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PubMed2003

Piezoelectric crystal immunosensor for the detection of staphylococcal enterotoxin B.

The work of fabricating a piezoelectric (PZ) immunosensor for the detection of staphylococcal enterotoxin B (SEB) is presented in this paper. Three different immobilization methods using anti-SEB antibody onto a gold electrode of the PZ have been conducted. The electrode coated with polyethyleneimine (PEI) has shown the best result. The fabricated PZ sensor can be used for SEB determination in the range of 2.5-60 microg/ml with a correlation coefficient of 0.997. Milk samples spiked with various concentrations of SEB gave an average of 111% recovery of the toxin. The SEB assay is specific. For example the presence of staphylococcal enterotoxin A (SEA) at 40 microg/ml gave 6.44% of the signal while staphylococcal enterotoxin D (SED) appeared to give no detectable signal. After regeneration with 1.2 M NaOH, the coated crystal could be reused three times with retention of 66% of the initial signal. The crystal has also been found to be stable for 3 days when stored at 4 degrees C in a dry atmosphere without appreciable loss of activity.

Animals↗

PubMed2000

Prevalence and expression of enterotoxins in Bacillus cereus and other Bacillus spp., a literature review.

Members of the Bacillus genus are ubiquitous soil microorganisms and are generally considered harmless contaminants. However, a few species are known toxin producers, including the foodborne pathogen, B. cereus. This species produces two distinct types of foodborne illness, the emetic (vomit-inducing) syndrome, associated with consumption of toxin in cooked rice dishes, and the diarrheal illness seen occasionally following consumption of contaminated meats, sauces, and certain dairy products. In the latter case, illness results from the production of enterotoxins by vegetative cells in the small intestine of the host. In dairy products, the occurrence of Bacillus spp. is inevitable, and the spore-forming ability of this organism allows it to easily survive pasteurization. Many strains have been shown to grow and produce enterotoxin in dairy products at refrigeration temperatures. Evaluation of toxin gene presence and toxin expression in Bacillus spp. other than B. cereus has not been thoroughly investigated. However, the presence of natural isolates of Bacillus spp. harboring one or more enterotoxin gene(s) and subsequent demonstration of conditions which may support toxin expression holds crucial importance in the food safety arena.

Bacillus↗

PubMed2003

Staphylococcal enterotoxin induced IL-5 stimulation as a cofactor in the pathogenesis of atopic disease: the hygiene hypothesis in reverse?

BACKGROUND: The incidence of Staphylococcus aureus (S. aureus) colonization on the skin of patients with atopic eczema/dermatitis syndrome (AEDS) is approximately 90% and a variety of evidence implicates epidermal staphylococcal infection as a pathogenic factor in atopic dermatitis. However, the mechanism(s) underlying the effects of this organism in the disease process are unclear. The cellular responses of AEDS suffers and asymptomatic atopic individuals to bacterial superantigens (SAg) were investigated in an attempt to elucidate the role of staphylococcal enterotoxin B (SEB) in atopic disease. METHODS: Peripheral blood mononuclear cells (PBMC) were isolated from normal nonatopic adults, asymptomatic atopic individuals, patients with active AEDS and patients with active allergic asthma. The cells were cultured for 24 or 96 h with house dust mite (HDM), SEB and phytohaemagluttinin (PHA), and the supernatants were assayed for cytokine levels. RESULTS: Staphylococcal enterotoxin B selectively stimulates the production of interleukin (IL)-5 in AEDS sufferers but not in asymptomatic atopics or nonatopics. Additionally, we observed comparable susceptibility to the IL-5-stimulatory effects of SEB in allergic asthmatics. CONCLUSIONS: Given the central role of IL-5-driven eosinophilia in progression from mild atopy to severe disease, these findings provide a plausible mechanism for the AEDS-promoting effects of staphylococcal SAg. Staphylococcal enterotoxin B may also have a similar role in atopic respiratory disease.

Adolescent↗

PubMedDate not supplied

[Bacterial enterotoxin receptors].

