Streptococcal Pyrogenic Exotoxin A Research Articles

Superantigen binding to a T cell receptor beta chain of known three-dimensional structure.

The three-dimensional structure of an unglycosylated T cell antigen receptor (TCR) beta chain has recently been determined to 1.7 A resolution. To investigate whether this soluble beta chain (murine V beta 8.2J beta 2.1C beta 1) retains superantigen (SAG)-binding activity, we measured its affinity for various bacterial SAGs in the absence of MHC class II molecules. Dissociation constants (KDs) were determined using two independent techniques: surface plasmon resonance detection and sedimentation equilibrium. Specific binding was demonstrated to staphylococcal enterotoxins (SEs) B, C1, C2, and C3 and to streptococcal pyrogenic exotoxin A (SPEA), consistent with the known proliferative effects of these SAGs on T cells expressing V beta 8.2. In contrast, SEA, which does not stimulate V beta 8.2-bearing cells, does not bind the recombinant beta chain. Binding of the beta chain to SAGs was characterized by extremely fast dissociation rates (> 0.1 s-1), similar to those reported for certain leukocyte adhesion molecules. Whereas the beta chain bound SEC1, 2, and 3 with KDs of 0.9-2.5 microM, the corresponding value for SEB was approximately 140 microM. The much weaker binding to SEB than to SEC1, 2, or 3 was surprising, especially since SEB was found to actually be 3- to 10-fold more effective, on a molar basis, than the other toxins in stimulating the parental T cell hybridoma. We interpret these results in terms of the ability of SEC to activate T cells independently of MHC, in contrast to SEB. We have also measured SE binding to the glycosylated form of the beta chain and found that carbohydrate apparently does not contribute to recognition, even though the N-linked glycosylation sites at V beta 8.2 residues Asn24 and Asn74 are at or near the putative SAG-binding site. This result, along with the structural basis for the V beta specificity of SEs, are discussed in relation to the crystal structure of the unglycosylated beta chain.

Major histocompatibility complex class II binding site for streptococcal pyrogenic (erythrogenic) toxin A.

Streptococcal pyrogenic exotoxin A (SPEA) is an important pathogenicity factor of group A streptococci. It is a member of the family of "superantigens" produced by Staphylococcus aureus and Streptococcus pyogenes and its T lymphocyte stimulating activity is involved into the pathogenesis of certain diseases caused by pyogenic streptococci. In this study we have produced and characterized recombinant SPEA molecules in Escherichia coli. These molecules are indistinguishable from natural SPEA in both T cell stimulatory and HLA class II binding activities. Human class II molecules are more efficient than mouse class II molecules in presenting SPEA to T cells. In binding tests to major histocompatibility complex class II-positive cells SPEA competes with staphylococcal enterotoxin B and A but not with toxic shock syndrome toxin-1.

Quantification of superantigen induced IFN-γ production by computerised image analysis — inhibition of cytokine production and blast transformation by pooled human IgG

A quantitative image analysis technique was developed to assess the cytokine content of immunocytochemically stained cytokine producing cells. Peripheral blood mononuclear cells were stimulated to induce cytokine production with the superantigen streptococcal pyrogenic exotoxin-A. We have developed a method based on indirect immunocytochemistry which identifies IFN-γ producing cells by a characteristic morphology generated by the accumulation of IFN-γ in the Golgi organelle. An image analysis technique permitted discrimination between these producer cells and IFN-γ binding target cells, which showed a different appearance, with staining restricted to the cell surface membrane. A semi-automated routine programme allowed the signal from a video camera to be processed by computerised image analysis methodology. This enabled us to measure the number of cytokine producing cells, the cytokine staining intensity in individual cells and the cell size expressed in actual cell area. The incidence of IFN-γ producing cells determined by image analysis measurement was compared to results obtained using manual microscopy. Cell size was assessed by the image analysis system as well as by flow cytometry. Administration of pooled human IgG for intravenous use (IVIg) to the superantigen stimulated cells significantly down-regulated IFN-γ production, both in terms of the numbers of producer cells and in terms of cytokine staining intensity in individual cells. In addition blast transformation of cells was substantially reduced. These effects, mediated by IVIg, were also evident following delayed IVIg administration 24 h after the initial cell stimulation.

Lymphokine production induced by streptococcal pyrogenic exotoxin-A is selectively down-regulated by pooled human IgG.

The influence of pooled human IgG preparations for intravenous use (IVIg) on cytokine production induced by streptococcal pyrogenic exotoxin-A (SPE-A) was studied at the single-cell level using cytokine-specific monoclonal antibodies and indirect immunofluorescence or immunohistochemical staining. Mononuclear cells from healthy adult blood donors were stimulated with SPE-A alone or in the presence of IVIg. IVIg was added either prior to stimulation or 24 h after initiation of cultures, in an attempt to evaluate whether IVIg treatment could influence an already established systemic streptococcal disease. Cells were harvested after 48 or 72 h of culture and stained for the following cytokines: interleukin(IL)-1 alpha, IL-1 beta, IL-1ra, IL-6, IL-8, IL-2, tumor necrosis factor interferon(IFN)-gamma and TNF-alpha and TNF-beta and granulocyte macrophage-colony-stimulating factor. Stimulation with SPE-A lead to extensive lymphokine and monokine production. With the addition of IVIg prior to stimulation there was a strong reduction of blast transformation and an almost complete inhibition of lymphokine production, in particular in the synthesis of IFN-gamma and TNF-beta while the synthesis of IL-1 and IL-8 was either unaffected or increased. Adding IVIg 24 h after SPE-A stimulation also resulted in reduced blast transformation and decreased synthesis of IFN-gamma and TNF-beta. These results indicate an immunomodulatory potential by IVIg on streptococcally induced T cell activation and lymphokine production.

