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PubMed1983

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.

Amino Acids↗
PubMed1983

Production of staphylococcal enterotoxin F and pyrogenic exotoxin C by Staphylococcus aureus isolates from toxic shock syndrome-associated sources.

A total of 136 isolates of Staphylococcus aureus were tested for production of staphylococcal enterotoxin F (SEF) and pyrogenic exotoxin C (PEC), both of which have been identified as reliable indicators of toxic shock syndrome (TSS)-associated strains. SEF and PEC production by isolates from TSS-associated and other sources was tested independently in two laboratories, after which the two sets of data were compared. A 100% concordance between SEF and PEC production was obtained. The TSS toxin candidates were produced by 30 of 136 isolates, and in all instances SEF and PEC were made concurrently by the same strains; in no case was one toxin made and not the other. In the five groups of S. aureus tested, toxins were detected as follows: 23 of 25 (92%) acute TSS isolates, 2 of 48 (4.2%) genital non-TSS isolates, 2 of 16 (12.5%) recovered TSS isolates, 1 of 23 (4.3%) clinical nongenital isolates, and 2 of 24 (8.3%) enterotoxigenic food outbreak isolates. Comparison of purified SEF and purified PEC by immunological and biochemical criteria by immunodiffusion, isoelectric focusing, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and Western blot analysis show that the toxins are immunologically identical and strongly suggest that the two nominal TSS toxins are in fact a single protein.

Bacterial Toxins↗
PubMed1987

Physiology of the potentiation of lethal endotoxin shock by streptococcal pyrogenic exotoxin in rabbits.

Streptococcal pyrogenic exotoxin (SPE) dramatically potentiates the lethal shock induced by gram-negative bacterial endotoxin. To provide further understanding of the mechanism underlying the potentiating effect, the physiological basis for the toxic synergism of the two toxins was investigated. Pretreatment of rabbits with an intravenous (i.v.) dose (10 micrograms/kg of body weight) of SPE greatly enhanced the endotoxin lethality and reduced the 50% lethal dose to less than 5 micrograms of endotoxin per kg. The SPE pretreatment dose caused severe pathophysiological changes in combination with a small i.v. dose of endotoxin (1 microgram/kg). These changes included transient hyperglycemia followed by profound hypoglycemia, elevation of the blood lipoperoxide level, and an acute increase in plasma beta-glucuronidase activity. These changes were comparable with those in animals given a large i.v. dose of endotoxin (100 micrograms/kg) alone. An injection of SPE alone did not alter any of the parameters described above. These results suggest that SPE renders rabbits more sensitive to extensive pathophysiologic effects of endotoxin, and the potentiating effect on endotoxin lethality may thus involve a general potentiation of physiologic failures. The SPE pretreatment depressed the vascular clearance of a large dose of endotoxin (100 micrograms/kg) but failed to affect that of a small dose of endotoxin (1 microgram/kg). The data suggest that the potentiating effect is not readily explained solely on the basis of the decreased clearance of endotoxin.

Animals↗
PubMed1992

Relative abilities of distinct isotypes of human major histocompatibility complex class II molecules to bind streptococcal pyrogenic exotoxin types A and B.

The relative ability of distinct isotypes of human leukocyte antigen class II molecules to bind streptococcal pyrogenic exotoxins A and B (SPE A and SPE B, respectively) was investigated by a direct-binding assay with 125I-labeled toxin for SPE A and by a functional assay system measuring the accessory cell activity of human leukocyte antigen class II transfectants in toxin-induced T-cell activation for SPE A and SPE B. SPE A binding was observed in L cells transfected with DQw1 genes. By contrast, it was not detected in L cells transfected with DR2, DR4, DPw4 or DP(Cp63) genes. All the transfectants supported SPE-induced interleukin-2 production by human T cells except the DP transfectants for SPE B. Levels of accessory cell activity were low in the DP transfectants induced by stimulation with SPE A and in the DR and DP transfectants induced by SPE B. The results indicate that SPE A and SPE B bind well to DQ molecules, less well to DR molecules, and very weakly to DP molecules.

