Activation of Escherichia coli heat-labile enterotoxins by native and recombinant adenosine diphosphate-ribosylation factors, 20-kD guanine nucleotide-binding proteins.

Escherichia coli heat-labile enterotoxins (LT) are responsible in part for "traveler's diarrhea" and related diarrheal illnesses. The family of LTs comprises two serogroups termed LT-I and LT-II; each serogroup includes two or more antigenic variants. The effects of LTs result from ADP ribosylation of Gs alpha, a stimulatory component of adenylyl cyclase; the mechanism of action is identical to that of cholera toxin (CT). The ADP-ribosyltransferase activity of CT is enhanced by 20-kD guanine nucleotide-binding proteins, known as ADP-ribosylation factors or ARFs. These proteins directly activate the CTA1 catalytic unit and stimulate its ADP ribosylation of Gs alpha, other proteins, and simple guanidino compounds (e.g., agmatine). Because of the similarities between CT and LTs, we investigated the effects of purified bovine brain ARF and a recombinant form of bovine ARF synthesized in Escherichia coli on LT activity. ARF enhanced the LT-I-, LT-IIa-, and LT-IIb-catalyzed ADP ribosylation of agmatine, as well as the auto-ADP ribosylation of the toxin catalytic unit. Stimulation of ADP-ribosylagmatine formation by LTs and CT in the presence of ARF was GTP dependent and enhanced by sodium dodecyl sulfate. With agmatine as substrate, LT-IIa and LT-IIb exhibited less than 1% the activity of CT and LT-Ih. CT and LTs catalyzed ADP-ribosyl-Gs alpha formation in a reaction dependent on ARF, GTP, and dimyristoyl phosphatidylcholine/cholate. With Gs alpha as substrate, the ADP-ribosyltransferase activities of the toxins were similar, although CT and LT-Ih appeared to be slightly more active than LT-IIa and LT-IIb. Thus, LT-IIa and LT-IIb appear to differ somewhat from CT and LT-Ih in substrate specificity. Responsiveness to stimulation by ARF, GTP, and phospholipid/detergent as well as the specificity of ADP-ribosyltransferase activity are functions of LTs from serogroups LT-I and LT-II that are shared with CT.

Crystal structure of a new heat-labile enterotoxin, LT-IIb.

BACKGROUND: Cholera toxin from Vibrio cholerae and the type I heat-labile enterotoxins (LT-Is) from Escherichia coli are oligomeric proteins with AB5 structures. The type II heat-labile enterotoxins (LT-IIs) from E. coli are structurally similar to, but antigenically distinct from, the type I enterotoxins. The A subunits of type I and type II enterotoxins are homologous and activate adenylate cyclase by ADP-ribosylation of a G protein subunit, G8 alpha. However, the B subunits of type I and type II enterotoxins differ dramatically in amino acid sequence and ganglioside-binding specificity. The structure of LT-IIb was determined both as a prototype for other LT-IIs and to provide additional insights into structure/function relationships among members of the heat-labile enterotoxin family and the superfamily of ADP-ribosylating protein toxins. RESULTS: The 2.25 A crystal structure of the LT-IIb holotoxin has been determined. The structure reveals striking similarities with LT-I in both the catalytic A subunit and the ganglioside-binding B subunits. The latter form a pentamer which has a central pore with a diameter of 10-18 A. Despite their similarities, the relative orientation between the A polypeptide and the B pentamer differs by 24 degrees in LT-I and LT-IIb. A common hydrophobic ring was observed at the A-B5 interface which may be important in the cholera toxin family for assembly of the AB5 heterohexamer. A cluster of arginine residues at the surface of the A subunit of LT-I and cholera toxin, possibly involved in assembly, is also present in LT-IIb. The ganglioside receptor binding sites are localized, as suggested by mutagenesis, and are in a position roughly similar to the sites where LT-I binds its receptor. CONCLUSIONS: The structure of LT-IIb provides insight into the sequence diversity and structural similarity of the AB5 toxin family. New knowledge has been gained regarding the assembly of AB5 toxins and their active-site architecture.

