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.     

Comparison of the glycolipid-binding specificities of cholera toxin and porcineEscherichia coli heat-labile enterotoxin: identification of a receptor-active non-ganglioside glycolipid for the heat-labile toxin in infant rabbit small intestine

The binding specificities of cholera toxin andEscherichia coli heat-labile enterotoxin were investigated by binding of¹²⁵I-labelled toxins to reference glycosphingolipids separated on thin-layer chromatograms and coated in microtitre wells. The binding of cholera toxin was restricted to the GM1 ganglioside. The heat-labile toxin showed the highest affinity for GM1 but also bound, though less strongly, to the GM2, GD2 and GD1b gangliosides and to the non-acid glycosphingolipids gangliotetraosylceramide and lactoneotetraosylceramide. The infant rabbit small intestine, a model system for diarrhoea induced by the toxins, was shown to contain two receptor-active glycosphingolipids for the heat-labile toxin, GM1 ganglioside and lactoneotetraosylceramide, whereas only the GM1 ganglioside was receptor-active for cholera toxin. Preliminary evidence was obtained, indicating that epithelial cells of human small intestine also contain lactoneotetraosylceramide and similar sequences. By computer-based molecular modelling, lactoneotetraosylceramide was docked into the active site of the heat-labile toxin, using the known crystal structure of the toxin in complex with lactose. Interactions which may explain the relatively high toxin affinity for this receptor were found.Abbreviations CT cholera toxin - CT-B B-subunits of cholera toxin - LT Escherichia coli heat-labile enterotoxin - hLT humanEscherichia coli heat-labile enterotoxin - pLT porcineEscherichia coli heat-labile enterotoxin - EI electron ionization     

Cloning, nucleotide sequence, and hybridization studies of the type IIb heat-labile enterotoxin gene of Escherichia coli.

Type IIb heat-labile enterotoxin (LT-IIb) is produced by Escherichia coli 41. Restriction fragments of total cell DNA from strain 41 were cloned into a cosmid vector, and one cosmid clone that encoded LT-IIb was identified. The genes for LT-IIb were subcloned into a variety of plasmids, expressed in minicells, sequenced, and compared with the structural genes for other members of the Vibrio cholerae-E. coli enterotoxin family. The A subunits of these toxins all have similar ADP-ribosyltransferase activity. The A genes of LT-IIa and LT-IIb exhibited 71% DNA sequence homology with each other and 55 to 57% homology with the A genes of cholera toxin (CT) and the type I enterotoxins of E. coli (LTh-I and LTp-I). The A subunits of the heat-labile enterotoxins also have limited homology with other ADP-ribosylating toxins, including pertussis toxin, diphtheria toxin, and Pseudomonas aeruginosa exotoxin A. The B subunits of LT-IIa and LT-IIb differ from each other and from type I enterotoxins in their carbohydrate-binding specificities. The B genes of LT-IIa and LT-IIb were 66% homologous, but neither had significant homology with the B genes of CT, LTh-I, and LTp-I. The A subunit genes for the type I and type II enterotoxins represent distinct branches of an evolutionary tree, and the divergence between the A subunit genes of LT-IIa and LT-IIb is greater than that between CT and LT-I. In contrast, it has not yet been possible to demonstrate an evolutionary relationship between the B subunits of type I and type II heat-labile enterotoxins. Hybridization studies with DNA from independently isolated LT-II producing strains of E. coli also suggested that additional variants of LT-II exist.     

Cloning and expression of the Salmonella enterotoxin gene.

This report examines the genetic basis for Salmonella typhimurium Q1 enterotoxin production. A 918-base-pair XbaI-HincII fragment of plasmid pJM17, composed of cholera toxin (CT) coding sequences (ctxAB), was used as a gene probe. With this probe, the S. typhimurium enterotoxin was identified on a 6.3-kilobase EcoRI-PstI fragment of chromosomal DNA from plasmidless strain Q1. We cloned this 6.3-kilobase fragment into Escherichia coli RR1. The genetic map of the cloned Salmonella enterotoxin (stx) gene was similar but not identical to the CT and E. coli heat-labile enterotoxin genes. By using synthetic oligonucleotides derived from the sequences of CT subunits A (ctxA) and B (ctxB), it was revealed that there were some conserved regions of DNA encoding the enterotoxins of strain Q1 and Vibrio cholerae. Expression of the cloned stx gene in minicells and subsequent Western blot (immunoblot) analysis with CT antitoxin demonstrated that the Salmonella enterotoxin had two or more subunits with molecular sizes of 45, 26, and 12 kilodaltons. Crude cell lysates of E. coli RR1(pCHP4), containing the cloned Salmonella enterotoxin gene, elicited fluid secretion in ligated rabbit intestinal loops and firm induration in rabbit skin. Both of these enterotoxic responses were neutralized by antisera specific for CT. Mucosal tissue from positive intestinal loops contained elevated levels of cyclic AMP. These data suggest some evolutionary relatedness between the enterotoxin genes of S. typhimurium and V. cholerae.     