Bacterial enterotoxin receptors. Enteric bacterial toxins display a great diversity in their structure, molecular weight and mechanism of action. The interaction of enterotoxins with the intestinal mucosa either leads to a direct effect on the cell membrane or an effect on signal transduction within eukaryotic cells. However, before a toxin can affect a cell, it must after its secretion by a microorganism, recognise and bind to a specific surface molecule, its receptor. Membrane receptors of bacterial enterotoxins have been identified as protein, glycoprotein or glycolipid in nature. The chemical nature of the molecules acting as receptors is crucial and during evolution they have been carefully selected. Some toxins, after their interaction with a receptor molecule, will transduce a signal across the cell membrane while remaining at the cell surface. Other toxins, after this initial binding step with a receptor, will be internalised. Others can form pores leading to leakage of cellular components and cell lysis. Receptors that have been identified often comprise a saccharidic chain that is directly involved in the recognition and binding of the toxin. Today, models explaining toxin-receptor interactions are more complex, including multistep events. This review summarises the knowledge of the interactions between bacterial toxins and membrane receptors present on intestinal mucosa.

Animals↗

PubMed1995

Staphylococcal enterotoxins bind H-2Db molecules on macrophages.

We screened a panel of monoclonal antibodies against selected macrophage cell surface molecules for their ability to inhibit enterotoxin binding to major histocompatibility complex class II-negative C2D (H-2b) macrophages. Two monoclonal antibodies, HB36 and TIB126, that are specific for the alpha 2 domain of major histocompatibility complex class I, blocked staphylococcal enterotoxins A and B (SEA and SEB, respectively) binding to C2D macrophages in a specific and concentration-dependent manner. Inhibitory activities were haplotype-specific in that SEA and SEB binding to H-2k or H-2d macrophages was not inhibited by either monoclonal antibody. HB36, but not TIB126, inhibited enterotoxin-induced secretion of cytokines by H-2b macrophages. Lastly, passive protection of D-galactosamine-sensitized C2D mice by injection with HB36 antibody prevented SEB-induced death. Therefore, SEA and SEB binding to the alpha 2 domain of the H-2Db molecule induces biological activity and has physiological consequences.

Amino Acid Sequence↗

PubMed1995

The Staphylococcus aureus enterotoxin B superantigen induces specific T cell receptor down-regulation by increasing its internalization.

Superantigens are able to stimulate T lymphocyte populations expressing T cell antigen receptors (TCR) belonging to particular V beta families. Moreover, the presence of these superantigens may induce long term unresponsiveness (anergy) of these sensitive cells. Some bacterial toxins are potent superantigens. We have analyzed in vitro the capacity of some Staphylococcus aureus enterotoxin superantigens to modulate T cell antigen receptor expression and the cellular mechanisms involved. Staphylococcus enterotoxin B (SEB) induced rapid down-regulation of surface T cell antigen receptors in V beta 3-expressing T lymphocytes, as assessed by flow cytometry. This phenomenon was a consequence of the direct interaction between the toxin and the TCR since it was observed in the absence of cells expressing major histocompatibility complex class II molecules. The cellular mechanism involved in SEB-induced down-regulation of TCR was further investigated. Immunofluorescence and confocal microscopy experiments showed that toxin B induced intracellular accumulation of TCR.CD3 in endocytic vesicles. Moreover, SEB induced an increase in T cell receptor endocytosis as measured using radiolabeled Fab fragments of an anti-CD3 monoclonal antibody. Taken together, our observations indicate that Staphylococcus enterotoxin B superantigen induced changes in the dynamics of surface T cell receptors, which resulted in the fast reduction of membrane receptor numbers.

CD3 Complex↗

PubMed1995

Generation of a monoclonal antibody that recognizes the amino-terminal decapeptide of the B-subunit of Escherichia coli heat-labile enterotoxin. A new probe for studying toxin assembly intermediates.

Cholera toxin and the related Escherichia coli heat-labile enterotoxin are hexameric proteins comprising one A-subunit and five B-subunits. In this paper we report the generation and characterization of a monoclonal antibody, designated LDS47, that recognizes and precipitates in vivo assembly intermediates of the B-subunit (EtxB) of E. coli heat-labile enterotoxin. The monoclonal antibody is unable to precipitate native B-subunit pentamers, thus making LDS47 a useful probe for studying the early stages of enterotoxin biogenesis. The use of LDS47 to monitor the in vivo turnover of newly synthesized B-subunits in the periplasm of E. coli demonstrated that (i) the turnover of unassembled B-subunits followed an apparent first order process and (ii) it occurred concomitantly with the assembly of native B-pentamers (k = 0.317 +/- 0.170 min-1; t1/2 = 2.2 min). No other proteins were co-precipitated with the newly synthesized B-subunits; a finding that implies that unassembled B-subunits do not stably associate with other periplasmic proteins prior to their assembly into a macromolecular complex. The use of overlapping synthetic peptides corresponding to the entire EtxB polypeptide demonstrated that the epitope recognized by LDS47 is located within the amino-terminal decapeptide of the B-subunit. From the x-ray structural analysis of the toxin (Sixma, T., Kalk, K., van Zanten, B., Dauter, Z., Kingma, J., Witholt, B., and Hol, W. G. J. (1993) J. Mol. Biol. 230, 890-918), this region appears to resemble a curved finger that clasps the adjacent B-subunit. Thus, this region might be expected to be exposed in the unfolded or unassembled subunit, but to become partially buried upon assembly and thus inaccessible to recognition by the monoclonal antibody.

