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PubMed1993

Purification and sequence determination of heat-stable enterotoxin elaborated by a cholera toxin-producing strain of Vibrio cholerae O1.

Four molecular species of heat-stable enterotoxins elaborated by a cholera toxin-producing strain of Vibrio cholerae O1 were isolated from its culture supernatant. The amino acid sequence of one of the enterotoxins was determined to be Phe-Ile-Lys-Gln-Val-Asp-Glu-Asn-Gly-Asn-Leu-Ile-Asp-Cys-Cys-Glu-Ile-Cys- Cys-Asn-Pro-Ala-Cys-Phe-Gly-Cys-Leu-Asn with three intramolecular disulfide linkages. The other enterotoxins had shorter amino acid sequences in the N-terminal regions, but possessed the same sequence in their C-terminal regions including the three disulfide linkages. The enterotoxins with the shorter N-terminal sequences showed more potent toxicities, and the minimum effective dose of the longest one with 28 amino acid residues was 10-folds of that of the shortest one.

Amino Acid Sequence↗

PubMed1988

Age-related differences in receptors for Escherichia coli heat-stable enterotoxin in the small and large intestine of children.

Escherichia coli that produce heat-stable enterotoxin are a worldwide cause of diarrheal disease, especially in children. We examined small and large intestinal specimens from children of various ages for the presence of E. coli heat-stable entero-toxin receptors and determined whether the number of receptors or the binding affinity of these receptors was related to the age of the child. We observed specific binding of 125I-heat-stable enterotoxin to all small intestinal and colonic specimens. However, a greater number of receptors per microgram of membrane protein were present in infants and the number of receptors rapidly decreased with increasing age. We also observed that increased heat-stable enterotoxin stimulation of guanylate cyclase was correlated with increased receptor density. We suggest that a greater number of gastrointestinal receptors for heat-stable enterotoxin, capable of activating more guanylate cyclase, may contribute to the increased severity of diarrhea noted in young children exposed to enterotoxigenic E. coli.

Adolescent↗

PubMed1990

The effect of Escherichia coli heat-stable enterotoxin on protein kinase activity.

Isolated rat enterocytes were incubated with E. coli heat-stable enterotoxin or buffer alone and the protein kinase activity and cyclic GMP level determined on the particulate fraction or cytosol, respectively. In the control cells, particulate protein kinase activity and cyclic GMP concentration were at a maximum after 20 sec and 1 min of incubation, respectively. In heat-stable enterotoxin-treated cells the particulate protein kinase activity was significantly increased (P less than 0.05) after 20 sec of incubation, but decreased (P less than 0.05) after 30 sec, 1 min and 2 min, when compared to the control reaction. During this time period the concentration of intracellular cyclic GMP increased 10-fold. The effect of heat-stable enterotoxin on particulate protein kinase activity and cyclic GMP concentration was dose-dependent. Analysis of radioactive membrane phosphorylation products indicate a role for phosphoproteins with a mol. wt of 25,000 and 120,000. These results suggest that the action of heat-stable enterotoxin may involve an effect on protein kinase.

Animals↗

PubMed1988

Cloning, sequencing, and expression in Ficoll-generated minicells of an Escherichia coli heat-stable enterotoxin gene.

The gene encoding a heat-stable enterotoxin of Escherichia coli was cloned as a 960-bp fragment from a plasmid isolated from a Mexican strain of human origin. Deoxyribonucleotide sequencing unveiled a 216-bp open reading frame similar to that of a previously sequenced ST-toxin gene. The gene is preceded by a proposed binding site for the cAMP-mediated positive regulator (CAP) that is part of a 23-bp inverted repeat. The proposed CAP site is followed by a 6A, 1T, and 6A deoxyribonucleotides. Minicells containing the toxin gene, which were isolated from Ficoll gradients, shown to preserve the localization of intracellular and periplasmic enzymes, allowed the detection of a biosynthetically radiolabeled polypeptide with an apparent Mr 8400. The data suggest that the enterotoxin genes estA2, estA3, and estA4 are very similar, even in clinical strains isolated from distinct geographical locations; that the transcription of heat-stable enterotoxin genes is controlled by the cAMP-mediated positive regulatory system, and that the heat-stable enterotoxins are initially synthesized as 72 amino acid precursors to yield the extracellular active 18-19 amino acid polypeptides.

