Bacteriophage conversion of heat-labile enterotoxin in Escherichia coli.

A temperate phage designated obeta1 (omicron beta) was mitomycin C induced and isolated from heat-labile enterotoxin (LT)-producing Escherichia coli E2631-C2. Phage obeta1 infected the nonlysogenic, nontoxigenic, mitomycin C-sensitive strain of E. coli K-12 (CSH38) and converted it to lysogeny and enterotoxigenicity. After the establishment of lysogeny, E. coli CSH38(obeta1) produced produced LT and phage particles at maximal levels following mitomycin C induction. The LT Tox+ character is carried by the temperate phage obeta1.     

Cellular location of heat-labile enterotoxin in Escherichia coli.

We demonstrated that both the A and B subunits of heat-labile enterotoxin from Escherichia coli are located in the periplasm. The toxin was shown to form aggregates in Tris-EDTA buffers which are routinely used for isolating membranes. The aggregates pellet upon centrifugation, and this may explain why several previous investigators have concluded that enterotoxin is associated with membranes.     

Release of heat-labile enterotoxin subunits by Escherichia coli.

Most of the heat-labile enterotoxin (LT) synthesized by Escherichia coli is cell associated; however, a small portion of LT (approximately 10%) is released by bacterial cells into the culture supernatant. The LT subunit B (LT-B) produced by a cloned LT-B gene (tox B) was released in amounts equal to the parent LT release. In contrast, no release of LT subunit A (LT-A) or its smaller derivatives was observed in strains containing cloned toxA genes. The data suggest that LT-B is necessary for the release of LT-A across the bacterial membrane.     

Our experiences with isolation of heat-labile enterotoxin from Escherichia coli

From the Escherichia coli strain isolated from a patient suffering from diarrhoea a homogenate and concentrated culture filtrate were prepared. From these materials the heat-labile enterotoxin was isolated after its elution with 0.2 M D-galactose from Sepharose 6B column. The obtained enterotoxin was positive in the rabbit ileal loop test up to a concentration of 1 microgram protein/ml. In the immunodiffusion test it reacted in a concentration up to 5 micrograms protein/ml with anticholeragen and in a concentration up to 30 micrograms protein/ml with its specific antiserum. This antiserum was prepared by intramuscular immunization of rabbits by enterotoxin with complete Freund's adjuvant.     

Sequence of heat-labile enterotoxin of Escherichia coli pathogenic for humans.

We determined the complete nucleotide sequence of the toxB gene (375 base pairs in length), which encodes the B subunit of heat-labile enterotoxin produced from Escherichia coli pathogenic for humans (hLT). The amino acid sequence of the B subunit of hLT was deduced from the nucleotide sequence. Consequently, it has become possible to study the homology between the B subunits of three similar toxins: hLT, LT produced from E. coli pathogenic for piglets (pLT), and cholera toxin (the latter two sequences have been reported by others). The three B subunits are all 103 amino acids in length. A comparison of the toxB gene and the eltB gene, which encodes the B subunit of pLT, showed a 98% homology at the nucleotide level and a 95% homology at the amino acid (of a precursor) level, indicating the possibility that the two genes share a common ancestor. With respect to the B-subunit sequences, the homologies between hLT and pLT, between hLT and cholera toxin, and between pLT and cholera toxin were 96, 81, and 79%, respectively. Several large common sequences are conserved by the three peptides. In contrast, no sequences are present in both pLT and cholera toxin but missing in hLT.     