Characterization of monoclonal antibodies that react with unique and cross-reacting determinants of cholera enterotoxin and its subunits.

Seventeen selected hybridoma cell lines that produced monoclonal antibodies against cholera enterotoxin (CT) were isolated and characterized. All of the monoclonal antibodies contained the kappa light chain; 14 were of the immunoglobulin G1 (IgG1) isotype and 3 were IgG2a. The 17 monoclonal antibodies were divided into a minimum of seven different specificity groups based on their abilities to bind to the following purified test antigens in solid-phase radioimmunoassays: CT, the A and B polypeptides of CT (CT-A and CT-B, respectively), and the heat-labile enterotoxins designated LTh and LTp from Escherichia coli. The binding of these antibodies to the following subunits and fragments of CT was also determined in Western blots: pentameric CT-B, monomeric CT-B, intact CT-A, and the A1 fragment of CT-A. Each of the monoclonal antibodies was tested for neutralization of CT and for precipitation with CT in immunodiffusion tests. Antigenic determinants were identified on CT that were not present either on CT-A or CT-B. One class was unique for CT and another was shared with LTh and LTp. Antibodies directed against these holotoxin-specific determinants had no neutralizing activity. Most of the monoclonal antibodies that reacted strongly with CT-A or CT-B also reacted strongly with CT holotoxin; however, one class of antibody reacted strongly with CT-A but weakly with CT. Among the monoclonal antibodies against CT-A or CT-B, some were specific for CT and others cross-reacted with LTh and LTp or with LTh only. The most potent neutralizing antibodies were against CT-B, and all of our monoclonal antibodies against CT-B had some neutralizing activity. In contrast, only some of the monoclonal antibodies against CT-A had neutralizing activity, and their specific activities were low. We found no direct correlation between the ability of monoclonal antibodies to neutralize CT and to cross-react with LTh or LTp. None of the epitopes recognized by our monoclonal anti-CT antibodies was present on CT-A and CT-B.

Purification and characterization of type II heat-labile enterotoxin of Escherichia coli.

Type II heat-labile enterotoxin (LT-II) of Escherichia coli has several biologic activities similar to cholera toxin (CT) and E. coli type I heat-labile enterotoxin (LT-I), but it is not neutralized by antiserum prepared against CT or LT-I. LT-II was purified from E. coli SA53 and from E. coli HB101(pCP3837), a strain that contains the cloned LT-II genes in a hybrid plasmid and produces up to 600 times more LT-II than does SA53. Purification involved sonic disruption of bacterial cells, ammonium sulfate fractionation, chromatography on Affi-Gel Blue, chromatofocusing, and gel filtration on Sephadex G-100. The LT-II purified to apparent homogeneity from HB101(pCP3837) had an isoelectric point of 6.8, induced increased vascular permeability in rabbit intracutaneous tests, caused rounding of cultured Y1 adrenal cells accompanied by increased intracellular cyclic AMP, and was 25 to 50 times more potent than CT or LT-I in the Y1 adrenal-cell assay. In contrast, purified LT-II did not cause secretion in ligated rabbit ileal segments at doses corresponding to CT controls that gave strongly positive reactions. LT-II was composed of two different polypeptides with MrS of 28,000 (A) and 11,800 (B); treatment of LT-II with trypsin cleaved the A polypeptide to fragments A1 (Mr, 21,000) and A2 (Mr, 7,000). The activity of LT-II was not blocked by ganglioside GM1 at concentrations that inactivated LT-I or CT. Antiserum against the LT-II from E. coli HB101(pCP3837) completely neutralized purified LT-II and the LT-II in crude extracts of SA53, but it did not neutralize purified LT-I or CT.

Purification and characterization of the diphtheria toxin repressor.

The diphtheria toxin repressor gene (dtxR) encodes a protein (DtxR) that regulates transcription of the diphtheria toxin gene (tox) by an iron-dependent mechanism. Cloned dtxR was expressed in Escherichia coli from the phage T7 gene 10 promoter, and DtxR was purified. Specific binding of DtxR to the tox+ operator was dependent on reduction of DtxR and the presence of ferrous ions. DtxR protected a sequence of approximately 30 nucleotide pairs, partially overlapping the tox promoter and containing a region of dyad symmetry, from digestion by DNase I. DtxR exhibited very little binding to the mutant tox-201 operator region and failed to bind to the promoter/operator region of the ferric uptake regulation (fur) gene of E. coli.