Animals↗
PubMed1992

Molecular population genetic evidence of horizontal spread of two alleles of the pyrogenic exotoxin C gene (speC) among pathogenic clones of Streptococcus pyogenes.

It has recently been demonstrated that the bacteriophage-borne gene (speC) encoding pyrogenic exotoxin C is harbored by phylogenetic lineages representing virtually the entire breadth of genomic differentiation present in the species Streptococcus pyogenes (J. M. Musser, A. R. Hauser, M. H. Kim, P. M. Schlievert, K. Nelson, and R. K. Selander, Proc. Natl. Acad. Sci. USA 88:2668-2672, 1991). To determine whether the speC genes occurring in association with divergent chromosomal genotypes (clones) are identical or represent a group of allelic variants, we sequenced speC from 23 S. pyogenes strains representing 15 clones identified by multilocus enzyme electrophoresis. Two alleles of speC are present in natural populations, and each allele occurs in clones that are well differentiated in overall chromosomal character; in one case, isolates of a single clone had different speC alleles. We interpret these patterns of toxin allele-clone distribution as evidence of occasional episodes of speC horizontal dissemination, presumably by bacteriophage-mediated gene transfer and recombination.

Alleles↗
PubMed1994

Similar cytokine induction profiles of a novel streptococcal exotoxin, MF, and pyrogenic exotoxins A and B.

The cytokine production induced by a newly discovered streptococcal exotoxin, MF, and the pyrogenic exotoxins SpeA and SpeB was determined by in vitro stimulation of peripheral blood mononuclear cells (PBMCs) obtained from healthy blood donors. The induction and kinetics of interleukin-1 alpha (IL-1 alpha), IL-1 beta, IL-1 receptor antagonist, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-10, gamma interferon, tumor necrosis factor alpha (TNF-alpha), TNF-beta, and granulocyte-macrophage colony-stimulating factor were studied at the single-cell level by use of cytokine-specific monoclonal antibodies and intracellular immunofluorescent juxtanuclear staining. The cytokine-producing cells, with the exception of IL-1-expressing cells, had a characteristic morphology generated by the accumulation of cytokines in the Golgi organelle. MF, SpeA, and SpeB induced a massive gamma interferon and TNF-beta response in 10 to 16% of the PBMCs after 48 to 96 h of cell stimulation. In contrast, IL-2 and TNF-alpha production was detected in only 1 to 3% of the PBMCs. The induction of a lymphocyte TH2 phenotype response, including production of IL-3, IL-4, IL-5, and IL-10, was weak. However, the monokines, IL-1 alpha, IL-1 beta, IL-1 receptor antagonist, and IL-8, were consistently found and gradually produced, peaking at 24 h in approximately 5 to 8% of the PBMCs. MF showed extensive cytokine- and proliferation-inducing capacities equal to those of SpeA and SpeB, which suggests that MF is also a superantigen. A marked interindividual variation could be noted both in the proliferative response and in the cytokine induction of lymphocytes isolated from different individuals, which may be one explanation for the varying clinical severity noticed during group A streptococcal infections.

Bacterial Proteins↗
PubMed1996

Temperature regulation of the streptococcal pyrogenic exotoxin A-encoding gene (speA).

The gene encoding the bacterial superantigen streptococcal pyrogenic exotoxin A is often found in streptococcal strains associated with the recently described streptococcal toxic shock syndrome. Here we demonstrate that this gene is expressed at approximate fourfold higher levels in cells grown at 37 degrees C when compared to cells grown at 26 degrees C. This suggests there is increased production of this toxin when Streptococcus pyogenes is found in infections of the soft tissues and bloodstream, as opposed to S. pyogenes that have not breached the epithelial layers and are living on the surface of the skin.

Exotoxins↗
PubMed1996

Analysis of the superantigenic activity of mutant and allelic forms of streptococcal pyrogenic exotoxin A.

