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PubMed1967

Gamma irradiation of staphylococcal enterotoxin B.

The inactivation of enterotoxin B by gamma irradiation was studied by use of single-and double-gel-diffusion assay techniques. Enterotoxin B (99+% purity) was suspended either in 0.04 m Veronal buffer (pH 7.2) or in milk, dispensed and heat-sealed in borosilicate glass vials, and irradiated essentially at 21 to 26 C with a cobalt-60 source. Parallel titrations of irradiated enterotoxin B in Veronal buffer were made by use of gel-diffusion and cat assay procedures to establish the relative sensitivity of these two assay procedures to irradiated enterotoxin. Results were identical. A dose of 5 Mrad was required to reduce an enterotoxin B concentration of 31 mug/ml in Veronal buffer to less than 0.7 mug/ml. When milk was used as a vehicle, a dose of 20 Mrad was needed to inactivate a 30 mug/ml concentration of enterotoxin B to less than 0.5 mug/ml. With Veronal buffer and milk as vehicles, the D values (dose required to inactivate 90%) for enterotoxin B inactivation were 2.7 and 9.7 Mrad, respectively.

Animals↗

PubMed1971

Effect of sodium chloride and pH on enterotoxin C production.

Growth and production of enterotoxin C by Staphylococcus aureus strain 137 in 3% + 3% protein hydrolysate powder N-Z Amine NAK broths with 0 to 12% NaCl and an initial pH of 4.00 to 9.83 were studied during an 8-day incubation period at 37 C. Growth was initiated at pH values as low as 4.00 and as high as 9.83 at 0% salt level as long as the inoculum contained at least 10(8) cells per ml. Rate of growth decreased as the NaCl concentration was increased gradually to 12%. Enterotoxin C was produced in broths inoculated with 10(8) cells per ml and above and having initial pH ranges of 4.00 to 9.83, 4.40 to 9.43, 4.50 to 8.55 and respective NaCl concentrations of 0, 4, and 8%. In the presence of 10% NaCl, the pH range supporting enterotoxin C production was 5.45 to 7.30 for an inoculum level of 10(8) cells per ml and 6.38 to 7.30 for 3.6 x 10(6) cells per ml. In repeated experiments in which the inoculum contained 10(8) cells per ml, we failed to demonstrate enterotoxin C production in broths with 12% NaCl and a pH range of 4.50 to 8.55 and concentrated up to 14 times. The effect of NaCl on enterotoxin C production followed the same pattern as its effect on enterotoxin B production. As the concentration of NaCl increased from 0 to 10%, yields of enterotoxin B and C decreased to undetectable amounts.

Culture Media↗

PubMed1975

Escherichia coli enterotoxin: stimulation of adenylate cyclase in broken-cell preparations.

The enterotoxin from cell-free filtrates of the enteropathogenic Escherichia coli strain P-263 was found to stimulate adenylate cyclase activity in broken-cell preparations from myocardial tissue. Particulate and detergent-solubilized fractions from cat heart were incubated with enterotoxin and assayed for adenylate cyclase activity. Adenylate cyclase activity was stimulated by enterotoxin; the extent of stimulation was proportional to the concentration of enterotoxin. The data demonstrate that stimulation of enterotoxin-sensitive adenylate cyclase in this system provides a sensitive in vitro assay, either as an accurate measure of enterotoxin concentration or as an assay for antitoxic titers in sera. A parallel comparison showed that stimulation of fluid production in rabbit intestinal loops by enterotoxin was less sensitive.

Adenylyl Cyclases↗

PubMed1977

Regulation of staphylococcal enterotoxin B.

