Streptococcal Pyrogenic Exotoxin Type Research Articles

T-cell receptor Vβ8 for detection of biologically active streptococcal pyrogenic exotoxin type C

Streptococcus pyogenes is an important human pathogen, commonly spread by airborne droplets but also by ingestion of contaminated food. Apart from causing infection, this pathogen produces 13 distinct types of streptococcal pyrogenic exotoxins (SPEs). The current method for detection cannot distinguish between the biologically active form of SPEs that has been reported to cause foodborne outbreaks and the inactivated toxin which poses no health risk. To measure the biological activity of SPE-C, one such toxin that was linked to foodborne outbreaks associated with milk and milk products, we developed a cell-based assay that can discern between biologically active and inactive SPE-C. To the best of our knowledge, this is the first showing that SPE-C activates T-cells expressing Vβ8. With this finding, we used a T-cell line natively expressing Vβ8 that was genetically engineered to also express the luciferase reporter gene under the regulation of nuclear factor of activated T-cells response element (NFAT-RE) in combination with a B-cell line to present the rSPE-C toxin via MHC class II to the Vβ8 TCR in an assay to detect and to discern between biologically active and inactive rSPE-C. By using this system, we demonstrated that SPE-C induced significant IL-2 secretion after 72 h and visible light emission after only 5 h, doubling by 24 h. We utilize this finding to assess the specificity of the assay and the effect of pasteurization on SPE-C activity. We observed no cross reactivity with SPE-B and significant loss of SPE-C biologically activity in spiked PBS while SPE-C spiked into milk is heat stable. Once SPE-C has formed, it is infeasible to eliminate it from milk by thermal treatment.

Seroreactivity against Streptococcal DRS (Distantly Related to SIC) Protein Is a Predictor for End-Stage Renal Failure

We hypothesized that immunoreactivity against antigens from nephritic strains of Streptococcus pyogenes may be elevated in patients with end-stage renal failure (ESRF). Additionally, we investigated whether a difference in seroreactivity exists between nonindigenous and indigenous (Aboriginal/Torres Strait Islander) patients. To examine these possibilities, antibodies against potentially nephritogenic proteins, streptokinase (Ska1) (from M1), streptococcal pyrogenic exotoxin type B (SpeB) (from M1), the streptococcal inhibitor of complement-mediated cell lysis (SIC) (from M1) and its two variants, closely related to SIC (CRS) (from M57) and distantly related to SIC (DRS) (from M12) were determined in 66 patients and 31 healthy controls by enzyme-linked immunosorbent assays. A significantly higher proportion of patients compared to controls were seropositive to Ska1 (P = 0.004), DRS (P = 0.0003), CRS (P = 0.001), and SIC (P = 0.018). Regression analysis showed that seroreactivity to DRS (r(2) = 0.85, P = 0.001) predicted the development of ESRF and that being diabetic was positively associated with being an ESRF patient (r(2) = 0.37, P < 0.0001) and being indigenous (r(2) = 0.47, P < 0.0001). These results suggest that these ESRF patients were exposed to strains of S. pyogenes that secrete Ska1, DRS, CRS, and SIC and may have pathological significance. No significant difference was observed between the indigenous patients and nonindigenous patients.

Molecular characterisation of group A streptococci from invasive and non-invasive disease episodes in Belgium during 1993-1994.