Amino Acid Sequence↗

PubMed1996

Assembly of the B subunit pentamer of Escherichia coli heat-labile enterotoxin. Kinetics and molecular basis of rate-limiting steps in vitro.

The B subunits of Escherichia coli heat-labile enterotoxin (EtxB) and cholera toxin (CtxB) assemble in vivo into exceptionally stable homopentameric complexes, which maintain their quaternary structure in a range of conditions that would normally be expected to cause protein denaturation. Recently, we showed that the simultaneous protonation of two of the COOH-terminal carboxylates in pentameric EtxB was required to cause its disassembly at pH values below 2.0 (Ruddock, L., Ruston, S. P., Kelly, S. M., Price, N. C., Freedman, R. B., and Hirst, T. R.(1995) J. Biol. Chem. 270, 29953-29958). Here, we investigate the influence of environmental parameters on the kinetics of reassembly of acid-generated EtxB monomers in vitro. Such monomers were found to undergo a further acid-mediated conformational change, with an activation energy of 76 +/- 2 J.mol-1.K-1, consistent with isomerization of the cis-proline residue at position 93, and which prevented subsequent EtxB reassembly. By using rapid neutralization of acid-generated monomers, a high proportion of the B-subunits adopted an assembly-competent conformation, which resulted in up to 75% of the protein reassembling into a stable pentameric complex, indistinguishable from native EtxB pentamers. The rate-limiting step in reassembly, over a concentration range of 50-200 microg/ml, was shown to be due to an intramolecular event, which exhibited a pH dependence with a pKa of 7.0. Modification of EtxB with amine-specific probes revealed that the protonation state of the NH2-terminal alanine residue was responsible for the pH dependence of reassembly. The implications of these findings for the biogenesis of Escherichia coli enterotoxin and related enterotoxins in vivo, are considered.

Administration, Oral↗

PubMed2002

Zinc-mediated dimerization and its effect on activity and conformation of staphylococcal enterotoxin type C.

Staphylococcal enterotoxins are superantigen exotoxins that mediate food poisoning and toxic shock syndrome in humans. Despite their structural and functional similarities, superantigens display subtle differences in biological properties and modes of receptor binding as a result of zinc atoms bound differently in their crystal structures. For example, the crystal structures of the staphylococcal enterotoxins in the type C serogroup (SECs) contain a zinc atom coordinated by one aspartate and two histidine residues from one molecule and another aspartate residue from the next molecule, thus forming a dimer. This type of zinc ligation and zinc-mediated dimerization occurs in several SECs, but not in most other staphylococcal enterotoxin serogroups. This prompted us to investigate the potential importance of zinc in SEC-mediated pathogenesis. Site-directed mutagenesis was used to replace SEC zinc binding ligands with alanine. SEC mutants unable to bind zinc did not have major conformational alterations although they failed to form dimers. Zinc binding was not essential for T cell stimulation, emesis, or lethality although in general the mutants were less pyrogenic. Thus the zinc atom in SECs might represent a non-functional heavy atom in an exotoxin group that has diverged from related bacterial toxins containing crucial zinc atoms.

Animals↗

PubMed2004

Suppression of Staphylococcal Enterotoxin B-induced Toxicity by a Nuclear Import Inhibitor.

Staphylococcal enterotoxin B and related toxins that target T cells have the capacity to elicit systemic inflammation, tissue injury, and death. Genes that encode mediators of inflammation can be globally inhibited by blocking the nuclear import of stress-responsive transcription factors. Here we show that cell-permeant peptides targeting Rch1/importin alpha/karyopherin alpha 2, a nuclear import adaptor protein, are delivered to T cells where they inhibit the staphylococcal enterotoxin B-induced production of inflammatory cytokines ex vivo in cultured primary spleen cells and in vivo. The systemic production of tumor necrosis factor alpha, interferon gamma, and interleukin-6 was attenuated in mice either by a cell-permeant cyclized form of SN50 peptide or by a transgene whose product suppresses the nuclear import of transcription factor nuclear factor kappa B in T cells. The extent of liver apoptosis and hemorrhagic necrosis was also reduced, which correlated with significantly decreased mortality rates. These findings highlight nuclear import inhibitors as a potentially useful countermeasure for staphylococcal enterotoxin B and other toxins that trigger harmful systemic inflammatory responses.