The effect of glucose and the glucose analogues 2-deoxyglucose and alpha-methyl-glucoside on the synthesis and regulation of staphylococcal enterotoxin B was examined. The attenuating effect of glucose on staphylococcal enterotoxin B synthesis was observed. However, when this effect was examined with analogues of glucose, contradictory responses were seen. alpha-Methylglucoside had a slight stimulatory effect on enterotoxin production and other extracellular proteins, whereas 2-deoxyglucose markedly inhibited enterotoxin production. beta-hemolysin and staphylococcal nuclease were also inhibited by 2-deoxy glucose but the synthesis of nuclease could be rescued by the addition of glucose to 2-deoxyglucose-containing cultures. Enterotoxin and beta-hemolysin synthesis were not subject to glucose rescue. The cells used in this study were permeable to cyclic 3',5'-adenosine monophosphate, but the addition of this compound did not reverse glucose repression or 2-deoxyglucose inhibition of enterotoxin B synthesis. We conclude from these data that the regulation of enterotoxin is not under catabolite control as previously reported.

Cyclic AMP↗

PubMed1981

Staphylococcal enterotoxins fail to disrupt membrane integrity or synthetic functions of Henle 407 intestinal cells.

The potential cytotoxic activity of purified staphylococcal enterotoxins for mammalian cells was evaluated. The effects of staphylococcal enterotoxins A (SEA) and B (SEB) on cell membrane integrity as measured by leakage of labeled cytoplasmic constituents ([3H]uridine), amino acid transport (lysine and aminoisobutyric acid), and macromolecular synthesis (protein, ribonucleic acid, and deoxyribonucleic acid) was evaluated for a human intestinal epithelial cell (Henle 407). No evidence of cytotoxicity by any of these criteria could be detected for cell monolayers incubated with SEA for periods of between 30 min and 24 h. Purified staphylococcal hemolysins (alpha- and delta-toxins) were shown to exert cytotoxicity by the leakage and amino acid uptake assays. In efforts to detect synergistic effects between enterotoxin and the staphylococcal cytotoxins, membrane functions were evaluated after sequential or combined treatment with enterotoxin and alpha-toxin or with enterotoxin and delta-toxin. In no instance could a contribution to cytotoxicity by the staphylococcal enterotoxin be detected. That the assays were sufficiently sensitive to detect synergistic effects was shown by the greater than additive effects achieved with a combination of alpha- and delta-toxins. The data, contrary to previous reports, showed that staphylococcal enterotoxins did not behave as bacterial cytotoxins.

Animals↗

PubMed1985

Penicillinase plasmid-linked genetic determinants for enterotoxins B and C1 production in Staphylococcus aureus.

The genes encoding for beta-lactamase (bla+) and resistance to metallic ions (cadmium, mercury, lead, arsenate, and arsenite) were located in a 56.2-kilobase plasmid, pZA10, isolated from a clinical strain, Staphylococcus aureus 6344. This strain produced enterotoxin B and enterotoxin C1. Elimination of pZA10 by either sodium dodecyl sulfate or heat treatment (43 degrees C) resulted in the loss of the capability of the bacteria to produce both enterotoxin B and enterotoxin C1. A physical map of pZA10 was constructed with BamHI, SalI and BglII restriction endonucleases. Penicillin-resistant, enterotoxin B- and C1-producing cotransformants were isolated by transformation with pZA10 DNA with either S. aureus RN450 or cured S. aureus 6344 as recipients. The transferred plasmids exhibited genetic instability shown by changes in restriction pattern and molecular size, loss of plasmid DNA, and addition of chromosomal DNA. Enterotoxin B production was related to a 18.1-kilobase pZA10 fragment carried by such a rearranged plasmid. Chromosomal cointegration of bla+ with genetic determinants for metallic ion resistance and enterotoxin B and C1 production were detected in heat-treated S. aureus 6344. Transformation employing chromosomal DNA containing the integrated plasmid resulted in excision and reestablishment of pZA10-related plasmids in the transformants. pZA10-linked resistance to cadmium, which was lost upon the integration of pZA10 into the host chromosome, reappeared in transformants carrying the excised plasmid.

Bacterial Proteins↗

PubMed1988

Molecular analysis of enterotoxin plasmids of enterotoxigenic Escherichia coli of 14 different O serotypes.

