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PubMed1972

Failure to detect cell-associated enterotoxin B in Staphylococcus aureus by immunofluorescence.

Enterotoxin B-producing and -nonproducing Staphylococcus aureus strains showed cell fluorescence when tested with fluoresceinisothiocyanate-labeled rabbit anti-enterotoxin B globulin, probably as a result of a protein A-immunoglobulin G (Ig G) interaction. No cell-bound enterotoxin B could be detected by immunofluorescence using F(ab(1))(2)-fragments of anti-enterotoxin B globulin. However, soluble enterotoxin B could be estimated by immunofluorescence. Approximately 1,000-fold more enterotoxin B was detected by immunodiffusion as an extracellular product in the media than could be detected in the cell fraction. The results show that intact Ig G is not suitable for the detection of antigens other than protein A on the cell surface of S. aureus in conventional immunofluorescence. For such purposes, the use of F(ab(1))(2)-fragments of Ig G is recommended.

Ammonium Sulfate↗

PubMed1975

Characterization of enterotoxin purified from Clostridium perfringens type C.

Enterotoxin produced by a sporulating culture of Clostridium perfringens type C, which had been isolated from a case of severe necrotic enteritis, was purified. The molecular weight was estimated to be 36,000 by gel chromatography on Sephadex G-100 and 33,400 by ultracentrifugation. The sedimentation coefficient S20,W was 2.92. The toxin protein exhibited unusual behavior on sodium dodecyl sulfate gels, and toxin aggregates having molecular weights of 68,000, 85,000, 105,000, and 140,000 were obtained. The purified enterotoxin often separated, apparently due to slight charge differences, into two protein bands on 7% polyacrylamide gels. Electrofocusing of enterotoxin on polyacrylamide gels gave an approximate isoelectric point of 4.3, with the enterotoxin being fractionated into four distinct protein bands. The specific toxicity of the enterotoxin was about 1,900 mouse mean lethal doses per mg of calculated nitrogen. The data obtained indicate that the enterotoxin from C. perfringens type C is identical in most respects to that obtained from type A strains. Whether or not this toxin plays a role in the necrotic enteritis caused by type C strains is unknown at present.

Bacterial Proteins↗

PubMed1977

Induction of Escherichia coli and Vibrio cholerae enterotoxins by an inhibitor of protein synthesis.

Enterotoxigenic Escherichia coli or Vibrio cholerae 569B (Inaba) grown in the presence of the antibiotic lincomycin, an inhibitor of protein synthesis, produced elevated levels of heat-labile enterotoxin or choleragen, respectively, as assayed by both vascular permeability factor and capacity to elicit fluid accumulation in rabbit ileal loops. This induction of enterotoxin did not reflect either a coupling of lincomycin resistance with increased enterotoxigenicity or an effect of lincomycin on cellular release of enterotoxin, since spontaneously isolated lincomycin-resistant mutants of both E. coli and V. cholerae still required lincomycin for induction, and large increases in E. coli permeability factor activity were found intracellularly as well as extracellularly. After the period of exponential growth, E. coli became refractory to induction by lincomycin, although most of the induced enterotoxin activity appeared only after this period. No increase in copy number of the enterotoxin plasmid in E. coli 711 (P307) was found in induced cells by analysis of deoxyribonucleic acid reassociation kinetics. These and other data suggest that synthesis of enterotoxin, or at least its accumulation, is normally limited by cellular factors whose synthesis is preferentially inhibited by lincomycin. A possible connection between this phenomenon and lincomycin-associated diarrhea is considered.

Animals↗

PubMed1978

Demonstration of shared and unique immunological determinants in enterotoxins from Vibrio cholerae and Escherichia coli.

Immunodiffusion and biological neutralization studies demonstrated that the heat-labile enterotoxin (LT) from Escherichia coli has antigenic determinants in common with each of the isolated subunits (A and B) of the enterotoxin (choleragen) from Vibrio cholerae. Each of the enterotoxins also possesses unique antigenic specificities. Monospecific antiserum to LT was prepared by immunization with antigens derived by immune precipitation of E. coli cell-free supernatant with isolated specific anticholeragenoid antibodies. This antiserum neutralized the biological acitivity of both LT and cholera enterotoxin and recognized antigens of both in immunodiffusion. This antiserum was adsorbed with choleragenoid to remove antibodies directed against the shared "B" immunological determinants. The neutralizing effect of the antiserum on cholera toxin was completely removed, but the neutralizing activity against the E. coli preparations was retained, although somewhat reduced. Antisera to the isolated subunits (A and B) of cholera enterotoxin neutralized the biological activity of cholera enterotoxin and LT. These antisera also recognized the homologous and heterologous antigens in immunodiffusion. Multiple forms or conformations of LT and its components may explain the diversity of the properties which have been reported for it.

