Takao Tsuji

A unique amino acid sequence of the B subunit of a heat-labile enterotoxin isolated from a human enterotoxigenic Escherichia coli

The purified B subunit of heat-labile enterotoxin produced from a human strain, 240-3, of enterotoxigenic Escherichia coli (LTh(240-3] was carboxymethylated, succinylated, digested with chymotrypsin and subjected to high performance liquid chromatography (HPLC), and the amino acid compositions of the peptide peaks from the column were analyzed and compared with the data reported by Yamamoto and Yokota (J. Bacteriol. 155, 728.1983), who deduced the amino acid sequence of LTh(H10407) from the DNA sequence of a human strain H10407. Only one fraction differed in amino acid composition from that reported by them. This fraction was found to consist of peptides with the sequences Arg-Asn-Thr-Gln-Ile-Tyr and Arg-Ile-Ala-Tyr. Yamamoto and Yokota reported the sequence of the latter peptide as Arg-Ile-Thr*-Tyr, which corresponds to the peptide from 73rd to 76th from amino (N-) terminus. Thus amino acid residue 75 from the N-terminus of LTh-B(240-3) is alanine, not threonine. The B subunit of cholera toxin also has alanine at position 75. LTh(240-3) appeared similar to LTh(H10407) in an Ouchterlony test, vascular permeability test and GMI ganglioside ELISA. These data show that substitution of threonine for alanine at position 75 from the N-terminus does not affect the immunological and biological characteristics of LTh.

Demonstration of enterotoxigenic Escherichia coli in diarrheic broiler chicks.

An investigation was made to survey the possible presence of enterotoxigenic Escherichia coli (ETEC) in the stools of diarrheal chicks.We analyzed two outbreaks of diarrhea in broiler chicks at two independent farms in the Philippines, from which no pathogens other than Escherichia coli were found. In one outbreak at Farm # 1, all 42 isolates produced heat-labile enterotoxin (LT), with 3 of these isolates also producing heat-stable enterotoxin (ST). The 0 serotypes of 15 strains tested randomly could not be identified as any known serotype (0-antigen; 1–170). In another outbreak at Farm #2, 7 out of 52 isolates produced only LT, their subtypes being identified as O-149 or O-8, common serotypes in pig ETEC. Strains from Farm #1 did not produce any pili usually found in human ETEC. We believe this to be the first isolation of ETEC from diarrheal chicks.

Studies on the binding substances on human erythrocytes for the heat-labile enterotoxin isolated from porcine enterotoxigenic Escherichia coli

The binding substance for the heat-labile enterotoxin (LTp) isolated from porcine enterotoxigenic Escherichia coli was studied by competitive binding assays. The binding of 125I-labeled LTp to neuraminidase-treated human type A erythrocytes was most effectively inhibited by ganglioside GM1 among inhibitors used. Mono-, di- and polysaccharides, glycoproteins and lectins were over 10(4)-times less potent inhibitors. Similar results were also obtained in competitive binding assays with 3H-labeled ganglioside GM1 and LTp-coupled Sepharose 4B. On the other hand, hemagglutination of neuraminidase-treated human type A erythrocytes by LTp was inhibited by methyl alpha-D-galactopyranoside, galactose, melibiose and some glycoproteins, but not effectively inhibited by ganglioside GM1 at the highest concentration used. Preincubation of LTp with an appropriate amount of ganglioside GM1 resulted in much higher hemagglutination than LTp alone. Although these findings show that there may be fundamental differences between interactions with ganglioside GM1 in hemagglutination compared to interactions with ganglioside GM1 in binding, the predominant binding substance for LTp on neuraminidase-treated human type A erythrocytes is suggested to be ganglioside GM1.