Synthesis of plasmid-coded heat-labile enterotoxin in wild-type and hypertoxinogenic strains of Escherichia coli and in other genera of Enterobacteriaceae.

The effect of host determinants on expression of plasmid-coded heat-labile enterotoxin (LT) was examined. A collection of LT plasmids was introduced into isogenic strains of Escherichia coli K-12 strains containing the wild type or hypertoxinogenic (htx-2) allele. For each plasmid tested, production of LT increased by approximately 1.5- to 3-fold in the host containing htx-2, indicating that the htx-2 allele affects a regulatory function for LT production that is common to many different enterotoxin plasmids. LT plasmids from E. coli were also introduced into strains of Shigella flexneri, Shigella sonnei, Citrobacter freundii, Enterobacter cloacae, Klebsiella pneumoniae, and Salmonella typhimurium. The plasmids were stably maintained and determined production of LT in those genera, although the amounts of LT produced varied by more than 50-fold. These observations demonstrate that host factors have an important role in determining the level of expression of plasmid-coded LT genes and support the hypothesis that interspecific, conjugal transfer of enterotoxin plasmids may confer enterotoxigenicity to a wide variety of potentially pathogenic enteric bacteria.

Monoclonal antibodies with an expanded repertoire of specificities and potent neutralizing activity for Escherichia coli heat-labile enterotoxin.

Nine selected hybridoma cell lines that produced monoclonal antibodies against the heat-labile enterotoxin encoded by a plasmid from an Escherichia coli strain of human origin (LTh) were characterized. Hybridomas that produced anti-LTh antibodies with previously unrecognized specificities or reactivities were selected for cloning. Each monoclonal antibody was tested for isotype and for binding to LTh holotoxin, the A and B subunits derived from LTh (LTh-A and LTh-B), holotoxin encoded by a plasmid from an E. coli strain of porcine origin (LTp), and cholera enterotoxin (CT). Binding was also tested in Western blots with the following antigens: pentameric LTh-B, monomeric LTh-B, LTh-A, and the A1 and A2 fragments produced from LTh-A by treatment with trypsin. These monoclonal anti-LTh antibodies and selected anti-LTh and anti-CT monoclonal antibodies described previously were tested for neutralization of LTh, LTp, and CT. Five of the nine new monoclonal antibodies gave detectable cross-reactions with LTp and CT. Four reacted with determinants of LTh that were not present on CT; one of these four did not react with LTp and was specific for a unique epitope of LTh. Three antibodies were specific for LTh-B. All three reacted with pentameric LTh-B, but only one reacted in Western blots with monomeric LTh-B. Six antibodies were specific for LTh-A. Three reacted in Western blots with LTh-A and its A1 fragment; the other three did not react in Western blots. All nine of the new monoclonal antibodies neutralized LTh but not CT; the eight that cross-reacted with LTp in binding assays also neutralized LTp. Of four neutralizing anti-CT monoclonal antibodies that bound to LTh, none had significant neutralizing activity against LTh.

Characterization of monoclonal antibodies to heat-labile enterotoxin encoded by a plasmid from a clinical isolate of Escherichia coli.

Eight selected hybridoma cell lines that produced monoclonal antibodies against heat-labile enterotoxin from an Escherichia coli strain of human origin (LTh) were characterized. Antibodies produced by these cell lines were tested for binding specificity in a series of solid-phase radioimmunoassays and Western blots by using as test antigens LTh, the A, A1, A2, and B polypeptides of LTh, the heat-labile enterotoxin from an E. coli strain of porcine origin, and cholera toxin. The monoclonal antibodies were also tested for isotype and ability to neutralize LTh. Two of the anti-LTh monoclonal antibodies cross-reacted with cholera toxin, and six were specific for determinants of LTh that were not present on cholera toxin. One was specific for a unique epitope of LTh that was not shared by the heat-labile enterotoxin from an E. coli strain of porcine origin or cholera toxin. Four antibodies specific for epitopes on the B subunit of LTh (LTh-B) reacted with pentameric LTh-B but did not react in Western blots with monomeric LTh-B. The remaining four antibodies were specific for epitopes on LTh-A; two of these antibodies bound to A1, one reacted with A2, and one recognized only intact LTh-A. Only one monoclonal antibody had detectable neutralizing activity, and it was specific for LTh-A.

