Kouichi Kimura

The complete nucleotide sequence of the gene encoding the nontoxic component of Clostridium botulinum type E progenitor toxin

We have analysed the genes borne on a 6.0 kb HindIII fragment cloned from the chromosome of Clostridium botulinum type E strain Mashike. This fragment, cloned within plasmid pU9EMH, contains part of the structural gene for botulinum toxin type E neurotoxin as well as the entire structural gene for a nontoxic component of botulinum type E progenitor neurotoxin gene, ent-120. ent-120 is transcribed in the same direction as the neurotoxin gene and consists of one open reading frame encoding 1162 amino acid residues. Western blotting with anti-nontoxic component sera demonstrates that ent-120 encodes a protein of 120 kDa which forms part of the nontoxic component. ent-120 is homologous to an analogous gene found in botulinum type C strains (69.3% identity at the nucleotide level and 56.1% at the amino acid level). Two stretches of amino acids at the N-terminus of the ent-120 protein are highly homologous to amino acid sequences within the type E neurotoxin. The stop codon of the ent-120 gene is situated 27 nucleotides upstream from the start codon of the neurotoxin gene.

A zinc-protease specific domain in botulinum and tetanus neurotoxins

Neurotoxins produced by Clostridium botulinum are classified into groups (A to G) based on their serological nature. They consist of two subunits, heavy and light chains, linked by one or more disulphide bridges. The light chain is responsible for the blocking of acetylcholine release. Amino acid sequences of light chains have already been reported for botulinum toxins types A, C, D and E. Five highly homologous regions are found between these four toxins. One of these homologous regions, sequence HELIHSL, shows strong similarity with the active site of zinc-proteases. We suggest that inhibition of acetylcholine release might be associated with this protease activity.

Binding of botulinum type C1, D and E neurotoxins to neuronal cell lines and synaptosomes

Clostridium botulinum 125I-labeled Cl neurotoxin bound to NG108 hybridoma cell line. Unlabeled type Cl neurotoxin inhibited the binding of the labeled Cl toxin but neither types D nor E toxin. 125I-labeled type D neurotoxin bound to rat brain synaptosomes but did not bind to NG108 cells. It is suggested that receptors for types C and D or E toxins on neuronal cell membranes are different.

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 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 gtl 1 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‐β‐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.

Cloning and Whole Nucleotide Sequence of the Gene for the Light Chain Component of Botulinum Type E Toxin from Clostridium butyricum Strain BL6340 and Clostridium botulinum Type E Strain Mashike

Chromosomal DNAs were extracted from Clostridium butyricum strain BL6340 and Clostridium botulinum type E strain Mashike. The 6.0 Kbp fragment coding for the entire light chain (L) component and the N‐terminus of heavy chain (H) component of botulinum type E toxin was obtained from each extracted DNAs after digestion with HindIII. The entire nucleotide sequences for the light chain components of these cloned genes were determined, and the derived amino acid sequences were compared to each other, and with those of botulinum type A, Cl, D, and tetanus toxins reported previously. The cleavage site of L and H components of type E toxin was presumed to be Arg‐422. In a total of 422 amino acid residues of L component, 17 residues were different between butyricum and type E toxins, and all these differences were found within 200 residues of N‐terminus of L component. On the contrary, five regions showing highly homologous sequences were found in L components among these six toxins, and one more region between botulinum type E and tetanus toxins.

Oligo-2',5'-adenylate synthetase activity in cells persistently infected with human T-lymphotropic virus type I (HTLV-I)

Spontaneous production of interferon‐gamma (IFN‐γ) was shown in several T‐lymphoblastoid cell lines persistently infected with human T‐lymphotropic virus (HTLV‐1). However, the produced IFN‐γ was not always associated with the induction of the antivirus state. The induction of oligo‐2′,5′‐adenylate synthetase (2‐5AS) by IFN was studied in five human T‐cell lines persistently infected with HTLV‐I (MT‐1, MT‐2, SMT‐1, HUT 102 and OKM‐2). Four cell lines are able to produce IFN‐γ spontaneously, while the OKM‐2 cell line is not. Poor induction of 2‐5AS was recognized in three (MT‐1, MT‐2 and SMT‐1) of the four cell lines producing IFN‐γ, though the poor induction was improved after long‐term cultivation of cells with IFN‐α. On the contrary, in the OKM‐2 cell line. significant activity of the enzyme was induced by IFN‐α. Induction of 2‐5AS was not correlated with cell growth inhibition, but with the antivirus state. Furthermore, an inverse relationship between IFN‐γ production and 2‐5AS induction was demonstrated in these cell lines with the exception of HUT 102 cells.