Crystal structures exploring the origins of the broader specificity of escherichia coli heat-labile enterotoxin compared to cholera toxin

Cholera toxin (CT) and Escherichia coli heat-labile enterotoxin (LT) are structurally and functionally related and share the same primary receptor, the GM1 ganglioside. Despite their extensive similarities, these two toxins exhibit distinct ligand specificities, with LT being more promiscuous than CT. Here, we have attempted to rationalize the broader binding specificity of LT and the subtle differences between the binding characteristics of LTs from human and porcine origins (mediated by their B subunit pentamers, hLTB and pLTB, respectively). The analysis is based on two crystal structures of pLTB in complexes with the pentasaccharide of its primary ligand, GM1, and with neolactotetraose, the carbohydrate determinant of a typical secondary ligand of LTs, respectively. Important molecular determinants underlying the different binding specificities of LTB and CTB are found to be contributed by Ser95, Tyr18 and Thr4 (or Ser4 of hLTB), which together prestabilize the binding site by positioning Lys91, Glu51 and the adjacent loop region (50-61) containing Ile58 for ligand binding. Glu7 and Ala1 may also play an important role. Many of these residues are closely connected with a recently identified second binding site, and there appears to be cross-talk between the two sites. Binding to N-acetyllactosamine-terminated receptors is further augmented by Arg13 (present in pLT and some hLT variants), as previously predicted.     

Common architecture of the primary galactose binding sites of Erythrina corallodendron lectin and heat-labile enterotoxin from Escherichia coli in relation to the binding of branched neolactohexaosylceramide

The heat-labile enterotoxin from Escherichia coli (LT) is responsible for so-called traveller's diarrhea and is closely related to the cholera toxin (CT). Toxin binding to GM1 at the epithelial cell surface of the small intestine initiates the subsequent diarrheal disease. However, LT has a broader receptor specificity than CT in that it also binds to N-acetyllactosamine-terminated structures. The unrelated lectin from Erythrina corallodendron (ECorL) shares this latter binding property. The findings that both ECorL and porcine LT (pLT) bind to lactose as well as to neolactotetraosylceramide suggests a common structural theme in their respective primary binding sites. Superimposing the terminal galactose of the lactoses in the respective crystal structures of pLT and ECorL reveals striking structural similarities around the galactose despite the lack of sequence and folding homology, whereas the interactions of the penultimate GlcNAcb3 in the neolactotetraosylceramide differ. The binding of branched neolactohexaosylceramide to either protein reveals an enhanced affinity relative to neolactotetraosylceramide. The b3-linked branch is found to bind to the primary Gal binding pocket of both proteins, whereas the b6-linked branch outside this site provides additional interactions in accordance with the higher binding affinities found for this compound. While the remarkable architectural similarities of the primary galactose binding sites of pLT and ECorL point to a convergent evolution of these subsites, the distinguishing structural features determining the overall carbohydrate specificities are located in extended binding site regions. In pLT, Arg13 is thus found to play a crucial role in enhancing the affinity not only for N-acetyllactosamine-terminated structures but also for GM1 as compared to human LT (hLT) and CT. The physiological relevance of the binding of N-acetyllactosamine-containing glycoconjugates to LT and ECorL is briefly discussed.     

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.     

Primary structure of heat-labile enterotoxin produced by Escherichia coli pathogenic for humans

Heat-labile enterotoxin of Escherichia coli pathogenic for humans (LTh) or for piglets (LTp) and Vibrio cholerae enterotoxin (CT) are structurally and functionally similar toxins. We have determined the complete nucleotide sequence of the toxA gene which encodes the subunit A of LTh (LTh A). The deduced amino acid sequence consists of 258 residues including a signal peptide of 18 residues. According to the previously completed LTh B sequence (103 residues), the predicted holotoxin (1A5B) of LTh comprises 755 residues and has Mr = 87,866. With respect to LTh A and LTh B, secondary structures, local hydrophilicity, and sites for antigenic determinants were predicted. Both codon usage and G + C content of the toxA gene and the LTh B gene (toxB) were markedly different from those observed with several E. coli chromosomal genes. Its relatively low G + C content was rather close to that of the V. cholerae chromosome. Although the toxA gene shares a common ancestor with the LTp A gene (eltA), the two genes are apparently distinguishable from each other in their sequences. Like LTh B reported previously, the predicted sequence of the catalytic fragment LTh A1 also showed more homology to that of CT A1 than did that of LTp A1. In contrast, unique sequences were found in LTh A2.     

