Purification and characterization of group A streptococcal pyrogenic exotoxin type C.

Group A streptococcal pyrogenic exotoxin (SPE) type C was partially purified by differential solubility in ethanol and acetate-buffered saline. Toxin prepared in this way consisted of protein and hyaluronic acid. After removal of hyaluronic acid, the toxin remained pyrogenic, enhanced susceptibility of rabbits to letahl endotoxin shock, was stable when treated with acid, base, or pepsin, but was inactivated by heat. Toxin further purified by thin-layer isoelectric focusing was pyrogenic and enhanced the susceptibility of rabbits to lethal endotoxin shock. Purified type C toxin appeared homogeneous when tested by Ouchterlony immunodiffusion and migrated as a single protein band in isoelectric focusing polyacrylamide gels (isoelectric point, 6.7) and sodium dodecyl sulfate-polyacrylamide gels (molecular weight, 13,200). The purified toxin was antigenically distinct from A and B SPE, and antisera raised against the purified toxin neutralized pyrogenic activity. The amino acid composition was determined.     

Heterogeneity of group A streptococcal pyrogenic exotoxin type B.

Streptococcal pyrogenic exotoxin type B purified from culture filtrates of either the NY-5 or T-19 strain of group A streptococcus was found to be heterogeneous in charge. Three protein fractions with isoelectric points of 8.0, 8.4, and 9.0 were isolated by differential solubility in ethanol and acetate-buffered saline followed by isoelectric focusing and shown to be antigenically identical to streptococcal pyrogenic exotoxin type B. The molecular weights of all three fractions were approximately 17,500, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with aggregates forming in the presence of hyaluronic acid. Only the pI 8.4 fraction showed the characteristic activities of streptococcal pyrogenic exotoxin in rabbits: pyrogenicity and ability to enhance susceptibility to lethal endotoxin shock. The pI 8.0 and pI 9.0 fractions were not pyrogenic, but could be used to immunize against pyrogenicity. These two fractions failed either to enhance lethal endotoxin shock or to immunize against enhancement activity. When the isolated fractions were electrofocused again they appeared heterogeneous, suggesting an instability of the B toxin molecular forms.     

Nucleotide sequence of streptococcal pyrogenic exotoxin type C.

The nucleotide sequence of the gene speC, encoding streptococcal pyrogenic exotoxin type C (SPE C), was determined. The gene encoded a mature protein of 208 amino acids, with a calculated molecular weight of 24,354. The mature amino acid sequence of SPE C was analyzed for homology with the amino acid sequences of streptococcal pyrogenic exotoxin type A, the staphylococcal enterotoxins, and toxic shock syndrome toxin-1. Of these, SPE C shared the greatest amount of homology with streptococcal exotoxin type A.     

Inhibition of ribonucleic acid synthesis by group A streptococcal pyrogenic exotoxin.

Group A streptococcal pyrogenic exotoxins (SPEs) A, B, and C and alpha-amanitin enhance host susceptibility to lethal endotoxin shock. The capacity of SPE C and alpha-amanitin to prepare rabbits for the enhancement phenomenon required pretreatment of the animals 1 to 2 h before giving endotoxin. Endotoxin clearance from the circulation of rabbits pretreated with either SPE C or alpha-amanitin was reduced. Even at the time of death, significant amounts of endotoxin remained in the circulation. It is proposed that the SPE and alpha-amanitin inhibit ribonucleic acid synthesis in Kupffer cells with concomitant alteration in reticuloendothelial clearnace function, allowing endotoxin to persist in the circulation and produce host injury. All three SPE types and alpha-amanitin inhibited ribonucleic acid synthesis by 50% or greater in whole liver cells. Kupffer cells, liver cell nuclei, and liver nuclear extracts; inhibition was observed liver cells from both mice and rabbits. The inhibitory effect by SPEs was dose dependent and was observed after as little as 15 min of preincubation with liver cells. The content of ribonucleic acid in liver nuclei of mice pretreated with either SPE C or alpha-amanitan was reduced, whereas total deoxyribonucleic acid and protein content remained unaltered.     

Reinterpretation of the Dick test: role of group A streptococcal pyrogenic exotoxin.

