Histopathological effect ofClostridium perfringens 8-6 enterotoxin on rabbit intestine

Intestinal damage caused by an enterotoxin from a coatless spore mutant ofClostridium perfringens type A (8-6) was identified by both light and scanning electron microscopy. Under the light microscope, damage to the epithelial layer of the villus and to the lamina propria was evident. Whole tissue viewed under the scanning electron microscope confirmed the two distinct forms of damage seen by light microscopy and showed that the action of the enterotoxin on an individual villus appears to occur in a specific sequence. The gross tissue damage observed contrasts with that found in previous studies of the action ofClostridium perfringens enterotoxin on rabbit ileal tissue; this suggests that the 8-6 enterotoxin may have a different mode of action on the cell, which subsequently leads to death and lysis.     

Mechanism of action of a cytotonic enterotoxin produced by Aeromonas hydrophila

In this study, we describe the mechanism of action of a cytotonic enterotoxin produced by two isolates of Aeromonas hydrophila. Isolates SSU and Ah65 are of different origin and both are capable of producing either a cytotoxic enterotoxin or aerolysin. A cytotonic enterotoxin produced by diarrheal isolate SSU, which was purified and characterized in our laboratory, elevated intracellular cAMP and PgE2 levels in cultured Chinese hamster ovary (CHO) cells. Likewise, enterotoxic activity expressed by a cytotonic enterotoxin was detected in the culture filtrate of a fish isolate (Ah65) after cytotoxic activity was neutralized with homologous aerolysin monoclonal antibodies. This cytotonic enterotoxin also elevated intracellular cAMP and PgE2 levels in CHO cells, suggesting a cholera toxin-like mechanism of action for Aeromonas cytotonic enterotoxins.     

Biosynthesis and metabolism of prostaglandins in the small intestine of rats exposed to cholera enterotoxin

The effect of cholera enterotoxin on biosynthesis and metabolism of prostaglandins in the rat small intestine was studied. It was shown that in the course of action of cholera enterotoxin maximal synthesis and metabolism of prostaglandins (PG) was observed within the first 30 minutes after enterotoxin administration into the isolated intestinal loop. It was found that cholera enterotoxin induced, on the one hand, the shift in the correlation of different types of prostaglandins synthetized in vitro and, on the other, differentially activated PG synthesis and metabolism after pretreatment with the PG-synthetase inhibitor indomethacin.     

In vitro stimulation of steroidogenesis in rat testis by cholera enterotoxin.

Cholera enterotoxin increases C-AMP production, and stimulates testosterone secretion in the rat testis $\text{in vitro}$. This gonadotropic action of cholera enterotoxin is potentiated by theophylline. It is suggested that cholera enterotoxin acts on Leydig cells directly to activate their adenyl cyclase, and consequently stimulates steroidogenesis in the rat testis. However, the receptor of cholera enterotoxin seems to be located at a different site from that of human chorionic gonadotropin.     

Protective effects of osmotic stabilizers on morphological and permeability alterations induced in Vero cells by Clostridium perfringens enterotoxin

Culture medium made hypertonic by the addition of osmotic stabilizers such as sucrose, poly(ethylene glycol), dextran and bovine serum albumin protected against changes in morphology and plasma membrane permeability induced by Clostridium perfringes enterotoxin. The protection did not appear to be due to binding inhibition. Results of these studies support an osmotic disruption mechanism for the action of the enterotoxin. A comprehensive model of the enterotoxin's action based on an osmotic disruption mechanism is proposed.     

Protective effects of osmotic stabilizers on morphological and permeability alterations induced in vero cells by Clostridium perfringens enterotoxin

Culture medium made hypertonic by the addition of osmotic stabilizers such as sucrose, poly(ethylene glycol), dextran and bovine serum albumin protected against changes in morphology and plasma membrane permeability induced by Clostridium perfringes enterotoxin. The protection did not appear to be due to binding inhibition. Results of these studies support an osmotic disruption mechanism for the action of the enterotoxin. A comprehensive model of the enterotoxin's action based on an osmotic disruption mechanism is proposed.     