Comparison of the carbohydrate-binding specificities of cholera toxin and Escherichia coli heat-labile enterotoxins LTh-I, LT-IIa, and LT-IIb.

The heat-labile enterotoxins of Vibrio cholerae and Escherichia coli are related in structure and function. They are oligomers consisting of A and B polypeptide subunits. They bind to gangliosides, and they activate adenylate cyclase. The toxins form two antigenically distinct groups; members of each group cross-react but are not necessarily identical. Serogroup I includes cholera toxin (CT) and type I heat-labile enterotoxin (LT-I) of E. coli. LTh-I and LTp-I are antigenic variants of LT-I produced by strains of E. coli from humans and pigs, respectively. Serogroup II contains the type II heat-labile enterotoxin (LT-II) of E. coli. Two antigenic variants designated LT-IIa and LT-IIb have been described. The binding of CT, LTh-I, LT-IIa, and LT-IIb to gangliosides was analyzed by immunostaining thin-layer chromatograms and by solid-phase radioimmunoassay. The four toxins have different glycolipid-binding specificities. LTh-I and CT bind strongly to ganglioside GM1 and less strongly to ganglioside GD1b. However, LTh-I, unlike CT, also binds weakly to GM2 and asialo GM1. LTh-I, like CT, probably binds to the terminal sugar sequence Gal beta 1-3GalNAc beta 1-4(NeuAc alpha 2-3)Gal . . ., where GalNAc is N-acetylgalactosamine and NeuAc is N-acetylneuraminic acid. LT-IIa probably binds to the same sugar sequence to which CT and LTh-I bind, with the additional contribution to binding of a second NeuAc as in GD1b and GD2. Also, LT-IIa must bind the Gal beta 1-3GalNAc . . . sequence in such a way that its binding is relatively unaffected by attachment of NeuAc to the terminal galactose residue as in GD1a, GT1b, and GQ1b. LT-IIb probably binds to the terminal sugar sequence NeuAc alpha 2-3Gal beta 1-4GalNAc . . ., as it binds to gangliosides GD1a and GT1b but not to GM1.

Variation in chemical properties and antigenic determinants among type II heat-labile enterotoxins of Escherichia coli.

Type II heat-labile enterotoxin (LT-II) from Escherichia coli 41 was purified and compared with prototype LT-II encoded by genes from E. coli SA53. Both toxins were oligomeric proteins consisting of polypeptides A (Mr, 28,000) and B (Mr, 11,800). The A polypeptides were cleaved by trypsin into fragments A1 (Mr, 21,000) and A2 (Mr, about 7,000). These two toxins were shown to belong to two different subclasses of LT-II. We propose to designate the prototype toxin LT-IIa and the new variant LT-IIb. The pI of LT-IIb was between 5.2 and 5.6, significantly lower than the pI of 6.8 for LT-IIa, and the behavior of LT-IIb during purification differed significantly from that of LT-IIa. The toxic dose of unnicked LT-IIb in the Y1 adrenal-cell assay was 94 pg, but trypsin-treated, nicked LT-IIb was toxic at about 3 pg. In contrast, the toxic dose of LT-IIa was previously shown to be 0.5 to 1 pg for several preparations that varied from unnicked to partially nicked, and treatment with trypsin was not required for full toxicity. The titer of LT-II antiserum in neutralization tests was 100-fold greater against LT-IIa than against LT-IIb. In immunodiffusion tests, LT-IIa and LT-IIb gave a reaction of partial identity. In a radioimmunobinding assay, the titer of LT-IIa antiserum against homologous LT-IIa was approximately 10-fold greater than against LT-IIb. The cholera-E. coli family of heat-labile enterotoxins has been divided into serogroup I, which includes cholera toxin and the antigenic variants of E. coli heat-labile toxin designated LTh-I and LTp-I, and serogroup II, which includes LT-IIa and LT-IIb. The type I and type II toxins do not cross-react in neutralization or immunodiffusion tests. By using very sensitive radioimmunobinding assays, it was possible to demonstrate common antigenic determinants between the type I and type II toxins. However, the titers of antibodies in hyperimmune sera that recognized these common determinants were very low.