Infections with Streptococcus pyogenes (group A streptococcus) can result in the recently described streptococcal toxic shock syndrome (STSS), which is characterized by rashes, hypotension, multiorgan failure, and a high mortality rate. S. pyogenes isolates associated with STSS usually produce streptococcal pyrogenic exotoxin A (SpeA), a bacterial superantigen capable of stimulating host immune cells. Most of the symptoms of STSS are believed to result from cytokine release by the stimulated cells. To better understand the pathogenesis of STSS, we began studies on the SpeA-immune cell interaction. We generated 20 mutant forms of SpeA1 (SpeA encoded by allele 1), and the mutant toxins were analyzed for mitogenic stimulation of human peripheral blood mononuclear cells, affinity for class II major histocompatibility complex molecules (DQ), and disulfide bond formation. Residues necessary for each of these functions were identified. There are four alleles of speA, and STSS strains usually contain either allele 2 or allele 3. The product of allele 2, SpeA2, had slightly higher affinity for the class II MHC molecule compared with SpeA1 but not significantly greater mitogenic activity. SpeA3, however, was significantly increased in mitogenic activity and affinity for class II MHC compared with SpeA1. Thus, we have evidence that the toxin encoded by some of the highly virulent S. pyogenes STSS-associated isolates is a more active form of SpeA.

Alleles↗
PubMed1997

The role of nitric oxide in experimental murine sepsis due to pyrogenic exotoxin A-producing Streptococcus pyogenes.

Nitric oxide (NO) produced by inducible NO synthase (iNOS) mediates hypotension in endotoxemia. In this study, NO induction by a toxin-producing Streptococcus pyogenes isolate, H250, and by recombinant streptococcal pyrogenic exotoxin A (rSPEA) has been examined, both in vitro and in vivo. Streptococcal supernatants, but not rSPEA, induce production of nitrite by murine macrophages when both are coincubated with gamma interferon. Intraperitoneal injection of rSPEA did not cause significant production of NO. However, an elevated level of nitrate in serum was detected in a model of streptococcal fasciitis due to live H250. iNOS was localized to Kupffer cells, hepatocytes, and renal tubular cells by immunostaining. Administration of a NOS inhibitor, N(G)-monomethyl-L-arginine (L-NMMA), reduced peak concentrations of nitrate in serum but did not affect survival. NO is induced by H250, both in vitro and in vivo, mainly via SPEA-independent mechanisms. In this model, iNOS is expressed predominantly in the liver. Furthermore, in this model L-NMMA is not protective.

Animals↗
PubMed1998

Role of streptococcal pyrogenic exotoxin B in the mouse model of group A streptococcal infection.

Streptococcal pyrogenic exotoxin B (SPE B) is a cysteine protease produced by Streptococcus pyogenes. In this study, the differences in virulence between protease-positive clinical isolates and their protease-negative mutants were examined in a mouse model. Isogenic protease-negative mutants were constructed by homologous recombination, using integrational plasmids to disrupt the speB gene. These mutants caused less mortality and tissue damage than protease-positive strains when inoculated into BALB/c mice via air pouch, suggesting that SPE B cysteine protease plays an important role in the pathogenesis of S. pyogenes infection. Reconstitution of SPE B in the air pouches increased the mortality of mice receiving the speB mutant strain. Infiltrated cell numbers in the exudates from the air pouches of mice infected with SPE B-producing S. pyogenes were higher than those from mice infected with protease-negative mutants at 12 h. However, despite pretreatment with vinblastine to deplete neutrophils, injection of protease-positive bacteria still resulted in severe tissue injury, indicating that neutrophil infiltration may not be the major factor involved in SPE B-enhanced tissue damage. The role of SPE B was further confirmed by demonstrating that SPE B immunization of mice conferred protection from challenge with a lethal dose of protease-positive bacteria.

Animals↗
PubMed1999

A two-component regulatory system, CsrR-CsrS, represses expression of three Streptococcus pyogenes virulence factors, hyaluronic acid capsule, streptolysin S, and pyrogenic exotoxin B.

