Coli Heat-labile Enterotoxin Research Articles

Rapid visual detection of Escherichia coli and Vibrio cholerae Heat-labile enterotoxins by nitrocellulose enzyme-linked immunosorbent assay.

A modification of the enzyme-linked immunosorbent assay for a sensitive and rapid visual detection of heat-labile enterotoxins from Escherichia coli and Vibrio cholerae is described. Small amounts of bacterial supernatant fluids are bound to nitrocellulose filters which are used as sorbents in the nitrocellulose enzyme-linked immunosorbent assay. The test is based on the immunological similarity between V. cholerae and E. coli heat-labile enterotoxins. Six isolates of V. cholerae and 48 isolates of E. coli were examined for heat-labile enterotoxins by the nitrocellulose enzyme-linked immunosorbent assay and the Vero cell bioassay. With some strains, the nitrocellulose enzyme-linked immunosorbent assay was found to be more sensitive for detection of E. coli heat-labile enterotoxin than the Vero cell test. A similar result was obtained by endpoint titration of heat-labile enterotoxin-positive E. coli H10407 culture fluid in both assays. The sensitivity of the nitrocellulose enzyme-linked immunosorbent assay for the detection of purified cholera toxin was at a total level of 1 ng, which is a good result when compared with other serological assays.

42] Toxin ADP-ribosyltransferases that act on adenylate cyclase systems

This chapter describes toxin ADP-ribosyltransferases that act on adenylate cyclase systems. cAMP plays a significant role in control of metabolism and function of many cells. The activity of adenylate cyclase, the enzyme responsible for cAMP synthesis, is regulated by both inhibitory and stimulatory molecules. Three bacterial toxins are known to exert their effects on cells through activation of the adenylate cyclase system. Choleragen and E. coli heat-labile enterotoxin, toxins involved in the pathogenesis of cholera and traveler's diarrhea, respectively, enhance cyclase activity by acting on the stimulatory arm of the system. Pertussis toxin, which may play a role in whooping cough, appears to increasecyclase activity by preventing the action of inhibitory ligands. An adenylate cyclase system generally consists of at least five components. The catalytic unit (C) is responsible for the conversion of ATP to cAMP. Stimulatory and inhibitory ligands act by binding to gonist-specific cell surface receptors. Stimulatory (Rs) and inhibitory (Ri) receptors are coupled to the catalytic unit through different GTP-binding regulatory proteins termed Gs and Gi, respectively. Demonstration of hormonal stimulation or inhibition of adenylate cyclase activity requires agonist, its specific receptor, the appropriate GTP-binding protein (Gs or Gi) and GTP. Toxin action on cells involves multiple steps from initial binding to the membrane through activation of adenylate cyclase and finally alterations in metabolism or function.

Local intestinal immune response to Escherichia Coli heat-labile (LT) enterotoxin: LT antitoxin levels in healthy, diseased and antigen-fed pigs

With purified LT toxin and IgA, specific anti-LT enterotoxin activity was demonstrated in small intestinal contents of 27 pigs. After 60 days of age, rise in intestinal LT antitoxin titer was observed. Feeding LT containing E. coli antigen increased LT antibody levels in the intestinal secretions, but decreased antibody titers in sera. In post-weaning E. coli diarrhea LT antibody levels in intestinal secretions and sera decreased significantly. This phenomenon can be related to the occurrence of the frequently observed post-weaning E. coli diarrhea.

Binding of Escherichia coli heat-stable enterotoxin to receptors on rat intestinal cells

This study was performed to determine whether receptors for Escherichia coli heat-stable enterotoxin (ST) exist on intestinal epithelial cells. Binding sites for 125I-ST were found on rat jejunal and ileal villus cells. Binding was rapid, reversible, linear with cell number, saturable, and temperature dependent. Significant degradation of 125I-ST occurred when incubated with cells at 37 degrees C but not at 25 degrees C. Binding was specific to ST since binding of 125I-ST was competitively inhibited by increasing concentrations of human or porcine ST but not by E. coli heat-labile, cholera, or staphylococcal enterotoxins. Addition of excess unlabeled ST to cells preincubated with 125I-ST resulted in dissociation of much but not all of the bound 125I-ST. Binding of 125I-ST to jejunal and ileal cells occurs with two affinities, and this is due to the phenomenon of negative cooperativity. The potency of ST for inhibiting the binding of 125I-ST was identical to the potency of ST in stimulating cGMP production. These data support the existence of receptors for ST on intestinal cells, and these receptors may be involved in the action of ST.