Antigens, Bacterial↗

PubMed1979

Production of heat-stable, methanol-soluble enterotoxin by Yersinia enterocolitica.

Seven isolates of Yersinia enterocolitica serotype 0:8, recovered during an outbreak of gastrointestinal illness, were examined for enterotoxin production. All seven strains were enterotoxigenic in the suckling mouse model, and three of five isolates tested produced keratoconjunctivitis in the guinea pig eye model (Sereny test). Enterotoxin was detected in broth supernatant fluid after 12 h of incubation at 25 degrees C. The toxin was not inactivated by exposure to 121 degrees C for 30 min or by storage at 4 or -40 degrees C for at least 5 months. The enterotoxin was also acid stable and methanol soluble. Methanol extraction did not affect its heat stability or enterotoxic activity in suckling mice. These physical characteristics plus limited ultrafiltration studies suggest that the enterotoxin is a low-molecular-weight substance. Y. enterocolitica enterotoxin resembles Escherichia coli heat-stable enterotoxin in heat and pH stability, methanol solubility, and enterotoxic activity in suckling mice. However, its role in the pathogenesis of human diarrhea has not been established.

Bacterial Toxins↗

PubMed1979

Mechanism of action of Yersinia enterocolitica enterotoxin.

Enterotoxin derived from three clinical isolates of Yersinia enterocolitica was compared with the heat-stable enterotoxin of Escherichia coli. Both toxins were biologically active in infant mice examined at 2 h and in ligated rabbit ileal loops at 6 h. Neither substance, however, produced changes in ligated ileal loops at 18 h or in Chinese hamster ovary or Y1 adrenal tissue cultures. In addition, both Y. enterocolitica enterotoxin concentrated approximately 20 times by ammonium sulfate precipitation and ultrafiltration and a similarly prepared sample of E. coli heat-stable enterotoxin stimulated the activity of guanylate cyclase but not that of adenylate cyclase in infant mouse intestine. These findings suggest that the role of enterotoxin in the pathogenesis of intestinal Y.enterocolitica infection may be similar to that of heat-stable enterotoxin in E. coli diarrhea.

Adenylyl Cyclases↗

PubMed1979

Purification and partial characterization of heat-stable enterotoxin of enterotoxigenic Escherichia coli.

Heat-stable enterotoxin was purified from a strain of enterotoxigenic Escherichia coli 53402 A-1 from human intestine. The cells were cultured in Casamino Acids-yeast extract-salts medium, and the purification procedure consisted of protamine sulfate treatment of the culture supernatant, ultrafiltration with an Amicon PM-10 membrane, diethylaminoethyl-cellulose column chromatography, hydroxyapatite column chromatography, Bio-Gel P-10 gel filtration, 90% ethanol extraction, and preparative polyacrylamide gel disc electrophoresis. About 300-fold purification was achieved, with a yield of about 12%. However, the homogeneity of the purified heat-stable enterotoxin was not rigorously demonstrated. The purified heat-stable enterotoxin had an absorption maximum at about 275 nm, and its isoelectric point was about 3.90. The molecular weight of the purified heat-stable enterotoxin was ca. 4,000 by Sephadex G-100 gel filtration. The minimum effective dose of purified heat-stable enterotoxin was about 2.5 ng in the suckling mouse assay. The purified heat-stable enterotoxin gave a positive reaction in not only the suckling mouse assay but also the mouse intestinal loop test and the guinea pig skin permeability test.

Electrophoresis, Polyacrylamide Gel↗

PubMed1971

Identification of enterotoxin E.

Identification of a new enterotoxin was accomplished by purification of the enterotoxin produced by staphylococcal strain FRI-326 and by preparation of specific antitoxin to the enterotoxin. Toxicity of the preparations was determined in rhesus monkeys, and specificity of the enterotoxin-antitoxin reaction was determined in gel diffusion plates. The enterotoxin was designated enterotoxin E.

Animals↗

PubMed1983

Stimulation of calcium uptake and cyclic GMP synthesis in rat basophilic leukemia cells by Escherichia coli heat-stable enterotoxin.

