Toshio Miwatani

Amino acid sequence of a heat-stable enterotoxin isolated from enterotoxigenic Escherichia coli strain 18D

A heat‐stable enterotoxin produced by a strain of enterotoxigenic Escherichia coli 18D was purified by ion‐exchange and reversed‐phase high‐pressure liquid chromatography. The amino acid sequence of the purified toxin was determined by Edman‐degradation and a combination of fast atom bombardment mass spectrometry and carboxypeptidase digestion to be Asn‐Thr‐Phe‐Tyr‐Cys‐Cys‐Glu‐Leu‐Cys‐Cys‐Asn‐Pro‐Ala‐Cys‐Ala‐Gly‐Cys‐Tyr.

Essential structure for full enterotoxigenic activity of heat-stable enterotoxin produced by enterotoxigenic Escherichia coli.

Several analogues of heat‐stable enterotoxins (STh and STP) produced by enterotoxigenic Escherichia coli were synthesized. Peptides (STh[6–18] and STP[5–17]) consisting of 13 amino acid residues from the Cys residue near the N‐terminus to the Cys residue near the C‐terminus and linked by three disulfide bonds had the same biological and immunological properties as native STh and STP, respectively. The results indicated that the sequence with the 13 amino acid residues and three disulfide linkages is essential for full biological activity of ST.

Production of paralytic shellfish toxins by a bacterium Moraxella Sp. isolated from Protogonyaulax tamarensis

A bacterium Moraxella sp. isolated from Protogonyaulax tamarensis was cultured in various conditions. Changes of toxicity and toxin components of the cells during culture were analyzed by bioassay and HPLC-fluorometric analysis. Toxin productivity of Moraxella sp. increased when it was cultured in nutrition-deficient environments. The main toxins produced by Moraxella sp. in these conditions were gonyautoxins (GTXs), mainly GTX 1 and 4 which are major toxins of P. tamarensis.

Mode of disulfide bond formation of a heat-stable enterotoxin (STh) produced by a human strain of enterotoxigenic Escherichia coli

To determine the modes of three disulfide linkages in the heat‐stable enterotoxin (STh) produced by a human strain of enterotoxigenic Escherichia coli, we synthesized STh(6–18), which consists of 13 amino acid residues and has the same intramolecular disulfide linkages as native STh [(1985) FEBS Lett. 181, 138–142], by stepwise and selective formation of disulfide bonds using different types of removable protecting groups for the Cys residues. Synthesis of the peptide with different modes of disulfide bond formation provided three peptides consistent with standard STh(6–18) in their physicochemical and biological properties, thereby indicating that the disulfide bonds in STh(6–18) are

Characterization of a new thermostable direct haemolysin produced by a Kanagawa-phenomenon-negative clinical isolate of Vibrio parahaemolyticus

The production of two haemolysins, thermostable direct haemolysin (Vp-TDH) and a Vp-TDH-related haemolysin (Vp-TRH), by clinical isolates of Vibrio parahaemolyticus has previously been reported. Here we describe a third type of haemolysin (named Vp-TDH/I), which is produced by a clinical isolate (strain TH012) that is Kanagawa phenomenon negative. Vp-TDH/I was purified by a series of column chromatographies on DEAE-Sephadex A25, hydroxyapatite, Sepharose 4B and Mono Q. By physicochemical, biological and immunological analyses, Vp-TDH/I was demonstrated to be similar, but not identical, to Vp-TDH and Vp-TRH. The gene encoding Vp-TDH/I was cloned and the deduced amino acid sequence of Vp-TDH/I confirmed that Vp-TDH/I has a sequence different from those of previously known Vp-TDH and Vp-TRH. Not only purified Vp-TDH/I but also live cells of the Vp-TDH/I-producing strain induced fluid accumulation in ligated rabbit intestine. We conclude that this clinical isolate produces a new type of Vp-TDH-related haemolysin, which may be involved in the pathogenesis of this organism.