Characterization of membrane permeability alterations induced in Vero cells by Clostridium perfringens enterotoxin

Alterations in plasma membrane permeability induced by Clostridium perfringens enterotoxin were studied using Vero (African green monkey kidney) cells which were radioactively labeled with four markers of different molecular size. The markers were alpha-amino[14C]isobutyric acid (Mr 103), 3H-labeled nucleotide (Mr approx. 300), 51Cr label (Mr approx. 3000) and [3H]RNA (Mr>25000). Over a 2h period, enterotoxin caused significant release of aminoisobutyric acid, nucleotides and 51Cr label but not RNA. The effects of enterotoxin on label release were dose- and time-dependent. The rate of release of markers was dependent upon their size. Permeability alterations could be detected within 15 min with a high dose of enterotoxin. Gel chromatography of released material was used to determine that markers of Mr 3000 but not 25000 leaked from permeabilized cells. It was concluded that enterotoxin is producing functional 'holes' of limited size in the membrane. Permeability changes due to enterotoxin treatment differed between confluent and nonconfluent (growing) cells. We propose that the primary action of the enterotoxin is to interact with the plasma membrane and produce functional 'holes' of defined size. The resultant alterations in membrane permeability cause the loss of essential cellular substances which inhibits processes such as macromolecular synthesis and eventually leads to cell deterioration and death.     

Calcium calmodulin in altered NaCl transport by heat-labile enterotoxin of Escherichia coli

The mucosal-to-serosal and serosal-to-mucosal fluxes of Na+ and Cl- were carried out in control and experimental groups treated with different doses of heat-labile enterotoxin in the presence or absence of Ca2+-ionophore, Ca2+ channel blocker and calmodulin inhibitor. There was net secretion of Na+ and Cl- in 16 and 32 units of heat-labile enterotoxin treated groups in comparison to net absorption in control group, however, in animals treated with 8 units of heat-labile enterotoxin, no change in Na+ and Cl- fluxes was found when compared to control. Ca2+- ionophore increased net secretion of Na+ and Cl- in 16 and 32 units of heat-labile enterotoxin treated groups and also caused secretion in control group instead of net absorption. Ca2+ channel blocker and calmodulin inhibitor partially reversed the effect of heat-labile enterotoxin. The effect of Ca2+-ionophore was more pronounced in the control group while that of Ca2+ channel blocker and calmodulin inhibitor was more pronounced in 16 and 32 units of heat-labile enterotoxin treated groups. The findings suggest the involvement of Ca2+ and calmodulin in the action of heat-labile enterotoxin of Escherichia coli in mice.     

Characteristics of the heat-stable enterotoxin of NAG vibrios

Cell-free culture filtrate boiled for 15 minutes has been found to retain its biological activity in various experimental models used for the determination of the toxicogenicity of cholera vibrio filtrates. During gel filtration of the concentrated filrate o. NAG vibrio, strain NO. 9852, through Sephadex G-75 toxic activity could be detected in the free volume of the column, which was indicative of the fact that the molecular weight of the thermostable enterotoxin was about 70,000 daltons and greater. The methods of gel diffusion and aggregated hemagglutination have been used to show that the thermostable enterotoxin of NAG vibrio No. 9852 is immunologically unrelated to cholerogen. Some data obtained in experimental models suggest that the thermostable enterotoxin probably differs from cholera enterotoxin in the mechanism of its action.     

Bacillus cereus enterotoxins

Data about Bacillus cereus different enterotoxins including hemolysin (HBL), nonhemolytic enterotoxin (NHE), enterotoxin (T), and emesis-inducing thermostable enterotoxin (ETE) are summarized in the article. Information about synthesis of different diarrhea-inducing and emesis-inducing enterotoxins, methods of their purification, structure, functions, and mechanisms of action are presented. Commercial kits for identification of B. cereus enterotoxins causing food-associated diarrhea are listed.     