Five hundred clinical group A streptococcal (GAS) isolates were collected in Belgium during the period 1 Nov. 1993 to 31 Oct. 1994. Clinical and laboratory data were recorded and isolates were characterised. The presence of the genes encoding streptococcal pyrogenic exotoxin types A (speA), B (speB), C (speC), F (speF) and streptococcal superantigen (ssa) were determined by PCR to target specific sequences. These isolates were also emm-typed and analysed by pulsed-field gel electrophoresis (PFGE) of genomic macrorestriction fragments with the enzyme SmaI. In total, 136 unrelated GAS PFGE types were identified and genetic diversity was clearly demonstrated. Two GAS PFGE types predominated; a first PFGE type comprised 66 (13.2%) emm1 isolates characterised by speA-, speB+, speC-, speF+ and ssa-; the second PFGE type comprised 44 (8.8%) emm12 isolates characterised by speA-, speB+, speC+ (or speC-), speF+ and ssa-. Indistinguishable PFGE types were observed among both invasive and non-invasive isolates. Ten different PFGE types were found among 11 streptococcal toxic shock syndrome (STSS) isolates, and five of these lacked speA. Twenty-five (34.7%) of 72 invasive isolates gave negative results for speA, speC and ssa. This retrospective study confirmed the observation that the dissemination of one specific clone cannot be associated with invasive GAS disease and posed a question regarding the role of SPE A as a major virulence factor. Other streptococcal virulence factors in conjunction with host factors may determine the outcome of invasive GAS infection.

Invasive group A streptococcal disease in Taiwan is not associated with the presence of streptococcal pyrogenic exotoxin genes.

We reviewed the clinical features of 44 patients with invasive group A streptococcal (GAS) disease who were treated at two teaching hospitals in southern Taiwan from 1991 to 1994. Genes encoding streptococcal pyrogenic exotoxin types A (speA), B (speB), C (speC), and F (speF) and serotypes of M1, M6, and M12 were determined by polymerase chain reaction to target specific sequences in the 44 isolates recovered from these patients and in 28 isolates recovered from upper respiratory sites in 28 additional patients during the study period. The protease activity of these isolates was tested by using the casein plate method. Of the 44 patients with invasive diseases, 25 (57%) had no obvious underlying diseases, and 14 (32%) had preexisting neoplastic diseases or had previously used steroids. Twenty-five patients (57%) presented with cellulitis or necrotizing fasciitis, 24 (55%) had bacteremia, and eight (18%) had streptococcal toxic shock syndrome (STSS). Eight patients (18%) died of invasive GAS disease; seven had STSS, and seven had underlying diseases. All eight patients died within 48 hours after hospitalization. The presence of speA, speC, or speF was not implicated in any particular clinical syndrome in patients with invasive GAS disease. High-level protease activity and the M1 serotype of the isolates were significantly associated with the clinical signs of STSS and with mortality. M1 serotype and protease activity, as well as host immune status, might play significant roles in the pathogenesis of invasive GAS disease in Taiwan.

Outbreak of scarlet fever at a hospital day care centre: analysis of strain relatedness with phenotypic and genotypic characteristics

An outbreak of scarlet fever involving 12 children occurred at a hospital day care centre from February to March 1996. Twenty-five throat isolates of Streptococcus pyogenes (GAS, group A streptococcus) available from 24 children, including 10 children with scarlet fever and 14 asymptomatic carriers, and one asymptomatic staff member were studied for the presence of genes encoding streptococcal pyrogenic exotoxin types A ( speA), B ( speB), and C ( speC) and for protease activity. Antimicrobial susceptibilities using the E-test, cluster analysis by cellular fatty acid composition and random amplified polymorphic DNA (RAPD) patterns by means of arbitrarily-primed polymerase chain reaction (APPCR) of the isolates were performed to investigate the outbreak. Only one isolate from an asymptomatic child possessed the speA gene. All isolates possessed the speB gene and 24 (96%) isolates were positive for the speC gene. There was no difference in protease activity between isolates from children with scarlet fever and from asymptomatic carriers. Thirteen isolates (10 recovered from children with scarlet fever, two from asymptomatic children, and one from the staff member) were considered to be the same strain according to the identical antimicrobial susceptibility profile and RAPD patterns and were also considered to be similar by cluster analysis of fatty acid composition. These findings suggest that the outbreak was caused by a unique clone of GAS. We conclude that RAPD typing and cluster analysis by cellular fatty acids composition both provide a powerful tool for epidemiological investigation of GAS infections.

