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PubMed1983

Inhibition of enterotoxin from Escherichia coli and Vibrio cholerae by gangliosides from human milk.

Inhibitory activity of enterotoxin from Escherichia coli and Vibrio cholerae was associated with the ganglioside fraction of human milk. Both the milk fat and skim milk contained gangliosides that inhibited the toxins. The most purified milk fraction contained three glycolipid components, of which two migrated close to ganglioside GM1 on thin-layer chromatography plates. A component with a slightly different mobility from GM1 appeared to be associated with the inhibitory activity. Milk ganglioside fraction, derived from 2 ml of human milk, contained 1 to 4 micrograms of lipid-bound sialic acid and completely inhibited 0.1 micrograms of cholera toxin in rabbit intestinal loop experiments. It is suggested that human milk gangliosides, although present only in trace amounts, may be important in protecting infants against enterotoxin-induced diarrhea.

Enterotoxins↗
PubMed1983

Cloning and molecular characterization of the B subunit of Escherichia coli heat-labile enterotoxin.

We have constructed a plasmid containing the gene for production of the B subunit of the heat-labile enterotoxin (LT-B) from a human isolate of Escherichia coli, strain H10407. The 0.8-kilobase gene fragment encoding synthesis of LT-B was cloned onto plasmid pBR322 after sequential digestion of the enterotoxin plasmid of strain H10407 with restriction endonucleases PstI and HindIII. LT-B was isolated by agarose affinity chromatography from cell lysates of recombinant clones expressing the B subunit. The B subunit was isolated in its oligomeric form, was structurally identical to native B subunit when examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, dissociated to monomeric B in the presence of 5 M guanidine, was immunologically identical to native B subunit in an enzyme-linked immunosorbent assay, and contained no demonstrable A subunit in any of the assays.

Antibodies, Bacterial↗
PubMed1983

Identification of enterotoxigenic Escherichia coli in patients with diarrhea in Asia with three enterotoxin gene probes.

A total of 984 enterotoxigenic Escherichia coli (ETEC) and 733 non-ETEC isolated from patients with diarrhea in Asia (one isolate per patient) were examined for homology with radiolabeled fragments of DNA encoding heat-labile toxin (LT) or heat-stable toxin of porcine (ST-P) or human (ST-H) origin. A total of 246 ETEC that produced LT and ST as determined by the Y-1 adrenal and suckling mouse assays were homologous with the LT probe. Of these 246 LTST ETEC, 156 (63%) were homologous with the ST-H, 46 (19%) were homologous with the ST-P, and 44 (18%) were homologous with both probes. A total of 401 LT ETEC were homologous with the LT probe. Of 337 ST ETEC identified by the suckling mouse assay, 244 (72%) were homologous with the ST-H, 84 (25%) were homologous with the ST-P, and 9 (3%) were homologous with both probes. None of the 733 isolates that were non-enterotoxigenic as determined by the Y-1 adrenal and suckling mouse assays was homologous with genes coding for enterotoxin. Four isolates (not included among the 984 ETEC examined) that were initially considered to produce LT because sterile culture supernatants produced rounding of Y-1 adrenal cells were not homologous with the LT probe. The sterile culture supernatants of these four isolates caused rounding after 8 h and subsequent destruction after 24 h of Y-1 adrenal tissue cultures. This effect was not inhibited by convalescent human cholera antiserum, Swiss Serum Institute cholera antitoxin, or antiserum to purified LT. These isolates were also negative in the Biken test previously used to identify LT-producing E. coli. The DNA hybridization technique with three enterotoxin gene probes was developed as a specific technique to identify ETEC in large numbers of specimens in Asia.

Animals↗
PubMed1983

Nucleotide sequence of the gene for heat-stable enterotoxin II of Escherichia coli.