A total of 104 isolates of enterotoxigenic Escherichia coli derived from diarrheal patients from more than 10 countries were examined for serotype and toxigenicity. The transferability and molecular structure of the enterotoxin plasmids from each isolate were also examined. Enterotoxin plasmids from serotypes such as O6, O25, O27, O126, O128, and O159, which are frequently associated with E. coli diarrhea (classical strains) generally did not transfer by conjugation from clinical isolates, whereas those from serotypes such as O7, O17, O80, O98, O139, O150, and O153, which are rarely associated with diarrhea (rare strains) transferred almost always from the clinical isolates by conjugation. Analyses of enterotoxin plasmids by restriction endonucleases and DNA-DNA hybridization with the enterotoxin probes revealed that the strains with the same O serotype and toxigenicity carry closely related enterotoxin plasmids. These results suggest that classical strains resulted from the dissemination of ancestral clones which received enterotoxin plasmids long ago, while the rare strains acquired the enterotoxin plasmids recently by conjugation and have not yet been spread to the same degree as the ancestral clones.

Cloning, Molecular↗

PubMed1998

Identification and characterization of staphylococcal enterotoxin types G and I from Staphylococcus aureus.

Staphylococcal enterotoxins are exotoxins produced by Staphylococcus aureus that possess emetic and superantigenic properties. Prior to this research there were six characterized enterotoxins, staphylococcal enterotoxin types A to E and H (referred to as SEA to SEE and SEH). Two new staphylococcal enterotoxin genes have been identified and designated seg and sei (staphylococcal enterotoxin types G and I, respectively). seg and sei consist of 777 and 729 nucleotides, respectively, encoding precursor proteins of 258 (SEG) and 242 (SEI) deduced amino acids. SEG and SEI have typical bacterial signal sequences that are cleaved to form toxins with 233 (SEG) and 218 (SEI, predicted) amino acids, corresponding to mature proteins of 27,043 Da (SEG) and 24,928 Da (SEI). Biological activities for SEG and SEI were determined with recombinant S. aureus strains. SEG and SEI elicited emetic responses in rhesus monkeys upon nasogastric administration and stimulated murine T-cell proliferation with the concomitant production of interleukin 2 (IL-2) and gamma interferon (IFN-gamma), as measured by cytokine enzyme-linked immunoassays. SEG and SEI are related to other enterotoxins of S. aureus and to streptococcal pyrogenic exotoxin A (SpeA) and streptococcal superantigen (SSA) of Streptococcus pyogenes. Phylogenetic analysis and comparisons of amino acid and nucleotide sequence identities were performed on related staphylococcal and streptococcal protein toxins to group SEG and SEI among the characterized toxins. SEG is most similar to SpeA, SEB, SEC, and SSA (38 to 42% amino acid identity), while SEI is most similar to SEA, SEE, and SED (26 to 28% amino acid identity). Polyclonal antiserum was generated against purified histidine-tagged SEG and SEI (HisSEG and HisSEI). Immunoblot analysis of the enterotoxins, toxic-shock syndrome toxin 1, and SpeA with antiserum prepared against HisSEG and HisSEI revealed that SEG shares some epitopes with SEC1 while SEI does not.

Amino Acid Sequence↗

PubMed2005

Increased sensitivity to staphylococcal enterotoxin B following adenoviral infection.

Staphylococcal enterotoxin B induces toxic shock and is a major virulence factor of staphylococcal diseases. We examined the effects of systemic adenoviral infection on responses to staphylococcal enterotoxin B in a murine model. We found that adenoviral infection markedly increases the severity of liver injury following exposure to staphylococcal enterotoxin B without d-galactosamine sensitization. In adenovirus-infected mice, staphylococcal enterotoxin B triggered a more profound hypothermia and increased apoptosis in the liver. Consistent with these observations, we also found that adenoviral infection primed for an increased production of gamma interferon in vivo and in vitro following stimulation with staphylococcal enterotoxin B. Gamma-interferon-knockout mice did not show increased sensitivity to staphylococcal enterotoxin B following adenoviral infection. These data suggest that a preexisting viral infection primes mice for subsequent staphylococcal enterotoxin B exposure, possibly via a gamma-interferon-mediated mechanism.

Adenoviridae Infections↗

PubMed1973

Comparison of the tissue receptors for Vibrio cholerae and Escherichia coli enterotoxins by means of gangliosides and natural cholera toxoid.