Characterization of Neurotoxigenic Clostridium butyricum Strain by DNA Hybridization Test and by in vivo and in vitro Germination Tests of Spores

The germination of spores of a neurotoxigenic Clostridium butyricum strain (BL 6340), which was isolated from infant botulism in Italy, and that of a non‐toxigenic C. butyricum type strain (NCIB 7423) were studied. The spores of BL 6340 strain were killed at 80 C for 10 min, and required the mixture of L‐alanine, L‐lactate, glucose and bicarbonate for their optimal germination. These characteristics are the same as those of Clostridium botulinum type E strain, but different from those of NCIB 7423 strain. In a hybridization test, however, the labeled DNAs extracted from NCIB 7423 strain highly (98%) hybridized to the DNAs of the BL 6340 strain, but little (45%) to the DNAs of C. botulinum type E strain. The biochemical properties of the BL 6340 and NCIB 7423 strains were identical, but different from those of C. botulinum type E. These data confirmed that the BL 6340 strain belongs to C. butyricum species, but that only its characteristics of toxin production, its minimum requirements for germination, and the behavior of its spores to heat treatment are the same as those of C. botulinum type E. When conventionally raised suckling mice were injected with 5 × 107 spores of BL 6340 strain intra‐ or orogastrically, botulism was not observed. However, 8‐ to 13‐day‐old mice had type E botulinum toxin in the large intestine 3 days after introduction of its spores.

Construction and Expression of the Genes for Neurotoxins and Non-Toxic Components in C. Botulinum Types C and E

Botulinum toxins are classified into seven groups (type A to G) based on their antigenicity. Type C 1 (or C) toxin exists in large molecular sizes of 12S (300 kDa) and 16S (500 kDa) in culture supernatants or in an acid condition, and type E toxin exists in 12S size (Fig. 1). These large (progenitor) toxins are formed by association of the 7S neurotoxin (150 kDa) with a nontoxic component(s). In an alkaline condition, the 12S and 16S toxins dissociate into neurotoxin and nontoxic components. The neurotoxin is produced as a single polypeptide chain and is separated into two fragments, designated as the heavy (H) chain (100 kDa) and the light (L) chain (50 kDa), by reduction of a disulfide bond. On the contrary, the molecular constitution of nontoxic components is not clear. The nontoxic component of the 16S toxin shows hemagglutinating activity, but that of 12S toxin does not. It has been postulated) that the nontoxic component of 16S toxin is made up by conjugation of the nontoxic component of 12S toxin (designated as nontoxic-nonHA) with hemagglutinin (HA), and that this conjugation is not separated in an alkaline condition. Molecular mass (Mr) of nontoxic-nonHA component is approximately 120 – 140 kDa in any progenitor toxins, but that of HA is not clear because the preparation of HA alone has not yet been isolated. Suzuki et al. 2 reported that Mr. of the nontoxic component of type C 16S toxin was 240 kDa and that it was dissociated into five components (27, 35, 55, 115, and 120 kDa), indicating that HA consisted of several subcomponents. Ohishi et al. 3 clarified that the nontoxic components were necessary to maintain the oral toxicity or to cause food poisoning because they prevented the neurotoxin from degradation by gastric juice at a low pH.

Inhibition of norepinephrine secretion from digitonin permeabilized PC12 cells by botulinum type D toxin.

Botulinum type D neurotoxin inhibited Ca(2+)-evoked norepinephrine secretion in digitonin permeabilized PC12 cells. Inhibition by the toxin required prior incubation with dithiothreitol (DTT). The inhibition was dependent on both concentration and incubation times of the toxin, and was affected by Ca2+ concentration. With less than 0.7 microM Ca2+ almost complete inhibition was observed; however, above 0.7 microM, Ca2+ stimulated additional norepinephrine release in a dose-dependent manner.