The effect of Escherichia coli heat-stable (ST) enterotoxin on calcium and cyclic nucleotide metabolism in rat basophilic leukemia cell cultures was investigated. Addition of ST enterotoxin to rat basophilic leukemia cell cultures resulted in dose- and time-dependent stimulation of calcium uptake and elevation of the intracellular cyclic GMP (cGMP) concentration. The effect of ST enterotoxin on calcium uptake (P less than 0.02) and cGMP synthesis (P less than 0.02) was demonstrated after 5 and 30 min of incubation at 37 degrees C, respectively. In further studies ST enterotoxin did not enhance calcium release or the intracellular concentration of cyclic AMP. The stimulation of calcium uptake and cGMP synthesis by ST enterotoxin was inhibited by pharmacological and chemical agents which block cellular calcium entry and prostaglandin synthesis. These results demonstrate that ST enterotoxin induces calcium uptake and cGMP synthesis in rat basophilic leukemia cell cultures.

Animals↗

PubMed1986

Oral immunization of mice with attenuated Salmonella enteritidis containing a recombinant plasmid which codes for production of the B subunit of heat-labile Escherichia coli enterotoxin.

We used Salmonella enteritidis serotype dublin strain SL1438, a nonreverting, aromatic-dependent, histidine-requiring mutant, as a recipient for a recombinant plasmid coding for production of the nontoxic B subunit of the heat-labile Escherichia coli enterotoxin. The S. enteritidis derivative EL23 produced heat-labile enterotoxin subunit B that was indistinguishable from heat-labile enterotoxin subunit B produced by strains of E. coli or Salmonella typhi harboring the same plasmid. Mice immunized orally with strain EL23 developed progressively increasing mucosal and serum antibody responses to both heat-labile enterotoxin subunit B and to the lipopolysaccharide of the vaccine strain. The mucosal antibody response was shown to be immunoglobulin A specific and to be capable of neutralizing the biological activities of both E. coli heat-labile enterotoxin and cholera enterotoxin in vitro.

Administration, Oral↗

PubMed1987

Isolation and function of a Clostridium perfringens enterotoxin fragment.

A fragment was obtained by treating Clostridium perfringens enterotoxin with 2-nitro-5-thiocyanobenzoic acid, a reagent which specifically cleaves the amino-terminal peptide bond of cysteine residues. The fragment (molecular weight, 15,000) was purified by high-performance liquid chromatography. The fragment had no cytotoxic effect on Vero cells but competitively inhibited enterotoxin-induced 51Cr release. Binding of 125I-labeled fragment to Vero cells was comparable to that of enterotoxin. Moreover, 125I-labeled fragment did not bind to FL cells, which lack receptor for enterotoxin. We conclude that the fragment contains the binding domain of enterotoxin. The amino acid composition of the fragment suggests that it is located on the carboxyl-terminal part of enterotoxin.

Amino Acids↗

PubMed1990

Proteolysis of Clostridium perfringens type A enterotoxin during purification.

The small satellite bands of enterotoxin frequently seen in polyacrylamide gels following purification of Clostridium perfringens enterotoxin were found to be due to endogenous protease activity and were not present if phenylmethylsulfonyl fluoride (PMSF; 1 mM) and EDTA (10 mM) were used in the purification protocol. The use of PMSF was avoided by passing gel filtration-purified enterotoxin material through DEAE-Sephacel. This modified protocol resulted in an 11.4-fold purification of enterotoxin and a 26.8% yield. Contrary to previous reports (B. R. Dasgupta and M. W. Pariza, Infect. Immun. 38: 592-597, 1982), if PMSF and EDTA were included during purification, we were unable to detect the novel enterotoxin ET-1 produced by strain NCTC 10240. C. perfringens proteases cleaved homogeneous enterotoxin into two additional fragments, suggesting that ET-1 was a product of endogenous protease action during purification.

Bacterial Toxins↗

PubMed1972

Purification and biochemical properties of Clostridium perfringens type A enterotoxin.

