Studies on the irradiation of toxins of Clostridium botulinum and Staphylococcus aureus

The effects of irradiation of Clostridium botulinum neurotoxin type A (BNTA) and staphylococcal enterotoxin A (SEA) in gelatin phosphate buffer and cooked mince beef slurries were investigated. Estimation of toxins by immunoassays showed that in buffer, toxins were destroyed by irradiation at 8.0 kGy; in mince slurries however, 45% of BTNA and 27–34% of SEA remained after this level of irradiation. At 23.7 kGy, over twice the dose of irradiation proposed for legal acceptance in the UK, 15% of BNTA and 16–26% of SEA still remained. Increasing concentrations of mince conferred increased protection against the effect of irradiation on both toxins. The biological activity of BNTA was more sensitive to irradiation than the immunological activity. Staphylococcal enterotoxin was more resistant to irradiation than BNTA. Irradiation should therefore only be used in conjunction with good manufacturing practices to prevent microbial proliferation and toxin production prior to irradiation.     

Lowering of bactericidal activity of mouse peritoneal macrophages under combined use of staphylococcal enterotoxin type A and endotoxin

The present study investigated the effect of staphylococcal enterotoxin type A (SEA) and endotoxin Serratia marcescens (LPS) on the phagocytosis and killing of Staphylococcus aureus by mouse peritoneal macrophages. Two hours after enterotoxin intraperitoneal injection phagocytic and bactericidal activity were depressed. 24 hours later there was increased functional activity of macrophages by SEA and LPS, apart. But when two toxins were administered together (LPS four hours later enterotoxin) marked inhibition of bacterial killing was observed. When peritoneal macrophages were treated in vitro for 24 hours with the same toxins they were also markedly suppressed in bactericidal activity.     

Production and characterization of anti-peptide monoclonal antibodies with specificity for staphylococcal enterotoxins A and B

A synthetic peptide containing selected epitopes from staphylococcal enterotoxin A (SEA) and enterotoxin B (SEB) was used to produce monoclonal antibodies (Mabs) to respective enterotoxins in a single fusion procedure. The peptide inhibited the reaction of polyclonal anti-SEA or anti-SEB antisera with their homologous enterotoxin, thus showing that the chosen epitopes are part of the antibody-inducing enterotoxin sequences. Two Mabs, Mab-A and Mab-B, reacted with both the peptide and with either SEA or SEB. Used in a double antibody sandwich ELISA, the Mabs were able to quantitate the native SEA or SEB toxins at nanogram levels.     

Staphylococcal toxins bind to different sites on HLA-DR

Staphylococcal enterotoxins (SE) and toxic shock syndrome toxin 1 (TSST-1) bind to MHC class II molecules and the toxin-class II complexes induce proliferation of T cells bearing specific V beta sequences. We have previously reported that these toxins display varying binding affinities for HLA-DR1. We now investigated whether these differences simply reflected differences in binding affinity for a single class II binding site or, at least in part, the engagement of different binding sites on the HLA-DR complex. Through competitive binding studies we show that SEB and TSST-1, which are not closely related by their amino acid sequences, bind to two different sites on HLA-DR. Both of these sites are also occupied by staphylococcal enterotoxin A (SEA), enterotoxin D (SED), and enterotoxin E (SEE) which exhibit more than 70% amino acid sequence homology. SEB and TSST-1 failed to inhibit SEA binding to HLA-DR. These studies suggest that there may be three distinct, although perhaps overlapping, binding sites on HLA-DR for these toxins. Further, although SED and SEE are similar to SEA in structure, and appear to bind the same sites on HLA-DR as SEA, they displayed significantly lower binding affinities. T cell proliferative responses to SED required a higher concentration of the toxin than SEA, probably reflecting its lower binding affinity. SEE, however, elicited T cell responses at very low concentrations, similar to SEA, despite its much lower binding affinity. Therefore, although the affinities of these toxins to MHC class II molecules appear to significantly influence the T cell responses, the effective recognition of the toxin-class II complex by the TCR may also contribute to such responses.     

Changes in the content of reduced glutathione in the liver of mice under the action of staphylococcal enterotoxin type A and lipopolysaccharide]

We investigated the level of reduced glutathione in mouse liver after injection of staphylococcal enterotoxin type A (SEA) and Serratia marcescens endotoxin (LPS). It was shown that SEA caused significant glutathione depletion. Glutathione level fell more under the combined treatment by both toxins. The data demonstrate a considerable role of antioxidant potential in the mechanism of enhanced sensitivity of animals to the lethal effect produced by LPS.     