Previously, the gene for heat-stable enterotoxin II (STII) has been mapped by transposon Tn5 insertion mutagenesis in the chimeric R-Ent plasmid pCG86 (Mazaitis, A. J., R. Maas, and W. K. Maas, J. Bacteriol. 145:97-105, 1981). DNA segments containing this gene were cloned from the wild-type and STII-insertion-mutant plasmid. The position of the Tn5 insertion was determined, and a 530-base-pair-long segment of the wild-type plasmid corresponding to the Tn5 insertion site was sequenced. An open reading frame for the STII gene was identified and is characterized by typical promoter and ribosome binding site sequences. The deduced STII structural gene codes for a protein 71 amino acids long, including a typical signal peptide of 23 amino acids and a mature protein of 48 amino acids. The size and overall structure of STII are similar to those of STI, but the amino acid compositions of the two heat-stable enterotoxins are completely different.

Amino Acids↗
PubMed1983

Effects of lincomycin and tetracycline on production and properties of enterotoxins of enterotoxigenic Escherichia coli.

Enterotoxigenic Escherichia coli grown in the presence of lincomycin and tetracycline produced an increased amount of heat-labile enterotoxin (LT). These antibiotics increased the production of not only extracellular LT but also intracellular LT. On the other hand, lincomycin did not stimulate the production of heat-stable enterotoxin by enterotoxigenic E. coli. The extracellular LTs produced in the presence of lincomycin and tetracycline were purified and analyzed by sodium dodecyl sulfate-polyacrylamide gel disc electrophoresis. Results showed that the A subunits of the purified LTs were not nicked, unlike that of extracellular LT produced in the absence of the antibiotics.

Anti-Bacterial Agents↗
PubMed1983

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.

Animals↗
PubMed1984

Effect of lodoxamide on the secretory response induced by Escherichia coli and Vibrio cholerae enterotoxins in infant mice.

The effect of lodoxamide tromethamine, a calcium antagonist, on intestinal fluid accumulation induced by Escherichia coli heat-stable (ST) and Vibrio cholerae (CT) enterotoxins in infant mice was investigated. The simultaneous administration of lodoxamide with ST or CT enterotoxin resulted in a significant (P less than 0.01) inhibition of the intestinal fluid response. A minimum concentration of 10(-7) or 10(-8)M lodoxamide caused an inhibition (P less than 0.01) of the ST- or CT-mediated fluid response, respectively. Treatment of infant mice with buffer or drug alone did not result in fluid accumulation. A significant inhibition of ST and CT enterotoxic activities was also observed when lodoxamide was administered 30 min before (P less than 0.02) or 30 min after (P less than 0.01) toxin challenge. These data suggest that calcium may be important in the ST- or CT-mediated induction of fluid accumulation. Further studies on the potential use of lodoxamide tromethamine in both the prophylaxis and treatment of diarrheal disease appear warranted.

Amino Acids↗
PubMed1984

Molecular homogeneity of heat-stable enterotoxins produced by bovine enterotoxigenic Escherichia coli.

Heat-stable enterotoxins (STs) from four strains of bovine enterotoxigenic Escherichia coli representing four serogroups were purified to homogeneity by utilizing previously published purification schemata. Biochemical characterization of the purified STs showed that they met the basic criteria for the heat-stable enterotoxins of E. coli. Amino acid analysis of the purified STs revealed that they were peptides of identical amino acid composition. This composition consisted of 18 residues of 10 different amino acids, 6 of which were cysteine. The amino acid composition of the four ST peptides was identical to that reported for the STs of human and porcine E. coli. In addition, complete sequence analysis of two of the ST peptides and partial sequencing of several others revealed strong homology to the sequences of STs from human and porcine E. coli and to the sequence predicted from the last 18 codons of the transposon Tn1681. There was also substantial homology to the sequence predicted from the ST-coding genetic element of human E. coli, which may indicate the existence of identical bioactive configuration among ST peptides of E. coli strains of various host origins. These data support the hypothesis that STs produced by human, bovine, and porcine E. coli are coded by a closely related genetic element which may have originated from a single, widely disseminated transposon.

Amino Acid Sequence↗
PubMed1984

Monoclonal antibodies to staphylococcal enterotoxins B and C: cross-reactivity and localization of epitopes on tryptic fragments.