Peroral Immunization of Rats with Escherichia coli Heat-Labile Enterotoxin Delivered by Microspheres

The antigenicity of the Escherichia coli heat-labile enterotoxin was not protected against the adverse effect of gastric acidity when the toxin was given together with bicarbonate for peroral immunization to rats, but immunization with the heat-labile enterotoxin encapsulated in pH-dependent microspheres aroused the same strong degree of serum and mucosal antitoxin responses and of protection against challenge as was achieved by peroral immunization after ablation of gastric secretions by pretreatment with cimetidine.

Biological similarity of enterotoxins of Vibrio cholerae serotypes other than type 1 to cholera toxin and Escherichia coli heat-labile enterotoxin.

Vibrio cholerae serotypes other than type 1, the so-called NAG vibrios, have been recognised as an important cause of diarrhoea. A few of them have been shown to produce an enterotoxin similar, immunologically and physiologically, to cholera toxin; and cholera toxin has been shown to be structurally, functionally and immunologically identical with Escherichia coli heat-labile toxin (LT). The present investigation has demonstrated biological similarities among cholera toxin, E. coli LT and enterotoxins produced by strains of V. cholerae of 59 serotypes other than 1, in the biological models, rabbit ileal loops and rabbit skin. Culture filtrates of almost all the strains were neutralised completely and all filtrates showed some neutralisation, in enterotoxic action and increase of permeability, by cholera antitoxin and E. coli LT antiserum. The partial neutralisation observed in a few strains was probably due to high concentrations of identical toxin rather than the presence of other toxic substances.

Specificity in Protein-Membrane Associations: The Interaction of Gangliosides with Escherichia Coli Heat-Labile Enterotoxin and Choleragen

Specificity in Protein-Membrane Associations: The Interaction of Gangliosides with Escherichia Coli Heat-Labile Enterotoxin and Choleragen

Protective effect of immunization of rats with holotoxin or B subunit of Escherichia coli heat-labile enterotoxin.

The relative immunogenicities of three forms of the Escherichia coli heatlabile enterotoxin (LT), the holotoxin, its B subunit, and the polymyxin-release form (PM LT) were compared by immunizing rats with various dosages of each given exclusively by the parenteral (IP/IP) or peroral (PO/PO) routes or by a combination of the two (IP/PO). The degree of protection was evaluated by challenge in ligated ileal loops, and the serum antitoxin response was determined by an enzyme-linked immunosorbent assay with homologous antigens. When given by the PO/PO route, each LT antigen provided only weak protection against the toxin and virtually none against viable LT-producing strains; serum antitoxin titers were not significantly increased. When the toxins were given after a parental primary immunization by either the IP/IP or the IP/PO routes, each LT antigen provided a dose-related increase in serum antitoxin titers and in the degree of protection against the toxin as well as against viable strains which produce LT alone (LT(+)/ST(-)) or in combination with the heat-stable toxin (LT(+)/ST(+)). The degree of protection against viable bacteria, particularly the LT(+)/ST(+) strain, was stronger in animals which received booster immunizations by the PO route. When expressed on the basis of molar equivalents, holotoxin provided significant protection (a protection index of >5 against toxin challenge and >50% reduced secretion with bacterial challenge) with 4 to 15 times fewer moles than PM LT and up to 50 times fewer moles than the B subunit. These observations indicate that, on the basis of molar equivalents, the holotoxin (which contains one A plus five or six B subunits) is a more potent immunogen than either PM LT (which contains one A and probably one B subunit) or the B subunit.

Isolation ofEscherichia coli heat-labile enterotoxin by affinity chromatography: Characterization of subunits

This paper describes the isolation ofEscherichia coli heat-labile enterotoxin (LT) by affinity chromatography on an anti-cholera toxin immunoglobulin-Sepharose column, and the subunit composition of crude and affinity-isolated LT. LT and its subunits were assayed with ganglioside (GM1)-ELISA, immunodiffusion, skin toxicity, and broken cell adenylate cyclase activation methods. The results show that the immunoaffinity method, applied to LT of different strains and batches, yielded about 100-fold purification with approximately 50% recovery of LT antigen. LT was shown to contain a GM1-ganglioside binding subunit as well as another subunit which does not bind to GM1 but activates adenylate cyclase. Immunodiffusion tests showed that the two LT subunits were immunologically related to but not identical with, respectively, the B and A subunits of cholera toxin. The LT “A” and “B” subunits were present in similar proportions in the affinity-isolated and crude LT preparations, but in the purified fraction they had only partially reassociated into holotoxin.

Protective effect of immunization with heat-labile enterotoxin in gnotobiotic rats monocontaminated with enterotoxigenic Escherichia coli.