Novel method for purification of staphylococcal enterotoxin A

A novel single-step procedure for the purification of staphylococcal enterotoxin A (SEA), namely, dye ligand affinity chromatography with the triazine dye Red A, was developed. SEA purified by this method produced a single band when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The yield from 5 liters of culture supernatant was 0.113 g, corresponding to an overall yield of 55%. In some instances, purification of SEA from culture supernatants by dye ligand affinity chromatography produced two enterotoxin peaks that could be eluted from the column with 300 and 500 mM phosphate buffer (pH 6.8). Enterotoxin from these peaks produced a single band when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but multiple bands were observed on isoelectric focusing gels. This method of purification represents a significant improvement in time, yields, and purity of enterotoxin over previously published purification methods.     

Binding of staphylococcal enterotoxin A to purified murine MHC class II molecules in supported lipid bilayers

The staphylococcal enterotoxins are a family of bacterial toxins that are thought to exert their pathogenic effects by the massive activation of T lymphocytes to produce lymphokines. Activation of T cells by these toxins is dependent on MHC class II+ APC. Recent studies from a number of laboratories have implicated MHC class II proteins as the APC surface receptor for a number of the staphylococcal enterotoxins. The present report shows that staphylococcal enterotoxin A, (SEA) binds to the purified murine MHC class II molecule I-Ed reconstituted in supported planar membranes, indicating that no other cell surface proteins are required for SEA binding. The Kd for SEA binding to I-Ed was determined to be 3.5 +/- 1.6 x 10(-6) M. Specific binding of SEA to I-Ad was also observed, but the interaction was of significantly lower affinity. Binding of SEA to purified I-Ed was blocked by antibodies against both the alpha- and the beta-chain of the I-Ed molecule, but not by antibodies specific for an unrelated MHC class II protein. Binding of SEA to I-Ad was blocked by an A beta d but not by an A alpha d-specific antibody. Planar membranes containing only lipid and purified I-Ed molecules were sufficient for activation of a V beta 1 expressing T hybrid by SEA. The T cells responded to as few as 180 toxin molecules per T cell.     

Novel method for purification of staphylococcal enterotoxin A.

A novel single-step procedure for the purification of staphylococcal enterotoxin A (SEA), namely, dye ligand affinity chromatography with the triazine dye Red A, was developed. SEA purified by this method produced a single band when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The yield from 5 liters of culture supernatant was 0.113 g, corresponding to an overall yield of 55%. In some instances, purification of SEA from culture supernatants by dye ligand affinity chromatography produced two enterotoxin peaks that could be eluted from the column with 300 and 500 mM phosphate buffer (pH 6.8). Enterotoxin from these peaks produced a single band when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but multiple bands were observed on isoelectric focusing gels. This method of purification represents a significant improvement in time, yields, and purity of enterotoxin over previously published purification methods.     

Partial purification and characterization of feline gamma-like interferon

Feline interferon (IFN) was produced from spleen cells stimulated by Staphylococcus enterotoxin A (SEA). The IFN was purified by a three-step procedure using controlled pore glass adsorption chromatography, concanavalin A (ConA)-agarose column chromatography and gel filtration. The final product of these procedures which had activity of an IFN appeared as a single peak of activity with molecular weight of approximately 50,000. It was sensitive to acid and heat, suggesting that the isolated material was a gamma IFN. The total recovery of feline gamma IFN was 8.2%. Specific activity was 2.95 X 10(4) unit/micrograms protein and was concentrated 2.8 X 10(4) times. This preparation of purified feline gamma IFN was destroyed completely by 0.1% sodium dodecyl sulfate within 20 min. As an inducer of feline gamma IFN, SEA appeared to produce a more uniform IFN product than did ConA. Further, the presence of 10% ethylene glycol in the sample applied to ConA-agarose column as well as its absence in the elution buffer was effective in reducing contaminating acid- and heat-resistant IFNs in the preparation.     

Partial Purification and Characterization of Feline Gamma-Like Interferon

Feline interferon (IFN) was produced from spleen cells stimulated by Staphylococcus enterotoxin A (SEA). The IFN was purified by a three-step procedure using controlled pore glass adsorption chromatography, concanavalin A (ConA)-agarose column chromatography and gel filtration. The final product of these procedures which had activity of an IFN appeared as a single peak of activity with molecular weight of approximately 50, 000. It was sensitive to acid and heat, suggesting that the isolated material was a gamma IFN.

The total recovery of feline gamma IFN was 8.2%. Specific activity was 2.95 × 10⁴ unit/μg protein and was concentrated 2.8 × 10⁴ times. This preparation of purified feline gamma IFN was destroyed completely by 0.1 % sodium dodecyl sulfate within 20 min. As an inducer of feline gamma IFN, SEA appeared to produce a more uniform IFN product than did ConA. Further, the presence of 10 7. ethylene glycol in the sample applied to ConA-agarose column as well as its absence in the elution buffer was effective in reducing contaminating acid- and heat-resistant INFs in the preparation.     