Active Transport, Cell Nucleus↗

PubMed2001

The effect of bacterial enterotoxins implicated in SIDS on the rabbit intestine.

The aim of this project was to characterise the type of damage caused to the intestine of the infant rabbit by bacterial enterotoxins implicated in sudden infant death syndrome (SIDS). Samples of the duodenum, jejunum, ileum, caecum and large intestine exposed to the toxins for up to 6 hours were examined by scanning (SEM) and transmission electron microscopy (TEM). The damage was quantitatively assessed (% villi damaged) by SEM and qualitatively by SEM and TEM. Clostridium perfringens enterotoxin, staphylococcal enterotoxin B and Clostridium difficile toxin A + toxin B combined all caused severe damage to the villi in the small intestine (80-90% damage). Clostridium difficile toxin B caused only slight damage (17% to the jejunum, 26% to the caecum). Clostridium perfringens alpha-toxin caused moderate damage to the small intestine (duodenum 34%, caecum 35%), and Escherichia coli STa caused significant damage to the small (53-70%) and large intestine (51%). The level of toxin damage increased with time, the small intestine being more susceptible generally to damage than the large intestine. Each toxin differed in its ability to damage the villi, microvilli, enterocytes and lamina propria.

Animals↗

PubMed1983

Evaluation of different cell cultures for the detection of heat-labile enterotoxins of Escherichia coli strains isolated from pigs.

The sensitivity of various cell cultures to heat-labile enterotoxins (LT) and Verocytotoxin (VT) of fifteen E. coli strains isolated from cases of pig colibacillosis in Poland was estimated and compared with the effect of enterotoxins of four standard E. coli strains. Of ten tested cell cultures, only the following were susceptible: CHO, Vero, GMK, and HeLa. Eight strains showed CTE in He La and CHO cells and five of these reacted in Vero cells. The results appear to suggest that some of the tested E. coli strains isolated from pigs produced VT enterotoxin. Morphological changes caused by the above mentioned E. coli toxins in Vero and GMK cells took the form of cell rounding, followed by cell dissolution.

Animals↗

PubMed1998

A mutational analysis of the binding of staphylococcal enterotoxins B and C3 to the T cell receptor beta chain and major histocompatibility complex class II.

The three-dimensional structure of the complex between a T cell receptor (TCR) beta chain (mouse Vbeta8.2Jbeta2.1Cbeta1) and the superantigen (SAG) staphylococcal enterotoxin C3 (SEC3) has been recently determined to 3.5 resolution. To evaluate the actual contribution of individual SAG residues to stabilizing the beta-SEC3 complex, as well as to investigate the relationship between the affinity of SAGs for TCR and MHC and their ability to activate T cells, we measured the binding of a set of SEC3 and staphylococcal enterotoxin B (SEB) mutants to soluble recombinant TCR beta chain and to the human MHC class II molecule HLA-DR1. Affinities were determined by sedimentation equilibrium and/or surface plasmon detection, while mitogenic potency was assessed using T cells from rearrangement-deficient TCR transgenic mice. We show that there is a clear and simple relationship between the affinity of SAGs for the TCR and their biological activity: the tighter the binding of a particular mutant of SEC3 or SEB to the TCR beta chain, the greater its ability to stimulate T cells. We also find that there is an interplay between TCR-SAG and SAG-MHC interactions in determining mitogenic potency, such that a small increase in the affinity of a SAG for MHC can overcome a large decrease in the SAG's affinity for the TCR. Finally, we observe that those SEC3 residues that make the greatest energetic contribution to stabilizing the beta-SEC3 complex ("hot spot" residues) are strictly conserved among enterotoxins reactive with mouse Vbeta8.2, thereby providing a basis for understanding why SAGs having other residues at these positions show different Vbeta-binding specificities.

Amino Acid Sequence↗

PubMed1975

Enterotoxins of Escherichia coli and vibriocholerae: tools for the molecular biologist.