The protective effect of active immunization with a purified preparation of the polymyxin-release form of Escherichia coli heat-labile enterotoxin (LT), administered using a parenteral prime and peroral boosts given after ablation of gastric secretion by means of cimetidine, was assessed in gnotobiotic rats which were challenged by monocontamination with enterotoxigenic strains of E. coli. Water transport was evaluated by the in vivo marker perfusion technique at weekly intervals over a 3-week period after contamination. Water transport in unimmunized control rats was consistently in absorption in those contaminated by a nontoxigenic strain, in secretion during only week 2 in those contaminated by an LT(+)/- strain, in secretion during weeks 2 and 3 in those contaminated by an LT(+)/ST(+) (heat-stable enterotoxin) strain, and consistently in absorption in those contaminated by an -/ST(+) strain. Rats immunized with a booster dosage of 250 mug had a significant increase (P < 0.001) in net water absorption as compared to unimmunized rats, with values in the borderline range of absorption, when challenged with either the LT(+)/- or LT(+)/ST(+) strains. Rats immunized with a 10-fold-higher boosting dosage had a significant increase (P < 0.001) in net water absorption as compared to those boosted at the lower dosage; water absorption was within the normal range. There was no difference between the ileal bacterial counts of unimmunized and immunized rats challenged by the various strains. These observations indicate that this immunization program provides complete protection in an animal model against challenge by intestinal contamination with enterotoxigenic strains of E. coli which produce LT, either alone or in combination with ST.

Influence of Route of Administration on Immediate and Extended Protection in Rats Immunized with Escherichia coli Heat-Labile Enterotoxin

The effect of route of administration, dosage, and number of boosts employed during immunization with the polymyxin-release form of Escherichia coli heat-labile (LT) enterotoxin on the degree and duration of protection afforded was evaluated in rats which were challenged by the ligated loop technique. Increasing the boosting dosage by fivefold from 50 to 250 mug resulted in a marked increase in protection against challenge with toxin in rats immunized either just by the parenteral route (i.p./i.p.) or by a parenteral prime, followed by peroral boosts (i.p./p.o.) in rats pretreated with cimetidine to ablate gastric secretions; such was not the case, however, even with a 50-fold increase in dosage in rats immunized just by the peroral route (p.o./p.o.). Four weekly peroral boosts were required to achieve the strongest degree of protection. Increasing the boosting dosage also increased the degree of protection against challenge with viable LT(+)/ST(-) and LT(+)/ST(+) strains (ST indicates heat-stable enterotoxin) in rats immunized by the i.p./p.o., but not by the i.p./i.p., route; no protection was evident against an LT(-)/ST(+) strain. Protection was lost within 3 weeks after immunization in rats immunized by the i.p./i.p. route. In contrast, protection was extended over the 3-month observation period in those immunized by the i.p./p.o. route; the degree of protection was enhanced in rats which received an additional boost at 2 months. These observations establish the fact that immunization with LT is similar to that with cholera toxin in that arousal of the local immune intestinal response by means of peroral immunization provides maximal extended protection.

Escherichia coli heat-labile enterotoxin: DNA-directed in vitro synthesis and structure.

Escherichia coli heat-labile enterotoxin was synthesized in a cell-free system directed by DNA of the plasmid P307. Synthesis of the toxin, assayed by the elongation induced in Chinese hamster ovary cells, was strongly stimulated by cyclic AMP and occurred at physiological levels of Mg2+ only when the polyamine spermidine was present. Activity was abolished by heat and antisera prepared against the enterotoxins of both E. coli P263 and Vibrio cholera. Tritium-labeled enterotoxin was purified by immunoprecipitation and examined by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. When gel slices were assayed for the ability to stimulate adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] activity in erythrocyte ghosts, two peaks were found, one at Mr 26,000 and frequently, but not always, another at Mr 23,000. Detection of radiolabeled protein by fluorography and scintillation counting of gel slices revealed three prominent polypeptides, two corresponding to the peaks having adenylate cyclase-stimulating activity and a further one of Mr 11,500, identical to that of the cholera subunit B. The data suggest that the E. coli heat-labile enterotoxin synthesized in the cell-free system has a subunit structure.

Immunological cross-reactivity betweenEscherichia coli heat-labile enterotoxin and cholera toxin A and B subunits

It was shown by immunoenzyme methods and neutralization tests thatEscherichia coli heat-labile entorotoxin cross-reacts immunologically with both the A and B subunits of cholera toxin. In addition to the shared structures, both toxins had unique antigenic determinants. The toxin-neutralizing capacity of anti-B antibody was considerably higher than that of anti-A antibody, as tested in the adrenal cell and rabbit skin assays. In intestinal loops, however, the neutralizing potencies of anti-A and anti-B were similar, possibly reflecting that in this system mere cross-linking of toxin by antibody inhibits its diffusion through mucus.