Induction of histidine decarboxylase in non-mast cells in the spleen of mice by injection of staphylococcal enterotoxin A

Injection of Staphylococcal enterotoxin A (SEA) into WBB6F1-W/WV mice genetically deficient in mast cells resulted in a 10-fold increase in the histidine decarboxylase [HDC, L-histidine carboxylase, EC 4.1.1.22] activity of their spleen. The nature of the spleen cells responsible for this increased HDC activity was studied. The HDC induction by SEA was abolished on day 1 after X-ray irradiation of the mice at 400 rad and restored by transplantation of bone marrow cells from normal WBB6F1-+/+ littermates into the X-ray irradiated WBB6F1-W/WV mice. Transplantation of cells from other organs of the normal mice, such as the thymus, mesenteric lymph node and spleen, did not restore the HDC increase significantly. Transplantation of cultured mast cells also did not restore the increase. Moreover, the high HDC activity of spleen cells induced by SEA was not affected by their treatment with anti-Thy-1,2 antibody and complement. Depletion of phagocytes from the spleen by treatment with carbonyl iron resulted in decrease in HDC activity. These results suggested that phagocytic cells derived from haemopoietic stem cells of the bone marrow were responsible for the increase in HDC activity induced by SEA.     

Determination of staphylococcal enterotoxin A in cheddar cheese produced without starter activity.

Three variants of the chloramine-T radioiodination method were used to iodinate staphylococcal enterotoxin A with 125I. Only one method consistently produced usable labels for radioimmunoassay. The iodine incorporation was 55 to 76%; the specific activity was 3.5 to 5.5 muCi/microgram of enterotoxin, and the label was extremely stable on storage at -20 degrees C. Determinations of the enterotoxin in extracts of cheddar cheese produced without starter activity were carried out with the radioimmunoassay system and protein A as antibody immunoadsorbent. The assay buffer used in this system significantly influenced the detected levels of enterotoxin in the cheese extracts. Phosphate buffer, but not tris(hydroxymethyl)aminomethane (Tris) buffer, caused gelling of cheese extract proteins, thus resulting in an incomplete separation of free from antibody-bound 125I enterotoxin. When Tris buffer was used, the results indicated a high degree of accuracy and precision for this radioimmunoassay. The lowest detectable enterotoxin concentration in cheese extract was 0.5 ng/ml.     

On the cross-reactivity of staphylococcal enterotoxins A, B, and C.

Strong cross-reactions were demonstrated for staphylococcal enterotoxins B (SEB) and C1 (SEC1) by antigen-binding capacity and by competitive binding ability. Both SEB and SEC1 combined completely with the heterologous antibody although requiring four times as much antiserum as the homologous enterotoxin and both displaced about one-third of the other enterotoxin from a heterologous antigen-antibody system. It is proposed that one of the three major antigenic determinants of these enterotoxins possesses a significant similarity but probably not an identity of structure. SEB and SEC1 did not combine with antiserum to enterotoxin A nor inhibit the reaction of SEA with anti-SEA. SEA had no intrinsic binding capacity for anti-SEB or anti SEC1 nor did it inhibit the binding of either enterotoxin to its own antibody. Affinity chromatography was employed to demonstrate that a small apparent binding of SEA to anti-SEB was due to antibody to SEA in the anti-SEB serum and that an almost complete displacement of SEC1 binding to anti-SEC1 was caused by contaminating SEC (about 0.01%) in preparations of enterotoxin A.     

Structural basis for abrogated binding between staphylococcal enterotoxin A superantigen vaccine and MHC-IIalpha

Staphylococcal enterotoxins (SEs) are superantigenic protein toxins responsible for a number of life-threatening diseases. The X-ray structure of a staphylococcal enterotoxin A (SEA) triple-mutant (L48R, D70R, and Y92A) vaccine reveals a cascade of structural rearrangements located in three loop regions essential for binding the alpha subunit of major histocompatibility complex class II (MHC-II) molecules. A comparison of hypothetical model complexes between SEA and the SEA triple mutant with MHC-II HLA-DR1 clearly shows disruption of key ionic and hydrophobic interactions necessary for forming the complex. Extensive dislocation of the disulfide loop in particular interferes with MHC-IIalpha binding. The triple-mutant structure provides new insights into the loss of superantigenicity and toxicity of an engineered superantigen and provides a basis for further design of enterotoxin vaccines.     

Immunological studies on staphylococcal enterotoxin D: production of murine monoclonal antibodies and immunopurification

Eight murine monoclonal antibodies (MAbs) against staphylococcal enterotoxin D (SED) were obtained by fusion of myeloma cells with mouse spleen cells immunized with SED only or a combination of SED and either enterotoxin A (SEA) or enterotoxin E (SEE). When only SED was used as an immunogen, six MAbs were specific for SED only, whereas one MAb was reactive with both SED and SEE when both SEs were used as immunogens. One MAb reacted with SEA, SED, and SEE when both SEA and SED were used as immunogens. A MAb with the highest reactivity to SED was used to prepare an immunosorbent for purification of SED by immunoaffinity chromatography. Approximately 70% of the partially purified SED was recovered in the eluate. The purified SED was electrophoretically and antigenically pure. Immunoaffinity chromatography proved useful in the purification of SED in terms of ease of purification, percent enterotoxin, and enterotoxin purity.     