Expression of T-cell receptor V beta 2 and type 1 helper T-cell-related cytokine mRNA in streptococcal pyrogenic exotoxin-C-activated human peripheral blood mononuclear cells.

Streptococcal pyrogenic exotoxin type C (SPE C) is a member of the bacterial superantigens that are potent stimulants of T cells. We expressed SPE C in Escherichia coli and characterized its selective stimulation properties on human T cells bearing specific V beta chains of T-cell receptors (TCRs). Cytokine profiles induced by SPE C were also examined. Recombinant SPE C significantly enhanced proliferation of human peripheral blood mononuclear cells (PBMCs) at concentrations as low as 10(-12)-10(-14)M. Reverse transcription of RNA, from SPE-C-stimulated PBMCs followed by polymerase chain reaction, revealed selective induction of TCR V beta 2 chain expression. SPE C raised the mRNA level of type 1 helper T cell (TH1) related cytokines, such as interferon gamma (IFN-gamma), interleukin 2 (IL-2), and tumor necrosis factor beta (TNF beta). The expression of TNF alpha was also increased. In contrast, the increase in mRNA levels of the p35 small fragment of IL-12 and type 2 helper T cell (TH2) related cytokines (i.e., IL-4 and IL-10) was not significantly affected by SPE C. The mRNA level of proinflammatory cytokine IL-6 was increased marginally. Consistent with the mRNA accumulation, protein concentrations of IFN gamma, IL-2, and TNF were increased in SPE-C-stimulated PBMCs, but IL-4 was not. From these results, we conclude that the stimuli of SPE C preferentially causes the TH1 responses in human T cells bearing TCR V beta 2.

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.

The Characterization of Two New Low Molecular Weight Proteins (LMPs) from Streptococcus pyogenes

Two novel extracellular mitogenic substances were isolated from Streptococcus pyogenes strain NY-5 and characterized. The purification steps involved an initial enrichment of the proteins from culture supernatant by silica gel adsorption, followed by ion exchange chromatography and gel filtration. The purified materials were homogeneous in SDS-PAGE, showed estimated molecular weights of 12 kD and isoelectric points of 4.7 and 4.3, respectively. Both proteins (LMP-12k-4.3pI and LMP-12k-4.7pI) demonstrated lymphocyte transformation activity at a concentration of 0.1 microgram/ml. The LMP-12k-4.7pI showed a 69.2% homology of the amino acid sequence with that of a phosphocarrier protein of Staphylococcus aureus and with a total identity in the active centre. The same protein was also isolated from streptococcal group C strain H46A with an N-terminal amino acid sequence being identical. The LMP-12k-4.7pI demonstrated biochemical properties identical with those of the earlier described streptococcal pyrogenic exotoxin type D. The LMP-12k-4.3pI did not show such a clear relation to other functional proteins.

Distinct T-cell receptor V beta gene usage by human T lymphocytes stimulated with the streptococcal pyrogenic exotoxins and pep M5 protein

A number of streptococcal products, including the streptococcal pyrogenic exotoxin (SPE) types A, B, and C as well as a 22-kDa fragment of M type 5 protein (pep M5), are potent stimulants of human T-lymphocyte blastogenesis and belong to the newly designated family of superantigens. The V beta usage of human T cells stimulated with these toxins was investigated by using the polymerase chain reaction. We demonstrate that SPE A, B, and C as well as pep M5 stimulate the proliferation of T cells in a dose-dependent manner. pep M5 stimulates cells bearing V beta 2, 4, and 8 elements of the T-cell receptor (TCR), whereas SPE A stimulates TCR V beta 2-, 12-, 14-, and 15-bearing cells. SPE B stimulated only cells expressing TCR V beta 8 elements, while SPE C stimulated cells expressing V beta 1, 2, 5.1, and 10. These studies reveal that the preferential usage of particular V beta elements is distinct for these different superantigens, which may be important in the pathogenesis of various streptococcal diseases.