Murine monoclonal antibodies reactive with staphylococcal enterotoxins B (SEB) and C1 (SEC1) were isolated by hybridoma techniques. Of the nine antibodies, three reacted only with SEB, two reacted with SEB and SEC1, three reacted with all subtypes of SEC, and one reacted only with SEC2 and SEC3. All of the antibodies reacted with protein blotted onto nitrocellulose from electrophoresis gels which corresponded to the enterotoxin band. Fragments of SEB and SEC1 were generated by limited digestion of the toxin with trypsin. With the immunoblot technique, four of the five antibodies reactive with SEB reacted with the tryptic fragment of molecular weight 17,000, and the five antibodies reactive with SEC1 reacted with the tryptic fragment of molecular weight 14,000.

Animals↗
PubMed1984

Preparation of procoligenoids from Escherichia coli heat-labile enterotoxins.

Heat-labile enterotoxins from Escherichia coli strains of porcine and human origin polymerize on heating to form high-molecular-weight aggregates, "procoligenoids," analogous to procholeragenoid derived from the cholera enterotoxin. This aggregation is accompanied by loss of biological activity (toxicity). Further heating results in the release of B-subunit oligomers, coligenoids, analogous to choleragenoid. Further studies are needed to determine whether, like procholeragenoid, the procoligenoids are superior antigens in stimulating gut immunity after parenteral administration.

Animals↗
PubMed1984

Purification of enterotoxins from Vibrio mimicus that appear to be identical to cholera toxin.

Cholera toxin-like (CT-like) enterotoxins produced by two strains of Vibrio mimicus, 61892 and 63616, isolated from diarrhea patients in Bangladesh, were purified, and their physicochemical, biological, and immunological properties were compared with those of CT produced by classical Vibrio cholerae 569B. The CT-like toxins were produced by lincomycin-resistant mutants grown in the presence of lincomycin at 200 micrograms/ml for strain 63616 and 250 micrograms/ml for strain 61892 and were purified by coprecipitation with hexametaphosphate followed by chromatography on phosphocellulose. The pure CT-like toxins were indistinguishable from 569B CT in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, rabbit intestinal loop and Y-1 adrenal cell assays, antiserum neutralization and binding inhibition assays, and Ouchterlony immunodiffusion, except that the CT-like toxins appeared to consist almost entirely of A subunit which was proteolytically unnicked. Trypsin nicking, however, resulted in fragments that appeared to be identical to those of 569B CT. These results indicate that at least one species of Vibrio other than V. cholerae can produce enterotoxins which are virtually identical to CT.

Biological Assay↗
PubMed1984

Effects of Escherichia coli heat-stable enterotoxin STb on intestines of mice, rats, rabbits, and piglets.

There are at least two classes of Escherichia coli heat-stable enterotoxins, STa and STb. Unlike STa, which is active in suckling mice and piglets, STb is inactive in suckling mice but active in piglets and older, weaned pigs. This study examined the activity of STb in several animals and its effect on intestinal histology and cyclic GMP levels in intestinal mucosal cells. STb did not cause fluid secretion in suckling mice up to 12 days old or in rat or rabbit intestinal-loop preparations. STb-induced fluid secretion in weaned-pig intestinal loops occurred by 30 min and became maximal by 3 to 6 h. STb did not disrupt intestinal histology and did not alter cyclic GMP levels in intestinal mucosal cells from piglet intestinal loops after 0.5- and 6-h incubations. Our studies support the concept that STb is a second heat-stable E. coli enterotoxin with properties and a mechanism of action unlike those of STa.

Animals↗
PubMed1984

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.

Antibodies, Bacterial↗
PubMed1985

Construction of a conjugative plasmid with potential use in vaccines against heat-labile enterotoxin.