Because of the association of the group A streptococcal pyrogenic exotoxins (SPEs) with erythrogenic toxin used in the classical Dick test, the involvement of the SPEs in production of erythematous skin reactions was assessed. Unless they had been presensitized, young adult rabbits failed to show skin reactions after intracutaneous challenged with SPEs. Rabbits presensitized to purified protein derivative exhibited enhanced skin reactivity when given purified protein derivative plus SPE C; the enhancement was neutralized by antiserum to SPE C. Rabbits sensitized to bovine serum albumin showed extensive red rash development resembling scarlet fever rashes when given bovine serum albumin containing SPE C. Desquamation occurred 5 to 10 days after injection. Animals sensitized to one SPE type showed enhanced skin reactivity to challenge with homologous or heterologous SPE types, indicating the presence of a cross-reactive determinant within the SPE molecules. Repeated challenge of SPE-sensitized animals with homologous toxin resulted in concomitant antitoxin production with reduction of the enhanced skin reactivities, until typical delayed-hypersensitivity skin reactions remained. The data indicate that, in addition to the toxic reaction previously described, SPEs enhance Arthus and delayed-hypersensitivity skin reactions. It follows that erythrogenic toxin represents the enhancement of acquired skin reactivity to streptococcal antigens by one or more SPE types. Therefore, the Dick test measures SPE-enhanced hypersensitivity to streptococcal products.     

Analysis of toxicity of streptococcal pyrogenic exotoxin A mutants.

Streptococcal pyrogenic exotoxin A (SPE A) is secreted by some strains of Streptococcus pyogenes and is strongly associated with streptococcal toxic shock syndrome (STSS), a severe and often fatal illness. SPE A possesses a number of biological properties, some of which are shared with a group of exotoxins of streptococcal and staphylococcal origins, the pyrogenic toxin superantigens (PTSAgs). SPE A's most extensively studied property is superantigenicity. Superantigenic activation of T cells and monocytes stimulates the release of cytokines such as tumor necrosis factors alpha and beta, interleukin 1, and gamma interferon. These endogenous mediators are considered to be the primary cause of capillary leak, hypotension, and shock, the most severe manifestations of STSS. However, several studies have suggested that other properties of SPE A, such as ability to greatly enhance host susceptibility to endotoxin and ability to interact directly with endothelial cells, may play substantial roles in the syndrome. In this work we generated single- and double-site mutations of SPE A at residues K16, N20, C87, C90, C98, K157, S195, N20/C98, and N20/K157. The mutant SPE A's were analyzed in vivo for their lethal activity and in vitro for their superantigenic ability. Our results indicate that SPE A's ability to induce lethality and endotoxin enhancement does not require superantigenicity, and conversely superantigenicity does not necessarily lead to lethality. Thus, these properties and their relative contributions to the onset of hypotension and shock may be separable. Furthermore, evidence is presented that certain mutant toxins may be suitable for use as vaccine toxoids.     

Further purification of group A streptococcal pyrogenic exotoxin and characterization of the purified toxin.

Streptococcal pyrogenic exotoxin (SPE) isolated from culture filtrates of strain NY-5 (type 10), and separated from other extracellular by differential solubility in ethanol and acetate-buffered saline, has previously been shown to exhibit a wide range of biological activities including erythrogenic activity, pyrogenicity, enhancement of susceptibility to endotoxin shock, blockage of the reticuloendothelial system immmunosuppression, and lymphocyte mitogenicity. Toxin prepared in this way was found to consist of hyaluronic acid and several proteins which could be distinguished by thin-layer polyacrylamide isoelectric focusing (IEF), SPE has been further purified by ion exchange chromatography on QAE-Sephadex columns. One of the fractions isolated from QAE-Sephadex, and shown to be a homogenous protein by thin-layer IEF and Ouchterlony with hyperimmune serum, was highly active erythrogenically, pyrogenically, and in enhancing susceptibility to endotoxin. This fraction was identified as exotoxin A. A second, less active fraction identified as SPE B showed similar activities, but differed from the other fraction antigenically and in net charge and molecular weight. These findings indicate that a single highly purified protein can mediate at least three of the biological activities attributed to SPE and NY-5 produces pyrogenic exotoxins A and B in vitro as well as in vivo.     