Binding and hemagglutinating properties of the B subunit(s) of heat-labile enterotoxin isolated from human enteroxigenic Escherichia coli

The binding and hemagglutinating activities of the B subunit(s) of the heat-labile enterotoxin (LTh-B) isolated from human enterotoxigenic Escherichia coli were investigated. The binding of 125I-labeled LTh-B to neuraminidase-treated human type B erythrocytes was most effectively inhibited by ganglioside GM1. A number of mono-, di- and polysaccharides, as well as several glycoproteins were at least 500 times less potent inhibitors. However, hemagglutination was effectively inhibited by galactose, melibiose and hog A + H but not by ganglioside GM1. Preincubation of the LTh-B with ganglioside GM1 gave much stronger hemagglutination than LTh-B alone. These results suggest that the predominant binding substance for LTh-B on neuraminidase-treated human type B erythrocytes is ganglioside GM1, but indicate that the interaction of LTh-B with ganglioside GM1 is different in hemagglutination.

Detection of IgA protease from Haemophilus influenzae by immunoblotting

IgA protease produced by various strains of Haemophilus infuenzae can digest serum IgA and yield its fragments which can react with anti-IgA serum. We assayed IgA protease activity by detecting the digests of IgA by SDS-PAGE and immunoblotting. The digests were separated with SDS-PAGE, transferrend to nitrocellulose membranes and detected with anti- (α chain of human IgA, its Fab and its Fc) immunoglobulin conjugated peroxidases.Using this method, we can determine which type of IgA protease is produced by various of H. infuenzae strains. All the 20 strains isolated from respiratory tracts produced IgA protease.

Crystallization and preliminary X-ray diffraction study of the heat-labile enterotoxin B subunit produced by enterotoxigenic Escherichia coli.

Heat-labile enterotoxin LT produced by enterotoxigenic Escherichia coli is composed of A and B subunits. The A subunit is enzymatically active; whereas, through the action of the B subunit, the toxin binds to the receptor, a GM1 ganglioside present on the cell surface. Crystals of the LT-B subunit were formed at room temperature by vapor diffusion with polyethylene glycol in the presence of the non-ionic detergent beta-octylglucoside. The crystals were characterized by X-radiation as orthorhombic, space group P2(1)2(1)2(1), with unit cell dimensions of a = 224.1 A, b = 65.3 A, c = 118.4 A. They diffract X-rays to a resolution of at least 2.5 A and are stable to X-rays.

Heterogeneity of Enterotoxins of Vibrio Cholerae and Enterotoxigenic Escherichia Coli

The molecular structure of heat-labile enterotoxin (LT) of enterotoxigenic Escherichia coli is similar to that of cholera enterotoxin (CT). Dallas and Falkow(1) showed that LT synthesized by minicells containing Ent plasmid DNA was composed of two distinct proteins with molecular weights of 25,500 and 11,500 daltons, values similar to those of the A and B subunits of CT. Clements and Finkelstein(2) demonstrated more directly that the subunit structure of purified LT was very similar to that of CT; that is LT consisted of two subunits, A and B, with molecular weights of 28,000 and 11,500, respectively. Moreover, similarities in the amino acid sequences of the A and B subunits of CT and LT were reported by Spicer et al.(3) and Dallas and Falkow(4), respectively.

Immunological and molecular heterogeneity of heat-labile enterotoxins from human and porcine enterotoxigenic Escherichia coli

The molecular structure of heat-labile enterotoxin (LT) of enterotoxigenic Escherichia coli is similar to that of cholera enterotoxin (CT). Dallas and Falkow (5) showed that LT synthesized by minicells containing Ent plasmid DNA was composed of two distinct proteins with molecular weights of 25,000 and 11,500 daltons, values similar to those of the A and B subunits of CT. Clements and Finkelstein (3) demonstrated more directly that the subunit structure of purified LT was very similar to that of CT; that is, LT consisted of two subunits, A and B, with molecular weights of 28,000 and 11,500, respectively. Moreover, similarities in the amino acid sequences of the A and B subunits of CT and LT were reported by Spicer et al. (17) and Dallas and Falkow (6), respectively.