The sporulation-specific enterotoxin of Clostridium perfringens type A, which is the toxin active in human food poisoning, has been purified from extracts of sporulating cells. Highly purified enterotoxin was obtained by treatment of crude cell extract with ribonuclease for 30 min, followed by sequential chromatography on Sephadex G-100, Cellex T cellulose, and hydroxylapatite. Recovery was 65 to 75% of the initial activity. Enterotoxin purity was > 99% as indicated by sedimentation velocity, sedimentation equilibrium, disc electrophoresis, and serological methods. Purified enterotoxin focused at pH 4.3 during isoelectric focusing. Molecular weights of 34,000 and 35,000 were obtained by Sephadex G-100 chromatography and sedimentation equilibrium, respectively. An S(20,w) of 3.08 was obtained for the purified enterotoxin. The enterotoxin precipitated heavily at its isoelectric point and at concentrations greater than 4 mg/ml.

Animals↗

PubMed1992

Characterization of hybrid toxins produced in Escherichia coli by assembly of A and B polypeptides from type I and type II heat-labile enterotoxins.

The genes encoding the individual A and B polypeptides of the type I enterotoxin LTp-I and type II enterotoxins LT-IIa and LT-IIb were cloned and tested for complementation in Escherichia coli. Each gene encoding an A polypeptide was cloned into pACYC184, and each gene encoding a B polypeptide was cloned into the compatible plasmid Bluescript KS+. In addition, operon fusions representing all combinations of A and B genes were constructed in Bluescript KS+. Extracts from strains of E. coli expressing each combination of A and B genes, either from compatible plasmids or from operon fusions, were tested for immunoreactive holotoxin by radioimmunoassays and for toxicity by Y1 adrenal cell assays. Biologically active holotoxin was detected in each case, but the toxicity of extracts containing the hybrid toxins was usually less than that of extracts containing the wild-type holotoxins. The ganglioside-binding activity of each holotoxin was tested, and in each case, the B polypeptide determined the ganglioside-binding specificity. The A and B polypeptides of the type II heat-labile enterotoxins were also shown to form holotoxin in vitro without exposure to denaturing conditions, in contrast to the polypeptides of the type I enterotoxins that failed to form holotoxin in vitro under comparable conditions. These findings suggest that type I and type II enterotoxins have conserved structural features that permit their A and B polypeptides to form hybrid holotoxins, although the B polypeptides of the type I and type II enterotoxins have very little amino acid sequence homology.

Bacterial Toxins↗

PubMed2004

The Type II heat-labile enterotoxins LT-IIa and LT-IIb and their respective B pentamers differentially induce and regulate cytokine production in human monocytic cells.

The type II heat-labile enterotoxins, LT-IIa and LT-IIb, exhibit potent adjuvant properties. However, little is known about their immunomodulatory activities upon interaction with innate immune cells, unlike the widely studied type I enterotoxins that include cholera toxin (CT). We therefore investigated interactions of LT-IIa and LT-IIb with human monocytic THP-1 cells. We found that LT-II enterotoxins were inactive in stimulating cytokine release, whereas CT induced low levels of interleukin-1beta (IL-1beta) and IL-8. However, all three enterotoxins potently regulated cytokine induction in cells activated by bacterial lipopolysaccharide or fimbriae. Induction of proinflammatory (tumor necrosis factor alpha [TNF-alpha]) or chemotactic (IL-8) cytokines was downregulated, whereas induction of cytokines with anti-inflammatory (IL-10) or mucosal adjuvant properties (IL-1beta) was upregulated by the enterotoxins. These effects appeared to depend on their A subunits, because isolated B-pentameric subunits lacked regulatory activity. Enterotoxin-mediated inhibition of proinflammatory cytokine induction in activated cells was partially attributable to synergism for endogenous production of IL-10 and to an IL-10-independent inhibition of nuclear factor kappaB (NF-kappaB) activation. In sharp contrast to the holotoxins, the B pentamers (LT-IIaB and, to a greater extent, LT-IIbB) stimulated cytokine production, suggesting a link between the absence of the A subunit and increased proinflammatory properties. In this regard, the ability of LT-IIbB to activate NF-kappaB and induce TNF-alpha and IL-8 was antagonized by the LT-IIb holotoxin. These findings support distinct immunomodulatory roles for the LT-II holotoxins and their respective B pentamers. Moreover, the anti-inflammatory properties of the holotoxins may serve to suppress innate immunity and promote the survival of the pathogen.

Bacterial Toxins↗

PubMed2005

Mucosal adjuvant properties of mutant LT-IIa and LT-IIb enterotoxins that exhibit altered ganglioside-binding activities.