The in vitro binding properties of enterotoxins of Vibrio cholerae and Escherichia coli to different pure gangliosides and related neutral glycosphin-golipids were analyzed with a sorbent assay utilizing plastic tubes to which the glycolipid substances had been coupled. It was found that the cholera toxin bound to G(M1) ganglioside better than to the other tested substances G(M3), G(M3)-NGN, G(M2), G(D1a), G(D1b), G(T), G(A1), tetrahexoside-GlcNac and globoside. With this assay using G(M1)-coated tubes it is possible to measure cholera toxin even at concentrations below 1 ng/ml. Also enterotoxin of various E. coli strains bound to G(M1), but the affinity was much less than for cholera toxin. The G(M1) ganglioside, in contrast to the other glycosphingolipids, effectively inactivated cholera toxin as determined with the intradermal and the ileal loop assays; approximately equimolar concentrations of the ganglioside in relation to toxin sufficed. Also, the skin and ileal loop activities of E. coli enterotoxins could be inhibited by G(M1); however, several orders more of the ganglioside were required for such inhibition than for inactivation of the cholera toxin, and the differences between G(M1) and the other substances were less pronounced for E. coli toxins. Preincubation of rabbit ileal loops with choleragenoid, a natural toxoid of V. cholerae which has binding properties to the G(M1) ganglioside similar to cholera toxin, made the loops resistant to subsequently added enterotoxin of V. cholerae. The responsiveness to enterotoxin of E. coli was not reduced by this toxoid. A likely interpretation of these data is that the G(M1) ganglioside constitutes or at least contains the structure of functional tissue receptors for the cholera toxin, whereas the weak binding to G(M1) by E. coli enterotoxins is probably a pathogenetically insignificant reflection of structural similarities between these toxins and cholera toxin. Consequently, the cholera toxoid by occupying functional intestinal G(M1) receptors for the cholera toxin could inhibit the ileal response to this toxin, but not the response to E. coli enterotoxin since the intestinal receptors for the latter toxin are not affected by the cholera toxoid.

Animals↗

PubMed1974

Action of Escherichia coli enterotoxin: adenylate cyclase behavior of intestinal epithelial cells in culture.

Heat-labile enterotoxin preparations obtained from two enteropathogenic strains of Escherichia coli of porcine and human origin were shown to stimulate adenylate cyclase activity of human embryonic intestinal epithelial cells in culture. Comparable results were also obtained when cholera toxin was used. The degree of enzyme stimulation was proportional to the concentration of enterotoxin. Similar preparations from two strains of non-enterotoxigenic E. coli had no effect on adenylate cyclase activity. Cells exposed to enterotoxin could be washed after 1 min of contact time without altering the subsequent course of maximum adenylate cyclase activity, which was maintained for at least 18 h at 37 C. During long periods (18 h) of tissue culture incubation, the determination of adenylate cyclase activity was 200- to 300-fold more sensitive than quantitating fluid accumulation in the adult rabbit ileal loop model. Decreasing the incubation time appreciably reduced the sensitivity of the epithelial cells to enterotoxin. E. coli enterotoxin is an effective activator of nonintestinal adenylate cyclase systems. Treatment of KB and HEp-2 cell lines with enterotoxin also resulted in significant enzyme stimulation. The intestinal epithelial cell tissue culture model provides a sensitive homogenous biological system for studying the response of intestinal adenylate cyclase to enterotoxin while eliminating the numerous cellular and tissue components present in the ligated ileal loop model.

Adenosine Triphosphate↗

PubMed1980

Plasmids coding for heat-labile enterotoxin production isolated from Escherichia coli O78: comparison of properties.