A conjugative plasmid with potential usefulness for vaccine strains was constructed. In the first step, a 5.9-kilobase DNA segment containing the two loci for the A and B subunits of heat-labile enterotoxin with a mutation in the gene for the A subunit was joined to the cloning vehicle pGA22, generating the nonconjugative plasmid pPMC4 with genes for resistance to tetracycline and chloramphenicol. In the second step, a segment of pPMC4 containing the genes for the A and B subunits, the gene for chloramphenicol resistance, and the replication genes of pGA22 was ligated to the genes for conjugal transfer of the F plasmid, generating the 54.9-kb plasmid pPMC5. Eleven porcine Escherichia coli isolates were tested as recipients for pPMC4 and pPMC5. For pPMC4, transformation and mobilization with a conjugative R plasmid were used to effect plasmid transfer. Only 1 of the 11 strains acted as a recipient in transformation. Mobilization with the R plasmid occurred with two strains, but the plasmids were altered during transfer. In contrast, pPMC5 was transferred with high frequency and unaltered to 9 of the 11 E. coli strains. Transconjugants from these nine matings produced high titers of the B subunit and no active heat-labile enterotoxin. Plasmid pPMC5 was stable in three porcine E. coli strains tested; plasmid pPMC4 was somewhat less stable in these strains. The method we describe for the construction of conjugative chimeric plasmids offers an opportunity for introducing genes with potential for immunization into bacterial strains that are suitable for colonizing the appropriate host sites.

Animals↗
PubMed1985

Immunological relationship of the B subunits of Campylobacter jejuni and Escherichia coli heat-labile enterotoxins.

The application of dissociation techniques, involving gel filtration in the presence of guanidine, to a semipurified preparation of Campylobacter jejuni heat-labile enterotoxin yielded a material whose functional and immunological properties resemble those of the B subunits of cholera toxin and Escherichia coli heat-labile toxin (LT). The C. jejuni toxin B subunit reacted with GM1 ganglioside in an enzyme-linked immunosorbent assay, but lacked the holotoxin's cytotonic activity in the Chinese hamster ovary tissue culture assay and its ability to cause fluid secretion in rat ileal ligated loops. The C. jejuni toxin B subunit showed lines of partial identity with the B subunits of both cholera toxin and LT in gel immunodiffusion; it appeared to be more closely related immunologically to the LT B subunit than to the cholera toxin B subunit in enzyme-linked immunosorbent assays that used antisera either to LT or to its B subunit. Rats immunized with LT B subunit were significantly protected against challenge with either the semipurified C. jejuni toxin or a viable enterotoxigenic strain of C. jejuni, although twice the immunization dosage was required to achieve protection comparable to that against the homologous toxin or viable bacteria. These observations indicate that the C. jejuni enterotoxin contains a B subunit that bears an immunological relationship with the B subunits of cholera toxin and LT.

Animals↗
PubMed1985

Evaluation of BW942C, a novel antidiarrheal agent, against enterotoxins of Escherichia coli and Vibrio cholerae.

BW942C, an enkephalin-like pentapeptide with anti-diarrheal activity, was tested against crude toxins of Escherichia coli and Vibrio cholerae in the Y-1 adrenal cell assay, rabbit ileal loop assay, and suckling mouse assay. The effects of BW942C on in vitro ion transport were measured in rabbit ileum mounted in Ussing chambers. In vitro, BW942C decreased basal short-circuit current (2.26 and 3.15 mueq cm-2 h-1 in experimental samples and controls, respectively; n = 7, P less than 0.05) and increased basal net Cl absorption (1.59 and 0.50 mueq cm-2 h-1 in experimental samples and controls, respectively; P less than 0.025). Net Na absorption was also increased, but not significantly. BW942C did not block the secretory response to a maximal dose of purified heat-stable toxin. BW942C directly enhanced intestinal fluid absorption. In the Y-1 adrenal cell assay, 5 mg of BW942C per ml inhibited the cytopathic effect caused by cholera toxin or heat-labile enterotoxin of E. coli. In the rabbit ileal loop assay, E. coli heat-stable toxin, E. coli heat-labile enterotoxin, and cholera toxin were inhibited 35 to 70% by administration of BW942C. With the suckling mouse model, the fluid accumulation caused by E. coli heat-stable toxin was ablated by prior treatment with BW942C. The drug is currently being evaluated in patients with acute secretory diarrhea to determine its effect on clinical symptoms.

Animals↗