Amino acid sequence of heat-stable enterotoxin produced by Vibrio cholerae non-01

The amino acid sequence of heat‐stable enterotoxin, produced by Vibrio cholerae non‐01 and isolated from its culture supernatant, was determined by both Edman degradation of native and reductively carboxy‐methylated enterotoxin and also a combination of fast atom bombardment mass spectrometry and carboxy‐peptidase Y digestion of native enterotoxin to be as follows: Ile‐Asp‐Cys‐Cys‐Glu‐Ile‐Cys‐Cys‐Asn‐Pro‐Ala‐Cys‐Phe‐Gly‐Cys‐Leu‐Asn. This sequence is very similar, but not identical, to those of heat‐stable enterotoxins produced by enterotoxigenic Escherichia coli and Yersinia enterocolitica.

Chemical synthesis of a highly potent and heat-stable analog of an enterotoxin produced by a human strain of enterotoxigenic Escherichiacoli

A shorter analog of a heat-stable enterotoxin produced by a human strain of enterotoxigenic Escherichia coli SK-1, consisting of 14 amino acid residues including 6 half-cystine residues, was synthesized by conventional methods. The peptide was evaluated for ability to induce intestinal secretion in suckling mice and for stability at high temperature under various conditions. The peptide was 2-5 times more potent than native toxin and was still toxic after heat-treatment at 120 degrees C for 30 min.

Production of monoclonal antibodies against thermostable direct hemolysin of Vibrio parahaemolyticus and application of the antibodies for enzyme-linked immunosorbent assay.

A total nine hybridoma cell lines that produced monoclonal antibodies against thermostable direct hemolysin (Vp-TDH), a possible pathogenic toxin, of Kanagawa phenomenon-positive Vibrio parahaemolyticus was isolated and characterized. These monoclonal antibodies (mAbs) were divided into a minimum of five different specificity groups, including mAbs specific to Vp-TDH and common to Vp-TDH and Vp-TRH, a Vp-TDH-related hemolysin produced by Kanagawa phenomenon-negative V. parahaemolyticus. An enzyme-linked immunosorbent assay (ELISA) using mAb-1-D, a mAb specific for Vp-TDH, was developed for specific detection of Vp-TDH. On the other hand, the ELISA using mAb-9-D, and mAb common to both Vp-TDH and Vp-TRH, could be used for detection of both Vp-TDH and Vp-TRH. Thus, by combining these two ELISAs differential detection of Vp-TDH and Vp-TRH can be performed. Hence, the two ELISAs were applied for various strains of V. parahaemolyticus and it was found that most Kanagawa phenomenon-positive and -negative clinical isolates produced Vp-TDH and Vp-TRH, respectively, but all environmental strains, that were Kanagawa phenomenon-negative, produced neither toxin.

Comparative analysis of the hemolysin genes of Vibrio cholerae non‐01, V. mimicus, and V. hollisae that are similar to the tdh gene of V. parahaemolyticus

The genes encoding the hemolysins similar to the thermostable direct hemolysin (tdh gene) of Vibrio parahaemolyticus were cloned from chromosomes of V. mimicus and V. hollisae. These cloned hemolysin genes and previously cloned tdh genes of V. parahaemolyticus and V. cholerae non-01 were compared by physical mapping and by hybridization with oligodeoxyribonucleotide probes. The nucleotide sequences in the coding regions of all the cloned hemolysin genes were very homologous and had only minor variations but the sequences flanking the homolysin genes were dissimilar, indicating that the hemolysin genes have a common ancestor and suggesting that they may have been transferred between Vibrio species as a descrete genetic unit.

Characterization of Purified Shiga Toxin from Shigella dysenteriae 1

Shiga toxin was purified from the culture supernatant of Shigella dysenteriae 1 by ammonium sulfate fractionation, DEAE‐cellulose column chromatography and repeated chromatofocusing column chromatography. About 1.6 mg of purified Shiga toxin was obtained from 15 liters of culture with a yield of about 27%. The molecular weight of purified Shiga toxin was estimated to be 62,000. The toxin consisted of A and B subunits with molecular weights of about 30,000 and 5,000–6,000, respectively. The isoelectric point of purified Shiga toxin was 7.0. Purified Shiga toxin showed the following biological activities: lethal toxicity to mice when injected intraperitoneally with an LD50 of 28 ng per mouse; cytotoxicity to Vero cells, killing about 50% of the cells at 1 pg and all of the cells at 10 pg; and fluid accumulation in rabbit ileal loops at a concentration of more than 1 μg.