Isolation of an avian erythrocyte protein possessing ADP-ribosyltransferase activity and capable of activating adenylate cyclase

An ADP-ribosyltransferase was purified approximately 500-fold from the supernatant fraction of turkey erythrocytes. The enzyme hydrolyzed [carbonyl-(14)C]NAD to ADP-ribose and [carbonyl-(14)C]nicotinamide at a low rate. Nicotinamide formation from NAD was enhanced by arginine methyl ester > D-arginine approximately L-arginine > guanidine; lysine, histidine, and citrulline were ineffective. Incubation of [adenine-U-(14)C]NAD and arginine methyl ester or arginine with the purified enzyme resulted in the formation of new compounds that contained (14)C, reacted with ninhydrin, and quenched background fluorescence of thin-layer plates viewed in ultraviolet light. Their mobilities on thin-layer chromatograms were indistinguishable from those of ADP-ribosylarginine methyl ester and ADP-ribosylarginine formed during incubation of choleragen with NAD and arginine methyl ester or arginine, respectively [Moss, J. & Vaughan, M. (1977) J. Biol. Chem. 252, 2455-2457]. The purified transferase also catalyzed the incorporation of label from [adenine-(14)C]-NAD into lysozyme, histones and polyarginine. When the (14)C-labeled lysozyme was incubated with snake venom phosphodiesterase, the radioactivity was released and, on thin-layer chromatograms, exhibited a mobility indistinguishable from that of 5'-AMP, as would be expected of an ADP-ribosylated protein, but not of a poly(ADP-ribosylated) product. The purified transferase activated rat brain adenylate cyclase and, as is the case with choleragen, activation was absolutely dependent on NAD. The presence in the avian erythrocyte of a protein that, like choleragen and Escherichia coli heat-labile enterotoxin, apparently activates adenylate cyclase and possesses ADP-ribosyl transferase activity is consistent with the view that the mechanisms through which the bacterial toxins produce pathology are not entirely foreign to vertebrate cells, at least some of which may possess and employ an analogous mechanism for activation of adenylate cyclase.

Identification ofEscherichia coli heat-labile enterotoxin by means of a ganglioside immunosorbent assay (GM1-ELISA) procedure

A sensitive, quantitative method for determination ofEscherichia coli heat-labile enterotoxin (LT) is presented. The assay is based on the specific binding of LT to polystyrene-adsorbed GM1 ganglioside and subsequent enzyme immunological demonstration of the bound toxin. Enterotoxin titers determined with this GM1 enzyme-linked immunosorbent assay (ELISA) method agreed closely with those obtained with the adrenal cell bioassay. The GM1-ELISA procedure was capable of demonstrating LT in allE. coli overnight cultures that gave positive adrenal cell results. The simplicity and high reproducibility of the described method should make it well suited for routine laboratory diagnosis of LT enterotoxigenicE. coli strains.

Immunological Study of the Heat-Labile Enterotoxins of Escherichia coli and Vibrio cholerae

Immunodiffusion experiments were conducted to associate a precipitin line with Escherichia coli heat-labile enterotoxin (LT). Wild strains of porcine and of human enteropathogenic E. coli as well as laboratory-derived enterotoxigenic variants of E. coli K-12 were used for LT antigen preparations. These were produced mainly by ultrafiltration and ammonium sulfate precipitation of broth culture supernatants. When antisera with anti-LT activity were reacted with antigen preparations from Ent(+) and Ent(-) variants of E. coli K-12, a line "a" was given by Ent(+) but not by Ent(-) preparations. Line "a" was removed by absorption of anti-LT serum with antigen preparation from an Ent(+)E. coli K-12, but was unaffected when the antigen preparation used to absorb the serum was from an Ent(-)E. coli K-12. A line identical to "a" was given by antigen preparations from wild strains of porcine enteropathogenic E. coli reacted with homologous or heterologous anti-LT sera. One human strain of enteropathogenic E. coli was shown to possess an antigen identical to that which gave rise to line "a." To demonstrate this line it was necessary to use high concentrations of gammaglobulin and high concentrations of the crude antigen preparations. LT preparations reacted with anticholera toxin to give a line "c," which showed a reaction of partial identity with line "b" produced by reaction of pure choleragenoid and anticholera toxin. Lines "a" and "c" gave reactions of identity.