Nineteen enterotoxigenic Escherichia coli strains of serogroup O78, isolated in different geographical areas from humans with diarrheal diseases, were tested for their ability to transfer enterotoxin production. All of the strains originally produced heat-labile enterotoxin, and 16 also produced heat-stable enterotoxin and colonization factor antigen I. Plasmids coding for the production of heat-labile enterotoxin only were transferred from 13 strains. Some properties of these plasmids were compared. All were fi+, but they could be divided into three groups on the basis of their incompatibility reactions, ability to restrict E. coli K-12 phages, and size. The three heat-labile enterotoxin plasmids isolated from African strains all belonged to one enterotoxin plasmid group. The heat-labile enterotoxin plasmids from the Asian strains were divided into two groups, those from serotype O78.H11 differing from those from serotype O78.H12.

Bacterial Toxins↗

PubMed1988

Nucleotide sequence of the type A staphylococcal enterotoxin gene.

We determined the nucleotide sequence of the gene encoding staphylococcal enterotoxin A (entA). The gene, composed of 771 base pairs, encodes an enterotoxin A precursor of 257 amino acid residues. A 24-residue N-terminal hydrophobic leader sequence is apparently processed, yielding the mature form of staphylococcal enterotoxin A (Mr, 27,100). Mature enterotoxin A has 82, 72, 74, and 34 amino acid residues in common with staphylococcal enterotoxins B and C1, type A streptococcal exotoxin, and toxic shock syndrome toxin 1, respectively. This level of homology was determined to be significant based on the results of computer analysis and biological considerations. DNA sequence homology between the entA gene and genes encoding other types of staphylococcal enterotoxins was examined by DNA-DNA hybridization analysis with probes derived from the entA gene. A 624-base-pair DNA probe that represented an internal fragment of the entA gene hybridized well to DNA isolated from EntE+ strains and some EntA+ strains. In contrast, a 17-base oligonucleotide probe that encoded a peptide conserved among staphylococcal enterotoxins A, B, and C1 hybridized well to DNA isolated from EntA+, EntB+, EntC1+, and EntD+ strains. These hybridization results indicate that considerable sequence divergence has occurred within this family of exotoxins.

Amino Acid Sequence↗

PubMed1989

Cloning, nucleotide sequence, and hybridization studies of the type IIb heat-labile enterotoxin gene of Escherichia coli.

Type IIb heat-labile enterotoxin (LT-IIb) is produced by Escherichia coli 41. Restriction fragments of total cell DNA from strain 41 were cloned into a cosmid vector, and one cosmid clone that encoded LT-IIb was identified. The genes for LT-IIb were subcloned into a variety of plasmids, expressed in minicells, sequenced, and compared with the structural genes for other members of the Vibrio cholerae-E. coli enterotoxin family. The A subunits of these toxins all have similar ADP-ribosyltransferase activity. The A genes of LT-IIa and LT-IIb exhibited 71% DNA sequence homology with each other and 55 to 57% homology with the A genes of cholera toxin (CT) and the type I enterotoxins of E. coli (LTh-I and LTp-I). The A subunits of the heat-labile enterotoxins also have limited homology with other ADP-ribosylating toxins, including pertussis toxin, diphtheria toxin, and Pseudomonas aeruginosa exotoxin A. The B subunits of LT-IIa and LT-IIb differ from each other and from type I enterotoxins in their carbohydrate-binding specificities. The B genes of LT-IIa and LT-IIb were 66% homologous, but neither had significant homology with the B genes of CT, LTh-I, and LTp-I. The A subunit genes for the type I and type II enterotoxins represent distinct branches of an evolutionary tree, and the divergence between the A subunit genes of LT-IIa and LT-IIb is greater than that between CT and LT-I. In contrast, it has not yet been possible to demonstrate an evolutionary relationship between the B subunits of type I and type II heat-labile enterotoxins. Hybridization studies with DNA from independently isolated LT-II producing strains of E. coli also suggested that additional variants of LT-II exist.

Amino Acid Sequence↗

PubMed1968

Pyrogenic responses to staphylococcal enterotoxins A and B in cats.