Novel binding site identified in a hybrid between cholera toxin and heat-labile enterotoxin: 1.9 A crystal structure reveals the details

A hybrid between the B subunits of cholera toxin and Escherichia coli heat-labile enterotoxin has been described, which exhibits a novel binding specificity to blood group A and B type 2 determinants. In the present investigation, we have determined the crystal structure of this protein hybrid, termed LCTBK, in complex with the blood group A pentasaccharide GalNAcalpha3(Fucalpha2)Galbeta4(Fucalpha3)GlcNAcbeta, confirming not only the novel binding specificity but also a distinct new oligosaccharide binding site. Binding studies revealed that the new specificity can be ascribed to a single mutation (S4N) introduced into the sequence of Escherichia coli heat-labile enterotoxin. At a resolution of 1.9 A, the new binding site is resolved in excellent detail. Main features include a complex network of water molecules, which is well preserved by the parent toxins, and an unexpectedly modest contribution to binding by the critical residue Asn4, which interacts with the ligand only via a single water molecule.     

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.     

Major outer membrane proteins in the phytopathogenic bacteria Erwinia carotovora subsp. carotovora and subsp. atroseptica

Abstract Immunological similarities of heat-labile Escherichia coli enterotoxins pathogenic for man (LTh) and piglets (LTp) and cholera enterotoxin (CT) were examined quantitatively by the reversed Mancini test. The following results were obtained by analysis of rabbit antisera against these toxins. (1) 86% and 61% of the immunoglobulins in anti-CT antisera were antibodies cross-reacting with LTh and LTp, respectively; (2) 77% and 66% of the immunoglobulins in anti-LTh antisera were antibodies cross-reacting with LTp and CT, respectively; (3) 75% and 59% of the immunoglobulins in anti-LTp antisera were antibodies cross-reacting with LTh and CT, respectively.     

Evolution and structure of two ADP-ribosylation enterotoxins, Escherichia coli heat-labile toxin and cholera toxin

Nucleotide sequence comparisons of the heat-labile enterotoxin (LTh) genes of E. coli pathogenic for humans with cholera toxin (CT) genes suggest that the two toxin genes have evolved from a common ancestry by a series of single base changes, while conserving the catalytic fragment A1 (ADP-ribose transferase). Based on the local hydrophilicity profiles of LTh and CT peptides, a transmembrane segment appears to be present in A1 in both toxins.     

Common antigenic determinant in the receptor binding sites of Escherichia coli enterotoxin and cholera toxin

Abstract A mutant (TUH No. 9) of a porcine strain of enterotoxigenic Escherichia coli (ETEC) produces as abnormal B subunit (B') of heat-labile enterotoxin (LT), which has aspartate instead of glycine at residue 33 from the N-terminus and does not bind to the receptor, GM1 ganglioside. The antigenicities of the receptor-binding site of LT were analyzed.
The antibody, which could not bind to the B' subunit in the anti-B subunit of porcine LT(LTp)-serum, could bind to cholera toxin (CT), LTp and LT produced by a human ETEC strain (LTh), suggesting that it recognizes a common epitope of LTp, LTh and CT. Thus glycine at residue 33 from the N-terminus in the B subunit of CT, LTh and LTp may be related to the common epitope of these three toxins. The bindings of CT, LTh and LTp to the antibody were inhibited by the GM1 ganglioside.
These data indicate that the antibody recognizes a common epitope in the receptor (GM1 ganglioside)-binding site of CT, LTh and LTp.     

Monoclonal antibodies to Escherichia coli heat-labile enterotoxins: neutralising activity and differentiation of human and porcine LTs and cholera toxin

Forty-four monoclonal antibodies (MAbs) prepared against heat-labile enterotoxins (LTs) from human (LTh) or porcine (LTp) E. coli isolates were characterised, especially with regard to their reactivity with epitopes shared with the heterologous LT and/or cholera toxin (CT), and their toxin neutralising activity. Of 24 MAbs against LTh (all directed against the B subunit portion) 12 cross-reacted with LTp and CT, 4 with LTp but not CT, and 1 with CT but not LTp; 7 MAbs reacted with LTh epitope(s) not shared by either LTp or CT. Among 20 MAbs against LTp (9 directed against the B subunits and 11 against the A subunit) 2 cross-reacted with LTh as well as CT, 13 with LTh but not CT, and 5 MAbs were specific for LTp. Irrespective of whether the anti-LT MAbs were directed against shared or unshared epitopes, or against the A or B subunits, they neutralised their homologous toxin in direct proportion to their toxin-binding titre. The results show how minute differences in enterotoxin primary structures e.g., the LTh and LTp B chains differ in only 4 of 103 amino acid residues, are associated with antigenic epitopes against which toxin-differentiating MAbs with neutralising activity can be produced. Such MAbs are promising tools for species-specific diagnostic detection of enterotoxins in clinical specimens.     