Amino Acid Sequence↗

PubMed1992

Occurrence of fimbriae and enterotoxins in Escherichia coli strains isolated from piglets in Poland.

1125 and 1146 E. coli strains isolated from suckling and weaned piglets with diarrhea, respectively, and 724 strains from healthy piglets were tested for the presence of fimbriae and production of enterotoxins. The fimbriae were determined by hemagglutination and slide agglutination tests, enterotoxins--by the use of ileal loop test in piglets (LT and STb enterotoxins) and suckling mouse assay (STa enterotoxin). It was found that 72.8 and 53.0% strains, isolated from diseased suckling and weaned piglets, respectively, possessed specific fimbrial hemagglutinins, in most cases with K88 antigen. Additionally, 987P fimbriae were detected in 14.0 and 0.7% strains isolated from piglets with diarrhea. Only 5 strains (0.7%) recovered from healthy piglets had specific fimbriae, usually with undetermined antigenic structure. F1 fimbriae (called common or unspecific) were found in strains isolated both from diseased (15.2 and 16.3% strains, respectively) and healthy piglets (27.1% strains). It was noted that the strains isolated from suckling and weaned piglets with diarrhea in most cases were enterotoxigenic (90.5 and 69.1% strains, respectively) and most frequently produced heat-labile toxin LT alone or with STb. 18.5% of enterotoxigenic strains isolated from healthy piglets produced STa toxin.

Animals↗

PubMed1982

Inhibition of protein synthesis by a tryptic polypeptide of Clostridium perfringens type A enterotoxin.

The biological activity of Clostridium perfringens enterotoxin can be tested more precisely and with a much higher sensitivity by using the inhibition of protein synthesis by Vero cells, rather than the guinea pig skin test. Tryptic peptides of the enterotoxin produced in the presence of different concentrations of sodium dodecyl sulfate (0-1%) have been tested for biological activity (Vero cells) and inhibitory effect on cell-free protein synthesis (rabbit reticulocyte lysate). A fraction of tryptic peptides, about 16,000 daltons, was able to inhibit the cell-free protein synthesis, while the native enterotoxin had no such effect. The 16 kDa fraction had, however, lost the ability to disrupt the Vero cells (normal biological activity). It is probable that the enterotoxin has the double function (A and B chain), known from several other toxins, confined in its single polypeptide chain.

Animals↗

PubMed1989

DOT-enzyme linked immunosorbent assay for detection of Clostridium perfringens type A enterotoxin.

A procedure, which we have termed DOT-ELISA, to detect Clostridium perfringens type A enterotoxin on nitrocellulose paper is described. Seventy eight preparations from 39 cultures of C. perfringens type A were tested simultaneously by this and by Plate-ELISA methods. The results were comparable. DOT-ELISA detected as little as 0.02 micrograms of purified enterotoxin and 0.13 micrograms of enterotoxin in cell-free culture supernatant. As little as 0.02 micrograms purified enterotoxin mixed with human faeces could be detected specifically. The method is simple and does not require an ELISA reader.

Clostridium perfringens↗

PubMed1995

Production of staphylococcal enterotoxin A in cream-filled cake.

Cakes were baked with normal ingredients and filled with cream, inoculated with different size enterotoxigenic-staphylococcal inocula. Samples of the cakes were incubated at room temperature and put in the refrigerator. Samples of cake and filling were taken at different times and analyzed for staphylococcal count and presence of enterotoxin. The smaller the inoculum, the longer the time required for sufficient growth (10(6)) to occur for production of detectable enterotoxin. Enterotoxin added to the cake dough before baking (210 degrees C, 45 min) did not survive the baking. The presence of enterotoxin in the contaminated cream filling indicated this as the cause of staphylococcal food poisoning from cream-filled cakes. Refrigeration of the cakes prevented the growth of the staphylococci.

Animals↗

PubMed1993

Recombinant enterotoxins as vaccines against Escherichia coli-mediated diarrhoea.