Blood group antigen recognition by Escherichia coli heat-labile enterotoxin

In a number of bacterial infections, such as Helicobacter pylori, Campylobacter jejuni and Vibrio cholerae infections, a correlation between the severity of disease and blood group phenotype of infected individuals has been observed. In the present investigation, we have studied the molecular basis of this effect for enterotoxigenic Escherichia coli (ETEC) infections. ETEC are non-invasive bacteria, which act through second messenger pathways to cause diarrhea. It has been suggested that the major virulence factor of ETEC from human isolates, i.e. the human heat-labile enterotoxin (hLT), recognizes certain blood group epitopes, although the molecular basis of blood group antigen recognition is unknown. The 2.5 A crystal structure of the receptor-binding B-subunit of hLT in complex with the blood group A antigen analog GalNAcalpha3(Fucalpha2)Galbeta4(Fucalpha3)Glcbeta provides evidence of a previously unknown binding site in the native toxin. The structure reveals the molecular interactions underlying blood group antigen recognition and suggests how this protein can discriminate between different blood group epitopes. These results support the previously debated role of hLT in the blood group dependence of ETEC infections. Similar observations regarding the closely related cholera toxin in V. cholera infections are also discussed.     

Hemagglutinating activity of the heat-labile enterotoxin isolated from porcine enterotoxigenic Escherichia coli

Abstract The hemagglutinating activity of the heat-labile enterotoxin (LT_p) isolated from porcine enterotoxigenic Escherichia coli was studied by hemagglutination inhibition. The hemagglutinating activity of LT_p was enhanced 64–512-fold with pronase- and neuraminidase-treated human erythrocytes although both intact human and sheep erythrocytes were not agglutinated by LT_p at the highest concentration used. No enhancement was found in hemagglutination of neuraminidase-treated sheep erythrocytes by LT_p. Hemagglutination of pronase-treated human type A erythrocytes induced by LT_p was inhibited by melibiose and galactose among mono-, di-, and polysaccharides used as inhibitors. Galactose was a slightly better inhibitor than melibiose. These findings suggest that LT_p is a bacterial lectin specific for galactose.     

Purification and characterization of heat-labile enterotoxin isolated from chicken enterotoxigenic Escherichia coli

Abstract The heat-labile enterotoxin (LTc) isolated from chicken enterotoxigenic Escherichia coli was purified to homogeneity and its molecular and antigenic properties were compared with those of purified LTs from porcine and human enterotoxigenic Escherichia coli (LTp, LTh). The A subunit of LTc was identical to that of LTp and the B subunit of LTc was identical to that of LTh but not that of LTp, in mobility on SDS-polyacrylamide gel electrophoresis. Ouchterlony tests demonstrated that LTc is antigenically identical to LTh but not with LTp. The p I point and amino acid composition of LTc were also compared and the results suggest that chicken enterotoxigenic E. coli produced an LT similar to LTh.     

NAD-dependent ADP-ribosylation of arginine and proteins by Escherichia coli heat-labile enterotoxin.

Escherichia coli heat-labile enterotoxin (labile toxin, LT) catalyzed the hydrolysis of NAD to ADP-ribose and nicotinamide and the ADP-ribosylation of arginine (Moss, J., and Richardson, S.H. (1978) J. Clin. Invest. 62, 281-285). Analysis of the product of the ADP-ribosylation of arginine by nuclear magnetic resonance spectroscopy indicated that the reaction was stereospecific and resulted in the formation of alpha-ADP-ribosyl-L-arginine. This reaction product rapidly anomerized to yield a mixture of the alpha and beta forms. In the presence of [adenine-U-14C]NAD, E. coli enterotoxin catalyzed the transfer of the radiolabel to proteins; the ADP-ribosylation of proteins was inhibited by arginine methyl ester, an alternative substrate. Digestion of the 14C-protein with snake venom phosphodiesterase released predominantly 5'-AMP. No product was obtained with a mobility similar to that of 2'-(5'-phosphoribosyl)-5'-AMP. This result is consistent with the covalent attachment by the enterotoxin of ADP-ribose rather than poly(ADP-ribose) to protein. Thus, LT is catalytically equivalent to choleragen, an enterotoxin of Vibrio cholerae, and activates adenylate cyclase through a similar stereospecific ADP-ribosylation reaction.     