Transfer of group A streptococcal pyrogenic exotoxin production to nontoxigenic strains of lysogenic conversion.

Production of group A streptococcal pyrogenic exotoxins (SPE) type A and C was transferred from toxigenic streptococcal strains to nontoxigenic strains by lysogeny. Lysogens were tested for SPE with Ouchterlony immunodiffusion on Todd-Hewitt agar plates; toxin diffusing from isolated colonies reacted with specific hyperimmune antisera to SPE. Phage prepared from strains T25(3) (T12gl) and 3GL16, both yielding SPE type A, formed plaques on T25(3) (NONLYSOGENIC) lawns. Over 90% of the colonies picked from the plaque centers yielded A toxin, suggesting SPE type A was transferred by lysogenic conversion. SPE type C formation was transferred to nontoxigenic strains T25(3) and K56 with supernatant fluids from mitomycin C-induced cultures of CS112, producing SPE types B and C. All lysogens tested were positive for SPE type C, indicating that C toxin induction also was transferred by lysogenic conversion. SPE type B formation was not transferable by lysogeny with the strains tested.     

Limulus amebocyte lysate reaction with streptococcal pyrogenic exotoxin.

Three immunologically distinct pyrogenic exotoxins derived from group A streptococci were found to cause coagulation of limulus amebocyte lysate. These exotoxins were shown to be several thousand times less active for lysate coagulation than gram-negative endotoxin.     

Effects of carrier ampholyte contamination on the biological and biochemical properties of streptococcal pyrogenic exotoxin type C.

Three streptococcal pyrogenic exotoxins (SPEs), designated as SPE A, B, and C, have been purified and characterized. Routine purification of the SPEs includes the technique of isoelectric focusing. An earlier study showed that the removal of commercial carrier ampholytes (Ampholines) from SPE was difficult. The physiochemical properties of SPE C were previously reported; however, the SPE C preparation used in those experiments was contaminated with Ampholines. As an alternative to Ampholines, we used simple buffers to generate the isoelectric focusing pH gradient and used this SPE C in a comparative study to evaluate the effects of Ampholine contamination on the biological and biochemical properties of this toxin. We found that Ampholine contamination overestimates protein concentration; consequently, the biological activity of SPE C was actually greater than reported. The most serious effect of Ampholines in SPE C was on amino acid analysis. The presence of Ampholines causes an apparent increase in neutral amino acids and a decrease in basic amino acids.     

Suppression of antibody response by group A streptococcal pyrogenic exotoxin and characterization of the cells involved.

The effect of purified streptococcal pyrogenic exotoxins (SPE) on the antibody response to sheep erythrocytes was studied in cultures of mouse spleen cells. Purified SPE types A, B, and C shared the ability to suppress the day 4 direct plaque-forming cell response when added to cultures. SPE A and C were most suppressive at concentrations of 0.1 to 1 ng per culture, while SPE B was active at 1 microgram per culture. Pretreatment of mice with SPE A, 3 h before removal of their spleens for culture, also produced suppression. Cell populations were separated from spleens of normal and toxin-treated mice and recombined in culture to test the cellular site of action of SPE immunosuppression. When nonadherent cells (lymphocytes) and adherent cells (macrophages) from control and SPE-treated mice were separated and recombined, the plaque-forming cell response depended on the source of lymphocytes. Macrophages from toxin-treated mice functioned normally in the presence of control lymphocytes. In a further experiment, toxin pretreatment failed to suppress the plaque-forming cell response of spleen cells that were T-cell depleted and reconstituted with control thymocytes. When the T lymphocytes were removed from toxin-treated spleen cell suspensions, the remaining cells were able to respond normally to antigen if normal helper T cells were provided. The results suggest that the suppressive activity of SPE on antibody production is mediated by altered activity of T lymphocytes.     

Immune cell lethality induced by streptococcal pyrogenic exotoxin A and endotoxin.