A fusion protein, comprising the B subunit of the heat-labile enterotoxin and a portion of the precursor to the heat-stable enterotoxin of Escherichia coli, has been created by recombinant genetic techniques. It is exported successfully to the bacterial periplasm and assembles into pentamers which retain the ability to bind to GM1 ganglioside. Native toxin epitopes are displayed and the molecule can be easily purified from periplasmic extracts of cells expressing the gene fusion. Although the protein carries the natural sequence of the heat-stable enterotoxin, it is greatly attenuated in toxicity. Systemic immunization of mice or oral administration of the fusion elicits antibody responses against both classes of E. coli enterotoxin.

Administration, Oral↗

PubMed1983

Immunological differences among the cholera/coli family of enterotoxins.

Pure enterotoxins from two strains of Vibrio cholerae and choleragen-related heat-labile enterotoxins (LTs) from strains of Escherichia coli of human and porcine origin were examined by Ouchterlony-type immunodiffusion assays and by neutralization tests in Y-1 adrenal cells using specific and immunopurified antisera. In accordance with previous findings, the results indicated that each of the toxins shared antigens with each of the others and that each, in addition, possessed unique antigenic determinants. The present study, however, indicates further that enterotoxin-type specific antibodies, in some instances, account for a major portion of the neutralizing activity. The full extent and importance of the antigenic drift in the cholera/coli family of ADP-ribosylating, adenylate cyclase-activating, heat-labile enterotoxins remains to be determined. The results will be pertinent to efforts to develop broad-spectrum antitoxic immunity as well as in the development of methods for the rapid detection and identification of enterotoxigenic strains.

Bacterial Toxins↗

PubMed1992

Expression and characterization of the cloned Salmonella typhimurium enterotoxin.

Earlier, our laboratory reported the cloning of a chromosomally encoded cholera toxin (CT)-like enterotoxin gene from Salmonella typhimurium Q1 into pBR322. Cell lysates from the plasmid clone pC1, containing a 4.8 kb EcoR1 DNA fragment from Salmonella, caused elongation of Chinese hamster ovary (CHO) cells and this biologic activity was neutralized by anti-CT. However, this cloned gene product did not elicit fluid secretion in the rabbit intestinal loop (RIL) model, because of poor expression. We report here, subcloning of a 4.8 kb EcoRI and a 2.7 kb HindIII/Eco Rl fragment into a high expression T7 RNA polymerase/promoter system. Cell lysates from these clones elicited fluid secretion in the RIL model, caused firm induration in rabbit skin and elongated CHO cells. These biological activities were neutralized by anti-CT. SDS-PAGE and subsequent fluorographic analysis of Escherichia coli, harboring recombinant plasmids in a T7 expression system, revealed the presence of two prominent 35S-labeled polypeptides of 25 and 12 kDa, which were immunoprecipitated with anti-CT. The enterotoxin appeared to be 125 kDa in size, based on chromatography on a P-300 column, had a pl of 6.6 to 6.8, and was heat-labile (60 degrees C/5 min). Unlike cloned CT and heat-labile enterotoxin (LT-l), which were localized in the periplasm, the Salmonella enterotoxin was cytoplasmic in nature.

Animals↗

PubMed1992

Pharmacologic action of Escherichia coli heat-stable (STa) enterotoxin.

Escherichia coli produces a heat-stable (STa) enterotoxin that belongs to a family of peptides that mediate several diarrheal diseases, including traveler's diarrhea and epidemic diarrhea in infants and newborns. The STa enterotoxin consists of 18 or 19 amino acids and is encoded by genes specified on a transposon. Intestinal secretion is induced by specific binding to high affinity receptors that reside on the brush border cell membrane of the small intestine. Receptor activation by STa enterotoxin induces a sequence of events that culminate in the release of fluid and electrolytes into the intestinal lumen. These events include the stimulation of particulate guanylate cyclae and subsequent increase of intracellular cyclic GMP, involvement of particulate protein kinase, elevation of intracellular calcium, and activation of the phosphatidylinositol pathway. The release of archidonic acid and production of prostaglandins and/or leukotrienes have also been implicated in the action of STa. Evidence indicates that the STa enterotoxin receptor may be a single multifunctional membrane protein.

Amino Acid Sequence↗

PubMed2004

Colony immunoblot assay for the detection of hemolysin BL enterotoxin producing Bacillus cereus.