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 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.

Type II heat-labile enterotoxin of Escherichia coli activates adenylate cyclase in human fibroblasts by ADP ribosylation.

Type II heat-labile enterotoxin (LT-II) from Escherichia coli causes characteristic morphological changes and accumulation of cyclic AMP in Y-1 adrenal cells, but it is not neutralized by antisera against choleragen (CT) or the classical type I heat-labile enterotoxin (LT-1) from E. coli. The action of purified LT-II on CT- and LT-I-responsive human fibroblasts was investigated and compared with that of CT. Fibroblasts incubated with LT-II or CT had an increased cyclic AMP content as well as a fourfold elevation of membrane adenylate cyclase activity. In membranes, activation of cyclase by toxin was enhanced by NAD, GTP, and dithiothreitol. The effect of LT-II on intact fibroblasts or membranes was increased by trypsin treatment of toxin. Since activation of adenylate cyclase by LT-II was stimulated by NAD, the ability of LT-II to catalyze the [32P]ADP-ribosylation of membrane proteins in the presence of [32P]NAD from control and LT-II- and CT-treated fibroblasts was investigated. Similar proteins were [32P]ADP-ribosylated in membranes exposed to LT-II or CT; LT-II- and CT-specific labeling was significantly decreased in membranes prepared from cells preincubated with either LT-II or CT. These studies are consistent with the hypothesis that LT-II, similar to CT and LT-I, increases cyclic AMP by activating adenylate cyclase through the GTP-dependent ADP-ribosylation of specific membrane proteins.

Isolation and characterization of hypertoxinogenic (htx) mutants of Escherichia coli KL320(pCG86).

The structural genes for heat-labile enterotoxin (LT) are present on plasmid pCG86. Escherichia coli KL320(pCG86), LT was found to be cell associated. LT was present as a soluble protein in sonic lysates of KL320(pCG86). Thirty-one mutants of KL320(pCG86) that produced increased amounts of extracellular LT were isolated. These hypertoxinogenic (htx) mutants were assigned to four phenotypically distinct classes based on the amounts of cell-associated and extracellular LT in early-stationary-phase cultures. Type 1 and type 2 htx mutants produced significantly increased amounts of cell-associated LT. Type 3 and type 4 htx mutants produced normal or decreased amounts of cell-associated LT was similar to that of the wild type. In the mutants of types 1, 3, and 4, the ratios of extracellular to cell-associated LT were higher than that of the wild type and were characteristic for each strain. Cell lysis or leakage of macromolecular cytoplasmic constituents appeared to be significant for release of LT by mutants of types 1, 3, and 4, because supernatants from cultures of these mutants also contained increased amounts of protein and of the cytoplasmic enzyme glucose 6-phosphate dehydrogenase. In all four representative htx mutants, the hypertoxinogenic phenotypes were dependent on chromosomal mutations. The resident pCG86 plasmids were eliminated from the htx mutants of types 2 and 3. After wild-type plasmid pCG86 was introduced into the cured strains by conjugation, their hypertoxinogenic phenotypes were restored. We conclude that chromosomal loci in E. coli KL320 are important in regulating expression of the LT structural genes of plasmid pCG86.

Production of type II heat-labile enterotoxin by Escherichia coli isolated from food and human feces.

Escherichia coli strains isolated in Sao Paulo, Brazil, from feces of patients with diarrhea and from food samples produced toxin(s) that was shown to be related both immunologically and genetically to the recently characterized type II heat-labile enterotoxin of E. coli. The new isolates of type II heat-labile enterotoxin-producing E. coli belonged to five different serotypes and did not represent a single clone.