With the independent discovery in several different laboratories that Vibrio cholerae and heat-labile Escherichia coli enterotoxins activated an enzyme (adenylate cyclase) in small intestinal epithealial cells to cause an enhanced intestinal secretion mediated by cyclic adenosine 3',5'-monophosphate (cyclic AMP), a molecular mechanism was provided for these disease states. As agents that also elevate intracellular concentrations of cyclic AMP in virtually every mammalian tissue tested, the enterotoxins are potentially invaluable tools in investigation of the molecular sequence of events of all cyclic AMP-related cellular phenomena. Cyclic AMP has been known for some time to be the central regulator in the cellular expression of the effects of hormones and has become known more recently as an agent that controls cellular growth by corrdinately influencing several biochemical processes related to rate of cell division. Successful application of the enterotoxins as cellular probes in the areas of regulation of cell division, determination of the reaction mechanism of adenylate cyclase, and elucidation of the relationship between prostaglandin and adenylate cyclase, both in this laboratory and in those of others, is reviewed.

Adenylyl Cyclases↗

PubMed1975

Enterotoxigenic intestinal bacteria in tropical sprue. III. Preliminary characterization of Klebsiella pneumoniae enterotoxin.

Cell-free broth filtrates of a strain of Klebsiella pneumoniae serotype 5 retained their capacity to induce fluid secretion in the rabbit ileal loop model after heating (100 C for 30 min), acid treatment (pH 4.4), and dialysis through viscose tubing. Sequential passage of an acetone precipitate of the broth filtrate through Diaflo membranes yielded an active fraction in the UM 2, but not the UM 10, retentate; this observation indicates that the molecular weight of the enterotoxin is in the range of 1,000-10,000. Klebsiella pneumoniae enterotoxin thus resembles the heat-stable enterotoxin of Escherichia coli in a number of respects.

Animals↗

PubMed1976

Effect of enterotoxins of Vibrio cholerae, Escherichia coli, and Shigella dysenteriae type 1 on fluid and electrolyte transport in the colon.

Experiments were designed to determine whether the enterotoxins of Vibrio cholerae, Escherichia coli, and Shigella dysenteriae type 1 alter the movement of fluid and electrolytes in the rat cecum. Net secretion of water and sodium were observed after incubation of 1.67 X10(-5) mug of purified cholera toxin (choleragen)/ml for 18 hr or of 50 mug/ml for 3 hr. The effect of choleragen on cecal transport of water and electrolytes was related to the dose. In addition, choleragen increased cecal mucosal content of adenosine 3':5'-cyclic phosphate but did not alter the histology of the cecum. The results demonstrate that the colon responds to choleragen in a manner similar to that of other tissues. In contrast, the enterotoxins of both E. coli and S. dysenteriae type 1 failed to affect cecal transport of water and electrolytes. These observations may explain several phenomena associated with the diarrhea produced by bacterial enterotoxins.

Animals↗

PubMed1977

Regional localization of activity of Clostridium perfringens type A enterotoxin in the rabbit ileum, jejunum, and duodenum.

Rabbit ileal, jejunal, and duodenal loops were exposed to purified enterotoxin from Clostridium perfringens type A and then perfused for comparative analysis of effects of the enterotoxin on each region of the intestine. Ileal loops responded with enhanced net secretion of fluid and sodium, inhibition of chloride and glucose uptake, and substantial sloughing of epithelial cells. The jejunum responded with fluid secretion, enhancement of sodium secretion only during the first 20 min, inhibition of chloride and glucose uptake, and substantial sloughing of epithelial cells. In the duodenum, transport of fluid, sodium, and chloride was significantly altered only during the first 20 min of perfusion, and significant inhibition of glucose uptake varied from one period to another. Epithelial damage was much less than that seen in the jejunum or ileum. Levels of fluid protein in all three sections corresponded closely to extent of tissue damage. In general, it was found that the severity of response to fixed doses of enterotoxin varied as follows: ileum greater than jejunum greater than duodenum.

Animals↗

PubMed1979

Evidence for enterotoxin production by a classic enteropathogenic serotype of Escherichia coli.

Isolates of classic enteropathogenic Escherichia coli O:128 that had been implicated in an outbreak of diarrhea in a hospital nursery were found to produce heat-stable enterotoxin after storage for six years. This finding indicated that enteropathogenicity and the ability to produce enterotoxin may coincide in E. coli, and further study of enteropathogenic strains that produce enterotoxin may help in elucidation of the relationship between enteropathogenic and enterotoxigenic E. coli.

Diarrhea, Infantile↗