Toxin gene profiling of enterotoxic and emetic Bacillus cereus

Very different toxins are responsible for the two types of gastrointestinal diseases caused by Bacillus cereus: the diarrhoeal syndrome is linked to nonhemolytic enterotoxin NHE, hemolytic enterotoxin HBL, and cytotoxin K, whereas emesis is caused by the action of the depsipeptide toxin cereulide. The recently identified cereulide synthetase genes permitted development of a molecular assay that targets all toxins known to be involved in food poisoning in a single reaction, using only four different sets of primers. The enterotoxin genes of 49 strains, belonging to different phylogenetic branches of the B. cereus group, were partially sequenced to encompass the molecular diversity of these genes. The sequence alignments illustrated the high molecular polymorphism of B. cereus enterotoxin genes, which is necessary to consider when establishing PCR systems. Primers directed towards the enterotoxin complex genes were located in different CDSs of the corresponding operons to target two toxin genes with one single set of primers. The specificity of the assay was assessed using a panel of B. cereus strains with known toxin profiles and was successfully applied to characterize strains from food and clinical diagnostic labs as well as for the toxin gene profiling of B. cereus isolated from silo tank populations.     

Expression of a Ricin Toxin B Subunit: Insulin Fusion Protein in Edible Plant Tissues

Onset of juvenile Type 1 diabetes (T1D) occurs when autoreactive lymphocytes progressively destroy the insulin-producing beta-cells in the pancreatic Islets of Langerhans. The increasing lack of insulin and subsequent onset of hyperglycemia results in increased damage to nerves, blood vessels, and tissues leading to the development of a host of severe disease symptoms resulting in premature morbidity and mortality. To enhance restoration of normoglycemia and immunological homeostasis generated by lymphocytes that mediate the suppression of autoimmunity, the non-toxic B chain of the plant AB enterotoxin ricin (RTB), a castor bean lectin binding a variety of epidermal cell receptors, was genetically linked to the coding region of the proinsulin gene (INS) and expressed as a fusion protein (INS–RTB) in transformed potato plants. This study is the first documented example of a plant enterotoxin B subunit linked to an autoantigen and expressed in transgenic plants for enhanced immunological suppression of T1D autoimmunity.     

Interference by Trypsin in the Interaction of Staphylococcal Enterotoxin B and Cell Cultures of Human Embryonic Intestine

The inhibitory effect of trypsin on the cytotoxicity of staphylococcal enterotoxin B acting with human embryonic intestine cell cultures was examined. Trypsin treatment of the cells rendered them resistant to enterotoxin for a period of 48 hr. The resistance increased proportionally with increased time of exposure of the cells to trypsin. Neither ethylenediaminetetraacetic acid nor scraping, which were used as alternate means of cell suspension, caused any resistance to the toxin. The effect is enzymatic and appears to be similar to the inhibitory action of trypsin and chymotrypsin on the attachment of polioviruses and coxsackieviruses to HeLa cells.     

Mode of action of heat-stable Escherichia coli enterotoxin. Tissue and subcellular specificities and role of cyclic GMP

