological studies showed that chelators, at concentrations relevant to antagonism of botulinum neurotoxin and tetanus toxin, did not enhance transmitter release.(ABSTRACT TRUNCATED AT 250 WORDS)

Nucleotide sequence of the gene coding for Clostridium barati type F neurotoxin: comparison with other clostridial neurotoxins.

The neurotoxin gene from Clostridium barati ATCC43756 was cloned as a series of overlapping polymerase chain reaction (PCR) generated fragments using primers designed to conserve toxin sequences previously published. The toxin gene has an open reading frame (ORF) of 1268 amino acids giving a calculated molecular mass of 141,049 Da. The sequence identity between the C. barati ATCC43756 and non-proteolytic C. botulinum 202F neurotoxins is 64.2% for the light chain and 73.6% for the heavy chain. This is much lower than reported identities for the type E neurotoxins from C. botulinum and C. butyricum (96% identity between light chains and 98.8% between the heavy chains). Previously identified conserved regions in other botulinal neurotoxins were also conserved in that of C. barati. An ORF upstream of the toxin coding region was revealed. This shows strong homology to the 3' end of the gene coding for the nontoxic-nonhemagglutinin (NTNH) component of the progenitor toxin from C. botulinum type C neurotoxin.

[Development of an animal model of autonomic over-activity in tetanus].

Although present day progressive respiratory management has decreased the tetanus mortality rate, tetanus-related autonomic overactivity remains a lethal condition. The cause, pathophysiology, and treatment of this condition are controversial and no animal model has ever been reported. So we tried to make a rabbit model of this condition. At first we injected 30 micrograms of purified tetanus toxin intravenously. Blood pressure oscillation occurred, but the degree of oscillation was smaller than that was seen in the clinical cases. The degree of oscillation had not changed by addition of 30 micrograms purified toxin 12 hours after first injection. Secondly, 7 days before the experiment we injected 0.5 micrograms of purified tetanus toxin intravenously. On the experiment day, we injected 30 micrograms of purified toxin again. By this method there occurred blood pressure oscillation as same as that was seen in the clinical cases. But no rabbits progressed to circulatory collapse by purified toxin. Then to the last group of rabbits, we injected crude tetanus toxin for purified toxin. The degree of blood pressure oscillation was same as that of cases in which purified toxin was injected, and all rabbits progressed to circulatory collapse.