Certain Tn916 insertions in the chromosome of an M1-type, nonmucoid Streptococcus pyogenes isolate (MGAS166) were previously shown to result in stable mucoidy with increased expression of the capsular synthetic genes. The transposon insertions in these strains are directly upstream of an apparent operon encoding a two-component regulatory system, designated csrR-csrS. Compared with MGAS166, these mucoid mutants are more hemolytic and cause significantly more tissue damage in a murine model of skin infection. To extend these observations, we constructed an in-frame deletion in the gene encoding the response regulator, csrR, and we evaluated the expression of other known S. pyogenes virulence factors. We discovered that csrR mutants have enhanced transcription of sagA, a gene associated with streptolysin S (SLS) and speB, the gene encoding pyrogenic exotoxin B (SpeB). The mutants also express substantially higher SLS activity and SpeB antigen in late-exponential-phase cultures. There is no change in expression of emm, scpA, sic, or cpa (genes encoding other S. pyogenes virulence factors). CsrR- strains but not the wild-type parental strain produce necrotizing lesions in a mouse model of subcutaneous infection. A double mutant with deletions in both csrR and the capsular synthesis genes caused fewer and smaller necrotic skin lesions than the csrR mutants. However, this nonmucoid csrR strain was more likely than the wild type to yield necrotic lesions, suggesting that mucoidy contributes to virulence in this model of infection but that there are other csrR-regulated factors involved in the production of necrotic lesions.

Animals↗
PubMed2000

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.

Animals↗
PubMed1973

Enhanced immune response after immunosuppression by Streptococcal pyrogenic exotoxin.

Streptococcal pyrogenic exotoxin (SPE) was shown previously to be a potent immunosuppressant. This paper presents data documenting that immunosuppression frequently was followed by elevated antibody and plaque-forming cell levels as noted in our earlier report. The data are interpreted as a differential effect of SPE upon suppressor T cells and secreting B cells.

Antibody Formation↗
PubMed2003

The zinc-dependent major histocompatibility complex class II binding site of streptococcal pyrogenic exotoxin C is critical for maximal superantigen function and toxic activity.

The cocrystal structure of streptococcal pyrogenic exotoxin C (SPE C) with HLA-DR2a (DRA*0101,DRB5*0101) revealed a zinc-dependent interaction site through residues 167, 201, and 203 on SPE C and residue 81 on the beta-chain of HLA-DR2a (DRA*0101,DRB5*0101). Mutation of these SPE C residues resulted in dramatically reduced biological activities. Thus, the zinc-dependent major histocompatibility complex II binding site is critical for maximal biological function of SPE C.

Animals↗
PubMed2004

Streptococcal pyrogenic exotoxin B-induced apoptosis in a549 cells is mediated by a receptor- and mitochondrion-dependent pathway.

It has been shown that streptococcal pyrogenic exotoxin B (SPE B) can induce cells to undergo apoptosis. The present study is to dissect the role of SPE B protease and SPE B protein in the apoptotic process of A549 cells and to elucidate the SPE B-induced apoptotic pathway. Recombinant SPE B (rSPE B) and C192S, a mutant of SPE B without protease activity, were expressed in Escherichia coli and purified by using an affinity column. The apoptosis of A549 cells was assayed by propidium iodide staining, followed by flow cytometry analysis. Our results showed that SPE B induced apoptosis in a dose-dependent manner, whereas C192S did not. When cells were pretreated with rSPE B (2 mug/ml) for as briefly as 5 min and then incubated with C192S of 28 kDa, an apoptosis that is proportional to the period of pretreatment was observed but not with C192S of 42 kDa. These results suggest that the extracellular protease activity of rSPE B is required for the initiation of apoptosis and that the size of SPE B is important for an effective induction of apoptosis. The time course analysis revealed that molecules activated in apoptosis were in the following order: caspase-8 (1.5 h), t-Bid (2.5 h), Bax (3 h), cytochrome c release (6 h), caspase-9 (7 h), and caspase-3 (8 h). The overexpression of Bcl-2 inhibited depolarization of mitochondrial membrane, cytochrome c release, and apoptosis. The results of the present study suggest that SPE B-induced apoptosis is mediated through a receptor-like mechanism and a mitochondrion-dependent pathway.