LT-IIa and LT-IIb, the type II heat-labile enterotoxins of Escherichia coli, are closely related in structure and function to cholera toxin and LT-I, the type I heat-labile enterotoxins of Vibrio cholerae and E. coli, respectively. Recent studies from our group demonstrated that LT-IIa and LT-IIb are potent systemic and mucosal adjuvants. To determine whether binding of LT-IIa and LT-IIb to their specific ganglioside receptors is essential for adjuvant activity, LT-IIa and LT-IIb enterotoxins were compared with their respective single-point substitution mutants which have no detectable binding activity for their major ganglioside receptors [e.g., LT-IIa(T34I) and LT-IIb(T13I)]. Both mutant enterotoxins exhibited an extremely low capacity for intoxicating mouse Y1 adrenal cells and for inducing production of cyclic AMP in a macrophage cell line. BALB/c female mice were immunized by the intranasal route with the surface adhesin protein AgI/II of Streptococcus mutans alone or in combination with LT-IIa, LT-IIa(T34I), LT-IIb, or LT-IIb(T13I). Both LT-IIa and LT-IIb potentiated strong mucosal and systemic immune responses against AgI/II. Of the two mutant enterotoxins, only LT-IIb(T13I) had the capacity to strongly potentiate mucosal anti-AgI/II and systemic anti-AgI/II antibody responses. Upon boosting with AgI/II, however, both LT-IIa(T34I) and LT-IIb(T13I) enhanced humoral memory responses to AgI/II. Flow cytometry demonstrated that LT-IIa(T34I) had no affinity for cervical lymph node lymphocytes. In contrast, LT-IIb(T13I) retained binding activity for T cells, B cells, and macrophages, indicating that this immunostimulatory mutant enterotoxin interacts with one or more unknown lymphoid cell receptors.

Adjuvants, Immunologic↗

PubMed2006

Control of enterotoxin gene expression in Bacillus cereus F4430/73 involves the redox-sensitive ResDE signal transduction system.

In contrast to Bacillus subtilis, the role of the two-component regulatory system ResDE has not yet been investigated in the facultative anaerobe Bacillus cereus. We examined the role of ResDE in the food-borne pathogen B. cereus F4430/73 by constructing resDE and resE mutants. Growth performances, glucose metabolism, and expression of hemolysin BL (Hbl) and nonhemolytic enterotoxin (Nhe) were analyzed in the three strains under distinct oxygenation and extracellular oxidoreduction potential (ORP) conditions. We show that growth and glucose metabolism were only moderately perturbed in both resDE and resE mutants under aerobiosis, microaerobiosis, and anaerobiosis generated under N(2) atmosphere (initial ORP = +45 mV). The major effects of resDE and resE mutations were observed under low-ORP anaerobic conditions generated under hydrogen atmosphere (iORP = -148 mV). These conditions normally favor enterotoxin production in the wild type. The resE mutation was more deleterious to the cells than the resDE mutation, causing growth limitation and strong deregulation of key catabolic genes. More importantly, the resE mutation abolished the production of enterotoxins under all of the conditions examined. The resDE mutation only decreased enterotoxin expression under anaerobiosis, with a more pronounced effect under low-ORP conditions. Thus, the ResDE system was found to exert major control on both fermentative growth and enterotoxin expression, and it is concluded that the ResDE system of B. cereus should be considered an anaerobic redox regulator. The data presented also provide evidence that the ResDE-dependent regulation of enterotoxins might function at least partially independently of the pleiotropic virulence gene regulator PlcR.

Aerobiosis↗

PubMed1979

Transposition of ampicillin resistance to an enterotoxin plasmid in an Escherichia coli strain of human origin.

We examined a strain of Escherichia coli, serotype O159.H34, of human origin which produced heat-stable and heat-labile enterotoxins, was resistant to ampicillin, and produced colicin. By conjugation and transformation experiments plasmids coding for enterotoxin production (Ent), enterotoxin production and ampicillin resistance (Ap-Ent), ampicillin resistance (Ap), and colicin production were isolated. Both the Ent and Ap-Ent plasmids were autotransferring and belonged to the F-incompatibility complex. However, the Apr Ent+ transconjugants showed differences in their levels of resistance and in their abilities to propagate F-specific phages and to transfer resistance. The results suggested there was transposition from the small Ap plasmid to the Ent plasmid. The Ap-Ent plasmids were larger than the enterotoxin factor and when treated with restriction endonuclease BamHI showed an additional fragment not present in the enterotoxin plasmid. The insertion of ampicillin resistance probably occurred at different sites on the enterotoxin plasmid, resulting in the observed variation in phenotype.

Ampicillin↗