Bacillus cereus strains involved in food poisoning cases of the diarrheal type may produce two different enterotoxin complexes. To facilitate the identification of hemolysin BL-enterotoxin complex (HBL) and/or the nonhemolytic enterotoxin (NHE) producing colonies a colony immunoblot procedure was developed, which allows a fast and easy identification of the respective colonies from blood agar plates. The enterotoxins were transferred from the blood agar medium to a nitrocellulose membrane and the immobilized toxins were probed with monoclonal antibodies. The antibodies 2A3 and 1A8 allowed the specific detection of the B component of HBL and the nheA component of NHE. The assay enabled the reliable identification of HBL expressing colonies and differentiation from NHE producing but HBL negative colonies.

Antibodies, Monoclonal↗

PubMed2005

Pomegranate extract inhibits Staphylococcus aureus growth and subsequent enterotoxin production.

In Brazil, pomegranate (Punica granatum L. (Punicaceae)) is widely used as a phytotherapeutic agent. This study evaluates the effect of pomegranate extract on Staphylococcus aureus FRI 722 growth and subsequent enterotoxin production. Bacterial susceptibility was determined by tube dilution method and production of enterotoxin was assessed using membrane-over-agar (MOA) plates. At a low extract concentration (0.01% v/v) bacterial growth was delayed, while a higher concentration (1% v/v) eliminated bacterial growth. Most interestingly, a 0.05% (v/v) concentration of extract was found to inhibit Staphylococcal enterotoxin (SE) A production. These data further implicate pomegranate extracts as potential antibacterial therapeutics with the added ability to inhibit enterotoxin production.

Anti-Bacterial Agents↗

PubMed2004

Amendments to the theory underlying Ussing chamber data of chloride ion secretion after bacterial enterotoxin exposure.

Bacterial enterotoxins may cause life-threatening diarrhoeal fluid loss in part because they stimulate enterocytes to secrete fluid into the small intestine as well as preventing normal fluid uptake. Abnormal chloride ion secretion is believed to provide the osmotic driving force for the inappropriate fluid movement. Evidence for enhanced chloride secretion consists of isotopic flux measurements in Ussing chambers, the standard apparatus for permeation studies. Flux from the lumen of the intestine is assumed to be determined solely by absorptive processes and flux towards the lumen solely by secretory processes. Bacterial enterotoxin increased flux towards the lumen is taken as an evidence of enhanced secretion. Examination of the flux equation solutions shows that the existing theoretical treatment of the Ussing chamber consists of the super-imposition of two contradictory unidirectional models. In contrast, the present analysis shows that a measured 'unidirectional' flux contains information both about absorptive and secretory processes, regardless of which flux is measured. Reciprocity is predicted for the fluxes, as decreases in the absorptive processes will cause increases in apparent secretory flux. Data from the literature show that mucosal-to-serosal chloride ion flux in rabbit ileum after exposure to secretagogues correlates inversely and highly significantly (r=0.74, n=17, p<0.001) with increases in serosal-to-mucosal chloride ion flux. As a category of evidence, flux data do not provide conclusive evidence of enhanced chloride secretion after exposure to enterotoxins, since an apparently enhanced serosal-to-mucosal flux would also be noted after inhibition of the mucosal-to-serosal flux. As interruption of absorptive processes can be misinterpreted as enhanced secretion in the Ussing chamber, this is a serious deficiency in the evidence for direct enterotoxin enhancement of the intestinal chloride ion channel as a basis for diarrhoeal disease.

Animals↗

PubMed2005

Pattern of enterotoxin genes seg, seh, sei and sej positive Staphylococcus aureus isolated from bovine mastitis.