Streptococcal pyrogenic exotoxin (SPE) A has many effects on the immune system, including immunolethality, which is characterized by a significant decrease in circulating immune cells as well as depletion of the spleen and lymph nodes prior to death of experimental animals. In this report, characterization of the mechanism of immunolethality has been undertaken. Synergistic induction of immunolethality was observed in vitro when human lymphocytes were treated with both SPE A and lipopolysaccharide (LPS). The same effect was demonstrated in the absence of a mitogenic response with the murine T-cell receptor, as well as in the absence of antigen-presenting cells and their secreted cytokines. The addition of antigen-presenting cells did not significantly affect lethality. SPE A directly interacted with LPS through interaction with ketodeoxyoctonate as demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and iodinated exotoxin overlays. This interaction was demonstrated to be important for immunolethality, since simultaneous addition of SPE A and LPS was required, whereas sequential addition of SPE A and LPS did not result in lethality. LPS appeared to be acting, in part, to enhance the cell-binding ability of SPE A, since SPE A could only be detected in A.E7 cell membrane preparations after simultaneous incubation with SPE A and LPS.     

Synergistic effects of streptolysin S and streptococcal pyrogenic exotoxin B on the mouse model of group A streptococcal infection

Streptococcus pyogenes is a group A streptococcus (GAS) and an important human pathogen that causes a variety of diseases. Streptococcal pyrogenic exotoxin B (SPE B) and streptolysin S (SLS) are important virulence factors involved in GAS infection, but it is not clear which one is more virulent. Using an air pouch infection model, the wild-type strain NZ131, its isogenic mutants, and complementary mutants were used to examine the effects of SPE B and SLS on GAS infection. The results of the skin lesion and mouse mortality assays showed that although SPE B and SLS had a synergistic effect on GAS infection, SPE B played a more important role in local tissue damage while SLS had a more prominent effect on mouse mortality. Surveys of the exudates from the air pouch revealed that the expression of inflammatory cytokines was significantly inhibited in the sagB/speB-double-mutant JM4-infected mice. Furthermore, in vivo and in vitro studies showed that the isogenic mutant strains were more susceptible to the immune cell killing than the wild-type strain and that the sagB/speB-double-mutant JM4 was the most sensitive among these strains. Moreover, infection with the sagB/speB-double-mutant JM4 strain caused the least amount of macrophage apoptosis compared to infection with the wild-type NZ131 and the other complementary strains, which express only SPE B or SLS or both. Taken together, these results indicate that both SPE B and SLS contributed to GAS evasion from immune cell killing, local tissue damage, and mouse mortality.     

Phage-host interactions and the production of type A streptococcal exotoxin in group A streptococci.

The infection of Streptococcus pyogenes nontoxigenic strain T 253 with bacteriophage T12 to form lysogen T 253 (T12) resulted in the production of type A streptococcal exotoxin (erythrogenic toxin or streptococcal pyrogenic exotoxin). Two lines of evidence indicated that lysogeny per se was not sufficient to promote toxigenic conversion of strain T 253. First, a virulent mutant of phage T12, unable to form stable lysogens, was able to affect type A exotoxin production by strain T 253. An unrelated virulent phage A25 did not affect type A exotoxin production after infection of strain T 253. Second, the temperate phage H4489A, which established stable lysogens with strain T 253 did not promote type A exotoxin production. These results suggest that there is a strain specificity to the phage-host interaction which affects type A exotoxin synthesis. Additional evidence is presented which indicates that type A streptococcal exotoxin was not a structural component of phage T12.     

Group A streptococcal pyrogenic exotoxin (scarlet fever toxin) type A and blastogen A are the same protein.

Group A streptococcal pyrogenic exotoxins A, B, and C (also known as scarlet fever toxins and erythrogenic toxins) were evaluated for relatedness to another streptococcus-derived lymphocyte mitogen, blastogen A. Streptococcal pyrogenic exotoxin A and blastogen A were immunologically cross-reactive and shared the same molecular weight, N-terminal amino acid sequence, and capacity to stimulate rabbit splenocyte proliferation nonspecifically.     

Alteration of clearance function by group A streptococcal pyrogenic exotoxin and its relation to suppression of the antibody response.