Escherichia coli heat-labile enterotoxin. Nucleotide sequence of the A subunit gene

We report the complete DNA sequence of the Escherichia coli elt A gene, which codes for the A subunit of the heat-labile enterotoxin, LT. The amino acid sequence of the LT A subunit has been deduced from the DNA sequence of elt A. The LT A subunit starts with methionine, ends with leucine, and comprises 254 amino acids. The computed molecular weight of LT A is 29,673. The A subunit of cholera toxin (CT A) has been shown to be structurally and functionally related to the LT A subunit. Comparison of the primary structure of LT A with the known partial amino acid sequence of CT A indicates that the 2 polypeptides share considerable homology throughout their sequences. The NH2-terminal regions exhibit the highest degree of homology (91%), while the COOH-terminal region, containing the sole cystine residue in each toxin is less conserved (approximately 52%). Alignment of homologous residues in the COOH-terminal regions of LT A and CT A indicates that a likely site for proteolytic cleavage of LT A is after Arg residue 188. The resulting A2 polypeptide would be 46 amino acids long, would contain a single cysteine residue, and have Mr = 5261. The elt A nucleotide sequence further predicts that the LT A protein is synthesized in a precursor form, possessing an 18-amino acid signal sequence at its NH2 terminus.     

Synthesis of a precursor to the B subunit of heat-labile enterotoxin in Escherichia coli.

Escherichia coli K-12 minicells were employed to investigate the biosynthesis of plasmid-encoded, heat-labile enterotoxin of E. coli. Two polypeptide species related to the B subunit of the toxin were expressed in the minicells. One of these polypeptides (molecular weight, 11,500) was immunoprecipitated by antiserum to cholera toxin. Because the B subunits of heat-labile enterotoxin and cholera toxin have common antigenic sites, we concluded that this species was the mature B subunit. The larger polypeptide (molecular weight, 13,000) is likely to be a precursor of the B subunit because it could be chased into the mature form. This conversion was inhibited by compounds which dissipate proton motive force, suggesting that processing requires energy.     

Escherichia coli heat-labile enterotoxin genes are flanked by repeated deoxyribonucleic acid sequences.

The enterotoxin regions of the heat-labile and heat-stable enterotoxin (LT+ ST+) plasmid, pJY11, originating in a clinically isolated Escherichia coli strain, have been isolated as various-sized deoxyribonucleic acid (DNA) fragments by using cloning vehicles. The structure of the LT+ region and its neighboring DNA regions was studied by utilizing these recombinant plasmids. The LT+ region consisted of at least two genes, toxA and toxB, which could complement each other in trans. The toxA- and toxB-encoded polypeptides (LT subunits A and B, respectively) were identified by their immunological cross-reactivity with Vibrio cholerae enterotoxin subunit A or B. These tox genes and the promoter(s) were localized with respect to the restriction endonuclease cleavage map. The LT+ region was flanked by repeated DNA sequences (designated as beta). Another tox gen(s), encoding ST (designated as toxS), which was also flanked by inverted, repeated DNA sequences (designated as alpha), was located between one of the beta sequences and the LT+ region. These novel DNA structures (beta-alpha-toxS-alpha-toxA-toxB-beta) suggest the possibility that the LT+ region is on a transposon containing an ST transposon within the structure.     

Cloning of genes that encode a new heat-labile enterotoxin of Escherichia coli.

The genes for a new enterotoxin were cloned from Escherichia coli SA53. The new toxin was heat labile and activated adenylate cyclase but was not neutralized by antisera against cholera toxin or E. coli heat-labile enterotoxin. Subcloning and minicell experiments indicated that the toxin is composed of two polypeptide subunits that are encoded by two genes. The two toxin subunits exhibited mobilities on polyacrylamide gels that are similar to those of cholera toxin and E. coli heat-labile enterotoxin subunits. A 0.8-kilobase DNA probe for the new enterotoxin failed to hybridize with the cloned structural genes for E. coli heat-labile enterotoxin.