Superantigens associated with staphylococcal and streptococcal toxic shock syndrome are potent inducers of tumor necrosis factor-beta synthesis.

The role of tumor necrosis factor-alpha (TNF alpha) in the pathogenesis of severe bacterial infections has been studied extensively. However, the role of TNF beta, a lymphokine with biologic activities similar to those of TNF alpha, has received little attention. Therefore, the purpose of this study was to examine the production of TNF beta by peripheral blood mononuclear cells in response to lipopolysaccharide (LPS) and the superantigens staphylococcal toxic shock syndrome toxin 1 (TSST-1) and streptococcal pyrogenic exotoxin A (SPEA). Though LPS was a more potent inducer of TNF alpha than was TSST-1 or SPEA, TSST-1 and SPEA were both more potent inducers of TNF beta. The superantigens TSST-1 and SPEA were more potent inducers of total TNF (TNF alpha and TNF beta) than was LPS. These data suggest that the induction of TNF beta synthesis may be a unique pathway by which superantigens associated with severe streptococcal and staphylococcal infections mediate shock and multiorgan failure characteristic of toxic shock syndrome.

Mutations affecting MHC class II binding of the superantigen streptococcal erythrogenic toxin A.

Streptococcal pyrogenic exotoxin A (SPEA) is an important pathogenicity factor of group A streptococci. It is a member of the family of 'superantigens' produced by Staphylococcus aureus and Streptococcus pyogenes, and its T lymphocyte stimulating activity is involved in the pathogenesis of certain diseases caused by pyogenic streptococci. In this study we have generated nine mutant SPEA molecules by substituting amino acids in the regions of homology between different streptococcal and staphylococcal superantigens. An additional mutant was created by deletion of the 10 N-terminal amino acids. The mutants were expressed as fusion proteins. Several mutations led to a loss of function due to a loss of class II-binding activity. Such loss mutations did not cluster to a certain region of the SPEA molecule. Rather, even a substitution of neighboring amino acids had opposite effects. None of the loss mutations affected the binding of neutralizing mAb and all loss mutants could be precipitated in Ouchterlony tests by a polyclonal anti-SPEA serum. We conclude that the functional activities of SPEA, and probably of other superantigens as well, cannot be attributed to a defined region of the molecule.

Streptococcal pyrogenic exotoxin A and streptolysin O enhance polymorphonuclear leukocyte binding to gelatin matrixes.

Autopsy data from cases of streptococcal toxic shock demonstrate accumulation of polymorphonuclear leukocytes (PMNL) within lung and soft tissue microvasculature. Because of the increased prevalence of streptococcal pyrogenic exotoxin A (SPEA)-producing strains associated with streptococcal toxic shock syndrome, experiments were done to determine whether SPEA or streptolysin O (SLO, a thiol-activated cytolysin produced by all group A streptococci) could stimulate PMNL-dependent adherence mechanisms in vitro. SPEA (0.01-10 micrograms/5.5 x 10(6) PMNL) only modestly enhanced PMNL adherence over the entire range of concentrations tested. In contrast, SLO-induced PMNL binding was highly dose dependent (maximal binding, 55.1 +/- 1.6% at 0.5 hemolytic units/5.5 x 10(6) PMNL) and was mediated by CD11/CD18 adherence glycoprotein.

Isolierung und Charakterisierung von erythrogenen Toxinen I. Untersuchung des von Streptococcus pyogenes, Stamm NY-5 gebildeten Toxins A

An erythrogenic toxin was isolated from culture supernatants of Streptococcus pyogenes, strain NY-5 (type 12) grown in yeast extract-pepton-dialysate medium. After concentration by evaporation, a crude material was obtained by precipitation with ethanol. This material was prepurified by ion exchange chromatography using a DEAE-Sepharose Cl-6B column. After precipitation of the active material with (NH4)2SO4 the redissolved precipitate was dialyzed against acetate buffer and rechromatographed on a CM-Sepharose Cl-6B column. The toxin was obtained by stepwise elution with 0.02 M acetate buffer, pH 5.0, and 0.05 M phosphate buffer, pH 6.5, followed by a last purification step on a Sephacryl S-200 column. The purified toxin behaved homogenously in SDS electrophoresis, the molecular weight being about 28 000. Amino acid analysis showed only one cysteine residue per molecule. The molecule was insensitive to 2-mercaptoethanol and alkylation. Isoelectric focusing yielded two narrow bands having an isoelectric point of 5.2. The toxin showed a serological reaction of identity with an antiserum against streptococcal pyrogenic exotoxin A (SPE-A) but was shown to have a different molecular weight. The purified material (erythrogenic toxin A) was pyrogenic and toxic for rabbits as well as mitogenic for human lymphocytes. A positive skin reaction in guinea pigs could be obtained by as little as 5 X 10(-8) mg.