The effect of purified group A streptococcal pyrogenic exotoxin (SPE) type A on the processing of and antibody response to sheep erythrocytes (SRBC) was studied in BALB/cWat mice. The rate of clearance of 51Cr-labeled SRBC from the bloodstream was decreased 3 or 24 h following a single intravenous injection of 1 or 10 microgram of SPE. Delayed uptake of label was observed in both the livers and spleens of SPE-treated mice, suggesting an inhibitory effect of the toxin on phagocytic cells of the reticuloendothelial system. Three daily intravenous injections of 0.1 or 1 microgram of purified SPE type A suppressed the early immunoglobulin response to SRBC. The role of altered macrophage function in producing the immunosuppression was tested in macrophage transfer experiments. SPE treatment suppressed the antibody response to SRBC transferred by normal macrophages, indicating that the immunosuppressive effect of the toxin was not due solely to altered antigen processing by macrophages.     

Toxoids of streptococcal pyrogenic exotoxin A are protective in rabbit models of streptococcal toxic shock syndrome

Streptococcal pyrogenic exotoxins (SPEs) are superantigens that have been implicated in causing streptococcal toxic shock syndrome (STSS). Most notably, SPE serotype A is made by nearly all M-protein serotype 1 and 3 streptococci, the M types most associated with the illness (these strains contain one or more other SPEs, and those proteins are likely also to contribute to disease). We have prepared double-, triple-, and hexa-amino-acid mutants of SPE A by PCR and other mutagenesis procedures. The sites chosen for mutation were solvent-exposed residues thought to be important for T-cell receptor (TCR) or major histocompatibility complex (MHC) class II interaction. These mutants were nonsuperantigenic for human peripheral blood mononuclear cells and rabbit and mouse splenocytes and were nonlethal in two rabbit models of STSS. In addition, these mutants stimulated protective antibody responses. Interestingly, mutants that altered toxin binding to MHC class II were more immunogenic than mutants altering TCR binding. Collectively, these studies indicate that multiple-site mutants of SPE A are toxoids that may have use in protecting against the toxin's effects in STSS.     

Cloning of the gene, speB, for streptococcal pyrogenic exotoxin type B in Escherichia coli.

The structural gene encoding streptococcal pyrogenic exotoxin type B, designated speB, was cloned in Escherichia coli and localized onto a 4.5-kilobase BamHI-BglII DNA fragment. Streptococcal pyrogenic exotoxin type B, partially purified from E. coli clones, was immunologically related to streptococcus-derived toxin. Also, toxin derived from either E. coli or Streptococcus pyogenes had similar lymphocyte mitogenic activity and molecular weight (29,300) and displayed comparable microheterogeneity when evaluated by isoelectric focusing.     

Histopathologic changes in kidney and liver correlate with streptococcal pyrogenic exotoxin B production in the mouse model of group A streptococcal infection

Previous studies show that isogenic mutants deficient in streptococcal pyrogenic exotoxin B (SPE B) cause less mortality and skin tissue damage than wild-type strains of Streptococcus pyogenes when inoculated into mice via an air pouch. In this study, the growth and dissemination of bacteria, pathologic changes in various organs, and their correlation with SPE B production were examined. Bacterial numbers in the air pouch from wild-type strain NZ131-infected mice increased at 48 h, while those from speB mutant SW510-infected mice continuously reduced. Mice infected with NZ131 developed bacteremia and greater dissemination in the kidney, liver, and spleen; those infected with SW510 showed either no or slight bacteremia and dissemination. Co-inoculation of SW510 with recombinant SPE B showed a higher bacterial count in the air pouch, bacteremia, and organ dissemination compared to co-inoculation with a C192S mutant lacking protease activity. The histopathologic changes examined showed lesions in kidney and liver in the NZ131-infected but not in SW510-infected mice. The elevation in sera of BUN, AST, and ALT correlated positively with renal and liver impairment. Taken together, SPE B produced during S. pyogenes infection plays a pathogenic role. A direct effect of SPE B on vessel permeability change was also demonstrated.     

A new procedure for the purification of streptococcal pyrogenic exotoxin A from Streptococcus pyogenes supernatant.

An important role in the pathogenesis of invasive group A streptococcal disease has been ascribed to the production of streptococcal pyrogenic exotoxin A. We present a new technique for the purification of streptococcal pyrogenic exotoxin A from Streptococcus pyogenes NY-5 supernate, which is highly efficient with respect to yield (35%), purity (> or = 99%), and time.