PCR detection of the genes encoding the newly described staphylococcal enterotoxins (SE) SEG, SEH, SEI and SEJ was carried out for 104 randomly selected Staphylococcus aureus field strains isolated from cases of bovine mastitis. Sixty-one (58.7%) isolates were positive for one or more of these novel enterotoxin genes. Thirty-six field strains were classified as carrier of seg, 22 of sei gene and 23 were positive for sej gene. None of the 104 investigated ruminant S. aureus strains carried the seh gene. Thirty-seven of these S. aureus strains showed a combination of genes encoding enterotoxin types SEA to SEE or toxic shock syndrome toxin 1 (TSST 1). Thirteen cultures harboured only one, 28 two, 12 three and 8 four enterotoxin genes. Among the 61 S. aureus field strains 14 (23.0%) were positive for the genes encoding SEJ and SED and 10 (16.4%) isolates for those encoding SEG and SEI. Isolates harbouring the sed/sej genes were further characterized by macrorestriction analysis and pulsed-field-gelelectrophoresis (Pfge). Macrorestriction analysis revealed six patterns. Nine of these14 S. aureus isolates (64.3%) exhibited two patterns with a high degree of relationship (>80%).

Animals↗

PubMed2003

An association of 27- and 40-kDa molecules with glycolipids that bind A-B bacterial enterotoxins to cultured cells.

It is well recognized that the Shiga-like toxins (Stxs) preferentially bind to Gb3 glycolipids and the cholera toxin (CT) and heat-labile enterotoxin (LTp) bind to GM1 gangliosides. After binding to the cell surface, A-B bacterial enterotoxins have to be internalized by endocytosis. The transport of the toxin-glycolipid complex has been documented in several manners but the actual mechanisms are yet to be clarified. We applied a heterobifunctional cross-linker, sulfosuccinimidyl-2-(p-azidosalicylamido)-1,3'-dithiopropionate (SASD), to detect the membrane proteins involved in the binding and the transport of A-B bacterial enterotoxins in cultured cells. Both Stx1 and Stx2 bound to the detergent-insoluble microdomain (DIM) of Vero cells and Caco-2 cells, which were susceptible to the toxin, but neither was bound to insusceptible CHO-K1 cells. Both CT and LTp bound to the DIM of Vero cells, Caco-2 cells, and CHO-K1 cells. In a cross-linking experiment, Stx1 cross-linked only with a 27-kDa molecule, while Stx2, which was more potently toxic than Stx1, cross-linked with 27- and 40-kDa molecules of Vero cells as well as of Caco-2 cells; moreover, no molecules were cross-linked with the insusceptible CHO-K1 cells. LTp was cross-linked only to the 27-kDa molecule of these three cell types but the CT, which was more toxic than LTp, was also cross-linked with 27- and 40-kDa molecules of Vero cells, Caco-2 cells, and CHO-K1 cells. The 27- and the 40-kDa molecules might play a role in the endocytosis and retrograde transport of A-B bacterial enterotoxins.

Animals↗

PubMed1997

Bromelain prevents secretion caused by Vibrio cholerae and Escherichia coli enterotoxins in rabbit ileum in vitro.

BACKGROUND & AIMS: Diarrhea is a major cause of illness and death in children and young animals. The aim of this study was to investigate the possible therapeutic effect of bromelain, a proteolytic extract obtained from pineapple stems on bacterial toxin and second-messenger agonist-induced intestinal secretion. METHODS: The effect of bromelain pretreatment on short-circuit responses to Escherichia coli heat-labile enterotoxin, heat-stable enterotoxin, and Vibrio cholerae cholera toxin was evaluated in rabbit ileum mounted in Ussing chambers. RESULTS: Bromelain was 62% effective in preventing heat-labile enterotoxin-induced secretion, 51% effective against cholera toxin, and 35% effective against heat-stable enterotoxin [corrected]. Bromelain also prevented secretory changes caused by prostaglandin E2, theophylline, calcium-ionophore A23187, 8-bromoadenosine 3':5'-cyclic monophosphate, and 8-bromoguanosine 3':5'-cyclic monophosphate, well-known intracellular mediators of ion secretion. The efficacy of bromelain was not caused by reduced tissue viability resulting from its proteolytic effects on enterocytes, indicated by experiments measuring uptakes of nutrients into intestinal cells and experiments measuring short-circuit responses to glucose. CONCLUSIONS: Bromelain prevents intestinal fluid secretion mediated by secretagogues that act via adenosine 3':5'-cyclic monophosphate, guanosine 3':5'-cyclic monophosphate, and calcium-dependent signaling cascades. It may be clinically useful as an antidiarrheal drug.

Animals↗