Staphylococcus aureus and staphylococcal enterotoxin A in breaded chicken products: detection and behavior during the cooking process

In this study we examined the presence of Staphylococcus aureus and staphylococcal enterotoxin A (SEA) in 20 industrial breaded chicken products obtained from different retail butchers and supermarket stores in Italy. The levels of contamination in the products analyzed were quite low, although the pH values and water activities (a(w)) in the samples considered were in ranges favorable for S. aureus growth. As demonstrated by phenotypic and molecular characterization, in spite of the high percentage of coagulase-positive Staphylococcus strains, only three strains could be referred to the species S. aureus. Moreover, all the strains were negative in PCR assays targeting staphylococcal enterotoxin genes (seA to seE, seG to seJ, and seM to seO), as well as the toxic shock syndrome toxin 1 gene, and no SEA was detected in the retail breaded chicken samples analyzed by a reversed passive latex agglutination assay or by Western blotting. Hence, we evaluated the thermal resistance of two strains of SEA-producing S. aureus in a laboratory-scale preparation of precooked breaded chicken cutlets. The heat treatment employed in the manufacture determined the inactivation of S. aureus cells, but the preformed SEA remained active during product storage at 4 degrees C. The presence of the staphylococci and, in particular, of S. aureus in the retail breaded chicken products analyzed is a potential health risk for consumers since the pH and a(w) values of these kinds of products are favorable for S. aureus growth. The thermal process used during their manufacture can limit staphylococcal contamination but cannot eliminate preformed toxins.     

Receptors for toxic shock syndrome toxin-1 and staphylococcal enterotoxin A on human blood monocytes.

Staphylococcal toxic shock syndrome toxin-1 (TSST-1) as well as staphylococcal enterotoxin A (SEA) and B (SEB) have recently been shown to bind directly to the class II major histocompatibility antigen, HLA-DR. Whereas others have characterized TSST-1 and SEA binding to HLA-DR on transfected L cells or B lymphoma cell lines, we sought evidence for direct binding of TSST-1 and SEA to HLA-DR on purified human monocytes. A single class of high-affinity receptors was found for both TSST-1 (dissociation constant (Kd) 40 nM, 3.4 x 10(4) receptors per cell) and SEA (Kd 12 nM, 3.2 x 10(4) receptors per cell) on normal human monocytes. Affinity cross-linking of 125I-labeled toxins to monocytes revealed the presence of two membrane protein subunits with molecular masses consistent with the alpha and beta chains of human HLA-DR (35 and 28 kDa, respectively). The anti-HLA-DR monoclonal antibody L243, but not L203 or 2.06, inhibited radiolabeled toxin binding to human monocytes and neutralized the mitogenic response of human T lymphocytes to both toxins. However, L243 was consistently more effective in blocking radiolabeled TSST-1 than SEA binding to human monocytes from the same donors, suggesting that TSST-1 and SEA may be binding to overlapping epitopes rather than to the same epitope on HLA-DR. Because TSST-1 and SEB bind to distinct epitopes on HLA-DR and because SEA cross competes with both TSST-1 and SEB on the HLA-DR receptor, we postulate that SEA occupies a binding site within HLA-DR that overlaps both TSST-1 and SEB.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

γ Irradiation of Staphylococcal Enterotoxin B

The inactivation of enterotoxin B by γ 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 μg/ml in Veronal buffer to less than 0.7 μg/ml. When milk was used as a vehicle, a dose of 20 Mrad was needed to inactivate a 30 μg/ml concentration of enterotoxin B to less than 0.5 μg/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.     

Production and characterization of murine monoclonal antibodies against staphylococcal enterotoxins A and E

Six murine monoclonal antibodies (MAbs) against staphylococcal enterotoxin A (SEA) and enterotoxin E (SEE) were prepared by fusion of myeloma cells with mouse spleen cells immunized with SEA and SEE. Of five MAbs to SEA tested, two MAbs were reactive with only SEA, whereas three were specific for both SEA and SEE. On the other hand, one MAb to SEE was found to be specific for only SEE. To study specificities of the combining sites of these MAbs, competitive binding assays with either SEA or SEE and horseradish peroxidase conjugated MAbs were performed using unconjugated MAbs as inhibitors. The results obtained in the assays suggest that different epitopes may be located on SEA and that some of them may be cross-reacting epitopes between SEA and SEE.