Some enteric strains of Escherichia coli release a heat-stable enterotoxin which, in contrast to cholera and heat-labile E. coli enterotoxins, stimulates guanylate cyclase (GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2). We have examined the tissue spcificity of its action and the relation of its action to those of the 8-bromo analogues of cyclic GMP and cyclic AMP. Heat-stable enterotoxin stimulated guanylate cyclase activity and increased cyclic GMP concentration throughout the small and large intestine. It increased transepithelial electric potential difference and short-circuit current in the jejunum, ileum and caecum but not in the duodenum or distal colon. This pattern of electrical responses was mimicked by 8-bromo-cyclic GMP. However, 8-bromo-cyclic AMP produced an electrical response in all intestinal segments. The enterotoxin failed to stimulate guanylate cyclase inliver, lung, pancreas or gastric antral mucosa. In the intestines, it stimulated only the particulate and not the soluble form of the enzyme. Preincubation of the toxin with intestinal membranes did not render it capable of stimulating pancreatic guanylate cyclase. Cytosol factors did not enhance the toxin's stimulation of intestinal guanylate cyclase. This study supports the role of cyclic GMP as intracellular mediator for heat-stable enterotoxin and suggests that the toxin affects a membrane-mediated mechanism for guanylate cyclase activation that is unique to the intestines.     

Effects of epinephrine, clonidine, L-phenylephrine, and morphine on intestinal secretion mediated by Escherichia coli heat-stable enterotoxin in pig jejunum

Perfusion of pig jejunum with Escherichia coli heat-stable enterotoxin (strain 1261) reversed net absorption of water and electrolytes to net secretion. Addition of the alpha-adrenergic agonists clonidine (5 X 10(-7) M) or L-phenylephrine (5 X 10(-6) M), or the opiate agonist morphine (3.6 X 10(-6) M) to the perfusate reduced the secretory response to enterotoxin and stimulated absorption in normal jejunum. Epinephrine (5 X 10(-5) M) did not stimulate absorption in controls but reduced chloride loss in the presence of enterotoxin. Mucosal sodium--potassium adenosine triphosphatase was unchanged but disaccharidase activity was decreased in the presence of enterotoxin. The results suggest that alpha-adrenergic agonists and opiate agonists may exert an antidiarrheal action by increasing net transport across intestinal epithelium.     

Enterotoxin of Clostridium perfringens type A forms ion-permeable channels in a lipid bilayer membrane

The enterotoxin of Clostridium perfringens type A was found to form ion-permeable channels in a lipid bilayer. A patch clamp technique was used to detect channel activities in an asolectin bilayer with incorporated enterotoxin. About 20% of the lipid bilayer patches examined showed rectangular or stepwise shift of membrane current. The shifts indicated the gating of ion-permeable channels in the patches. The channels showed high conductance (40-450 pS), no rectification in current-voltage curves and occasional long-lasting events. The significance of these findings is discussed in relation to the mechanism of action of the toxin.     

Mode of action of heat-stable Escherichia coli enterotoxin Tissue and subcellular specificities and role of cyclic GMP

Some enteric strains of Escherichia coli release a heat-stable enterotoxin which, in contrast to cholera and heat-labile E. coli enterotoxins, stimulates guanylate cyclase (GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2). We have exmined the tissue specificity of its action and the relation of its action to those of the 8-bromo analogues of cyclic GMP and cyclic AMP. Heat-stable enterotoxin stimulated guanylate cyclase activity and increased cyclic GMP oncentration throughout the small and large intestine. It increased transepithelial electric potential difference and short-circuit current in the jejunum, ileum and caecum but not in the duodenum or distal colon. This pattern of electrical responses was mimicked by 8-bromo-cyclic GMP. However, 8-bromo-cyclic AMP produced an electrical response in all intestinal segments. The enterotoxin failed to stimulate guanylate cyclase in liver, lung, pancreas or gastric antral mucosa. In the intestines, it stimulated only the particulate and not the soluble form of the enzyme. Preincubation of the toxin with intestinal membranes did not render it capable of stimulating pancreatic guanylate cyclase. Cytosol factors did not enhance the toxin's stimulation of intestinal guanylate cyclase. This study supports the role of cyclic GMP as intracellular mediator for heat-stable enterotoxin and suggests that the toxin affects a membrane-mediated mechanism for guanylate cyclase activation that is unique to the intestines.     