Amino acid sequence of heat-labile enterotoxin from chicken enterotoxigenic Escherichia coli is identical to that of human strain H 10407

Abstract The DNA sequence of heat-labile enterotoxin from the chicken enterotoxigenic Escherichia coli 21d strain was determined by direct dideoxy sequencing of polymerase chain reaction (PCR)-amplified DNA and was compared with those of heat-labile enterotoxins from porcine and human enterotoxigenic E. coli strains EWD 299 and H 10407. The structural genes of the A and B subunits of chicken heat-labile enterotoxin were identical to those of human heat-labile enterotoxin from the human H 10407 strain. Moreover, 67 base pairs of the upstream and 60 base pairs of the downstream region of the chicken heat-labile enterotoxin gene were also identical to those of the human heat-labile enterotoxin from strain H 10407. However, the patterns of plasmids from the 21d and H 10407 strains were different. The 21d strain had no band corresponding to the 42-MDa plasmid of the H10407 strain encoding the heat-labile enterotoxin gene but it had a smaller plasmid. These data suggest that although the DNA sequence of chicken heat-labile enterotoxin is identical to that of human heat-labile enterotoxin, the plasmid encoding the chicken heat-labile enterotoxin gene in the chicken might be different from that encoding the human heat-labile enterotoxin gene in the H10407 strain.     

Crystal structure of heat-labile enterotoxin from Escherichia coli with increased thermostability introduced by an engineered disulfide bond in the A subunit.

Cholera toxin (CT) produced by Vibrio cholerae and heat-labile enterotoxin (LT-I), produced by enterotoxigenic Escherichia coli, are AB5 heterohexamers with an ADP-ribosylating A subunit and a GM1 receptor binding B pentamer. These toxins are among the most potent mucosal adjuvants known and, hence, are of interest both for the development of anti-diarrheal vaccines against cholera or enterotoxigenic Escherichia coli diarrhea and also for vaccines in general. However, the A subunits of CT and LT-I are known to be relatively temperature sensitive. To improve the thermostability of LT-I an additional disulfide bond was introduced in the A1 subunit by means of the double mutation N40C and G166C. The crystal structure of this double mutant of LT-I has been determined to 2.0 A resolution. The protein structure of the N40C/G166C double mutant is very similar to the native structure except for a few local shifts near the new disulfide bond. The introduction of this additional disulfide bond increases the thermal stability of the A subunit of LT-I by 6 degrees C. The enhancement in thermostability could make this disulfide bond variant of LT-I of considerable interest for the design of enterotoxin-based vaccines.     

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.     

Demonstration of cholera-like enterotoxin production by Campylobacter jejuni

Abstract By means of a newly developed medium, cholera-like enterotoxin production by Campylobacter jejuni could be shown in 25 C. jejuni strains isolated from diarrheic cases. This new medium was found to yield a higher amount of enterotoxin than the two previously reported media for this purpose. Neutralization of the activity of the toxin to cause morphological changes of Chinese hamster ovary (CHO) cells by antisera against cholera toxin and heat-labile enterotoxins of Escherichia coli (LT_h and LT_p) was also demonstrated, indicating a close immunological relation of these toxins.     

Inactivation of the Escherichia coli heat-labile enterotoxin by in vitro mutagenesis of the A-subunit gene

In vitro mutagenesis of the LTA gene, encoding the A subunit of the Escherichia coli heat-labile enterotoxin, has been used to obtain A subunits deficient in enzymic activity. One inactive A-subunit mutant which contained two amino acid substitutions, was shown to associate with native B subunits to form a holotoxoid lacking toxin activity. A serine to phenylalanine mutation appears to be responsible for the loss of toxicity.     

Immunological properties of rat phosphoglycerate mutase isozymes

In mammalian tissues three phosphoglycerate mutase (D-phosphoglycerate 2,3-phosphomutase, EC 5.4.2.1) isozymes result from the homo-dimeric and hetero-dimeric combinations of two subunits (types M and B). Whereas rabbit antisera against type M subunit (purified from rat muscle) and against type BB isozyme (purified from rat brain) possessed a high degree of specificity, both antisera reacted with type BB and MM isozymes, as demonstrated by immunoneutralization and ELISA. Both the M subunit and B subunit were more immunoreactive than their respective dimeric isozymes. Subunits type M and B may possess common antigenic determinants, and some of these determinants may be sterically hindered in their dimeric structures.