Pyrogenic responses, ranging up to 4.8 F, were induced in cats by oral administration of highly purified staphylococcal enterotoxin B in doses from 10 to 100 mug/kg. Fever was a more sensitive indicator of intoxication than was emesis. Highly purified preparations of enterotoxin A, whether administered intravenously (0.01 to 1.0 mug/kg), orally (10 to 25 mug/kg), or into the cerebral ventricles (0.005 to 0.020 mug in 0.20 ml), were also pyrogenic in cats. Tolerance to the pyrogenic activity was produced by repeated intravenous injection of a given dose of enterotoxin A but not by repeated intracerebroventricular injection. Enterotoxin A was more potent than enterotoxin B after intravenous injection in causing both fever and emesis. Cross-tolerance could not be demonstrated between enterotoxin A and enterotoxin B or Salmonella typhosa endotoxin. This lack of cross-tolerance plus the inability of large oral doses (100 to 4,700 mug/kg) of endotoxin to cause fever or emesis indicate that the reported responses were attributable to the specific toxins administered and not to contamination by other pyrogens.

Animals↗

PubMed1984

Rapid detection of Clostridium perfringens type A enterotoxin by enzyme-linked immunosorbent assay.

Clostridium perfringens type A enterotoxin was specifically detected and readily quantified by indirect and four-layer sandwich enzyme-linked immunosorbent assays (ELISAs). With the indirect ELISA, enterotoxin was detected in quantities of as low as 2.5 ng (25 ng/ml). When the more sensitive sandwich ELISA procedures was used, 100 pg (1 ng/ml) of enterotoxin was detected. The sandwich ELISA procedure specifically detected enterotoxin in human fecal extracts. Additionally, the sandwich ELISA specifically differentiated enterotoxin-positive strains from enterotoxin-negative strains of C. perfringens. Both the indirect and sandwich ELISA procedures described for C. perfringens enterotoxin in this report are rapid, specific, sensitive, and easily adaptable for large-scale use by clinical or research laboratories.

Antibodies, Bacterial↗

PubMed1987

Production of Escherichia coli STa-like heat-stable enterotoxin by Citrobacter freundii isolated from humans.

Citrobacter species are often present in the stools of children and are generally considered a normal component of the intestinal microflora. Previous reports suggested that they might act as enteric pathogens. Aiming at defining the role of Citrobacter species in inducing diarrhea, we looked for their presence in the stools of 328 children with diarrhea and in 108 controls. Citrobacter strains were isolated from 46 patients (14%) and 7 controls (6.5%) (P less than 0.05). All isolates were tested for heat-stable (ST) and heat-labile (LT) enterotoxin. No LT-producing organisms were found. Three C. freundii strains, all isolated from children with diarrhea, elaborated an enterotoxin detected by the suckling mouse assay. A crude extract was prepared by acetone precipitation and a sequential ultrafiltration technique. The enterotoxin was heat stable, and its estimated molecular weight was between 2,000 and 10,000. Citrobacter enterotoxin was soluble in methanol and stable at acid and neutral pHs but not above pH 8, and its activity was destroyed by treatment with 2-mercaptoethanol. Citrobacter enterotoxin was inactive in the 18-h rabbit ileal loop test. All these characteristics closely resemble STa produced by Escherichia coli. The time course of Citrobacter enterotoxin-induced intestinal secretion in suckling mice was similar to that of E. coli STa. The enterotoxin produced by C. freundii cross-reacted with monoclonal antibodies raised against E. coli STa. These results suggest that C. freundii is capable of inducing diarrhea through the production of an E. coli-like STa, and its presence in the stools of patients with diarrhea should be considered as that of a possible etiologic agent.

Bacterial Toxins↗

PubMed1977

Enterotoxin formation by Clostridium perfringens type A studied by the use of fluorescent antibody.

Fluorescein isothiocyanate-conjugated antibody to purified enterotoxin of Clostridium perfringens was used to study the intracellular formation of enterotoxin by this organism. Enterotoxin was detected at 4 h of growth at the end of the cell containing forespore. With the development of the spore, enterotoxin accumulation continued and involved the entire length of the cell until its lysis with the release of enterotoxin and mature spore. The spores did not contain demonstrable enterotoxin. Only a certain number of the sporulated cells of the enterotoxigenic strains studied produced this toxin. The amount of enterotoxin produced varied with sporulation percentage, and between strains and individual cells.

Clostridium perfringens↗