Purification and partial characterization of a cytotonic enterotoxin produced by Aeromonas hydrophila

This report describes the purification and partial characterization of a cytotonic enterotoxin produced by a human diarrheal isolate (SSU) of Aeromonas hydrophila. The extracellular enterotoxin was purified by (NH4)2SO4 precipitation, hydrophobic column chromatography, and chromatofocusing. The highly purified enterotoxin exhibited a molecular mass of 44 kDa and an isoelectric point in the range of 4.3 - 5.5 as determined by chromatofocusing. Western blot analysis using Aeromonas anti-enterotoxin revealed a single band at 44 kDa; however, cholera antitoxin failed to detect the enterotoxin antigen. This non-cholera toxin cross-reactive (non-CTC) enterotoxin was biologically active in vivo as determined by rabbit ligated ileal loop and rabbit skin vascular permeability assays. Biological activity also was in vitro by this toxin as measured by the elongation of Chinese hamster ovary (CHO) cells. The enterotoxic activity associated with this molecule was neutralized completely by homologous antibodies but not by cholera antitoxin. The purified toxin preparation was free of hemolytic and cytotoxic activities as determined by its inability to lyse rabbit red blood cells or damage CHO cells, respectively. Furthermore, this toxin induced the elevation of cAMP in CHO cells suggesting thereby that the mechanism of action of Aeromonas non-CTC enterotoxin may be similar to heat-labile enterotoxins of Escherichia coli and Vibrio cholerae.     

High-level expression and secretion of a lysine-containing analog of Escherichia coli heat-stable enterotoxin.

The mechanism of action of the heat-stable enterotoxin STa secreted from enterotoxigenic forms of Escherichia coli has remained elusive, in part due to a tedious, low-yield purification procedure. We report here a method for obtaining large amounts of a biologically active lysine-containing analog of STa. Initial attempts to express the toxin using an expression vector that did not encode a signal sequence resulted in no biologically active material being recovered either from lysed cells or as a secretory product. However, use of the secretion vector pJAL36, which contains the STII enterotoxin signal sequence, allowed large amounts of an STa derivative containing the additional sequence Ser-Thr-Lys at the amino terminus of the mature enterotoxin to be readily purified from culture supernatants. This enterotoxin analog, known as KSTa-1, was equal in biological and receptor binding activity to the native toxin STa. The lysine residue present in KSTa-1 promises to be useful as a reactive amino acid that is readily derivatized to allow coupling of the enterotoxin to supports for affinity chromatography and antigenic conjugates. Additionally, the insertion of the lysine residue carboxy terminal to the Ser-Thr sequence adds a reversible "handle" to the toxin sequence in that the Ser-Thr-Lys segment can be removed by treatment with trypsin, releasing the native form of STa.     

High-level expression and secretion of a lysine-containing analog of Escherichia coli heat-stable enterotoxin'

The mechanism of action of the heat-stable enterotoxin STa secreted from enterotoxigenic forms of Escherichia coli has remained elusive, in part due to a tedious, low-yield purification procedure. We report here a method for obtaining large amounts of a biologically active lysine-containing analog of STa. Initial attempts to express the toxin using an expression vector that did not encode a signal sequence resulted in no biologically active material being recovered either from lysed cells or as a secretory product. However, use of the secretion vector pJAL36, which contains the STII enterotoxin signal sequence, allowed large amounts of an STa derivative containing the additional sequence Ser-Thr-Lys at the amino terminus of the mature enterotoxin to be readily purified from culture supernatants. This enterotoxin analog, known as KSTa-1, was equal in biological and receptor binding activity to the native toxin STa. The lysine residue present in KSTa-1 promises to be useful as a reactive amino acid that is readily derivatized to allow coupling of the enterotoxin to supports for affinity chromatography and antigenic conjugates. Additionally, the insertion of the lysine residue carboxy terminal to the Ser-Thr sequence adds a reversible “handle” to the toxin sequence in that the Ser-Thr-Lys segment can be removed by treatment with trypsin, releasing the native form of STa.