Apoptosis↗
PubMed2004

Proapoptotic effect of proteolytic activation of matrix metalloproteinases by Streptococcus pyogenes thiol proteinase (Streptococcus pyrogenic exotoxin B).

Streptococcus pyogenes thiol proteinase, also known as streptococcal pyrogenic exotoxin B (SpeB), has been suggested to be a major virulence factor in S. pyogenes infection. SpeB was reported to induce apoptosis of host cells, but its mechanism of action is not yet fully understood. In this study, we examined the involvement of matrix metalloproteinases (MMPs) in SpeB-induced apoptosis. We first developed a large-scale preparation of recombinant SpeB and precursors of human MMP-9 and -2 (proMMPs) by using Escherichia coli Rosetta (DE3)pLysS and baculovirus-insect cell expression systems, respectively. Treatment with SpeB induced effective proteolytic activation of both proMMP-9 and -2. When RAW264 murine macrophages were incubated with SpeB-activated proMMP-9, the level of tumor necrosis factor alpha (TNF-alpha) in conditioned medium (CM), assessed by an enzyme immunoassay, was elevated. This increase was completely inhibited by addition of the MMP inhibitor SI-27 to the cell culture. The CM also produced marked induction of apoptosis of U937 human monocytic cells. Similarly, soluble Fas ligand (sFasL) was detected in CM of cultures of SW480 cells expressing FasL after treatment with SpeB-activated proMMPs; this CM also induced apoptosis in U937 cells. SpeB had a direct effect as well and caused the release of TNF-alpha and sFasL from the cells. SpeB-dependent production of MMP-9 and -2 and proapoptotic molecules (TNF-alpha and sFasL) was evident in a murine model of severe invasive S. pyogenes infection. These results suggest that SpeB or SpeB-activated MMPs contribute to tissue damage and streptococcal invasion in the host via extracellular release of TNF-alpha and sFasL.

Animals↗
PubMed1986

Bacteriophage involvement in group A streptococcal pyrogenic exotoxin A production.

Lysogenic conversion has been suggested as a mechanism of control of group A streptococcal pyrogenic exotoxin type A production. Digestion of DNA from two converting bacteriophages, 3GL16 and T12, with a variety of restriction endonucleases yielded identical DNA fragments upon electrophoresis in agarose gels. Several known A toxin-positive strains that did not appear to produce converting phage upon induction were analyzed for toxin and phage DNA. Strains, including NY5, 594, and C203S, were shown by hybridization studies to carry the A toxin gene (speA) adjacent to chromosomally inserted phage fragments, homologous to phage T12 DNA, which may represent defective converting phages. The phage T12 att site mapped adjacent to speA. These data suggest that phage T12 acquired the A toxin gene from the bacterial genome. All streptococcal strains tested that were A toxin negative by Ouchterlony immunodiffusion failed to show any hybridization to speA-specific probes.

Bacterial Proteins↗
PubMed1989

Bacteriophage association of streptococcal pyrogenic exotoxin type C.

A gene encoding streptococcal pyrogenic exotoxin type C (SPE C) was isolated from bacteriophage DNA derived from Streptococcus pyogenes CS112. The gene, designated speC2, was shown to reside near the phage attachment site of phage CS112. A restriction endonuclease map of the CS112 phage was generated, and the location and orientation of the speC2 gene were determined. Hybridization analyses of eight SPE C-producing strains revealed restriction fragment length polymorphism of the speC gene-containing DNA fragments and further showed that each speC was linked to a common CS112 phage-derived DNA fragment.

Bacterial Toxins↗