Molecular analysis of pyrogenic exotoxins from Streptococcus pyogenes isolates associated with toxic shock-like syndrome

Toxic shock-like syndrome (TSLS) is characterized by hypotension or shock, fever, multiorgan system involvement, and a concurrent group A streptococcal infection. We analyzed 34 streptococcal strains isolated from patients with clinically well-documented TSLS for their pyrogenic toxin profiles and M-protein types. Although strains of nine different M types were represented in the sample, 74% of the isolates were of either M type 1 or 3. It was determined that 53% produced streptococcal pyrogenic exotoxin type A under in vitro growth conditions and that 85% contained the gene encoding this toxin. These values are in contrast to the published value of 15% for the incidence of this gene in a sample of general group A streptococcal isolates. As has been found with all group A streptococci examined to date, regardless of disease association, 100% of TSLS-associated isolates contained the gene encoding pyrogenic exotoxin type B. This toxin was detectably produced by 59% of isolates. The gene encoding pyrogenic toxin type C was found in only 21% of isolates. We conclude that the pyrogenic exotoxin type A gene is associated with group A streptococcal strains isolated from patients with TSLS and may play a causative role in this illness. However, other factors are also likely to be important, since not all strains from patients with TSLS contained the A toxin gene.

Activation of murine T cells by streptococcal pyrogenic exotoxin type A. Requirement for MHC class II molecules on accessory cells and identification of V beta elements in T cell receptor of toxin-reactive T cells.

We investigated the mechanisms of murine T cell activation by streptococcal pyrogenic exotoxin type A (SPE A), focusing on the role of MHC class II molecules on accessory cells (AC) and V beta usage in alpha beta TCR of SPE A-reactive T cells in comparison with staphylococcal enterotoxin B-reactive T cells. L cells transfected with I-Ab genes functioned as effective AC for SPE A-induced responses by C57BL/6 T cells, proliferation, and IL-2 production, but control L cells were not effective AC. Anti-I-Ab mAb inhibited the SPE A-induced responses. Staphylococcal enterotoxin B-induced C57BL/6 T cell blasts were composed of cells bearing V beta 3, members of the V beta 8 family, and V beta 11. Most of the SPE A-induced T cell blasts (about 80%) bore V beta 8.2. mAb reactive to V beta 8.2 markedly inhibited SPE A-induced T cell responses. Apparently, SPE A activates mainly T cells bearing V beta 8.2 in physical association with MHC class II molecules expressed on AC. We also discuss the pathogenic activities of SPE A in relation to toxic shock syndrome.

Nucleotide sequence of the streptococcal pyrogenic exotoxin type B gene and relationship between the toxin and the streptococcal proteinase precursor

The streptococcal pyrogenic exotoxin (SPE) type B-encoding structural gene, speB, was subcloned from a 4.5-kilobase streptococcal DNA insert onto a 2.4-kilobase insert, which was then sequenced. Studies indicated that a 1,194-base-pair open reading frame encoded a 398-amino-acid protein. Removal of the putative signal peptide resulted in a mature protein with 371 residues (molecular weight, 40,314), which was subsequently proteolyzed to yield a 253-residue breakdown product (molecular weight, 27,588). This processing was confirmed by amino-terminal sequencing of both the 40,314-molecular-weight protein and the breakdown product. Monte Carlo analysis indicated that SPE B was relatively dissimilar to other members of the pyrogenic toxin family that also includes SPEs A and C, toxic shock syndrome toxin 1, and the staphylococcal enterotoxins. Comparison with the published amino acid sequence of streptococcal proteinase precursor as well as DNA hybridization experiments indicated that SPE B is a variant of this protein even though the particular gene sequenced did not encode a proteolytically active molecule.

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.

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.

Complete secretion of activable bovine prochymosin by genetically engineered L forms of Proteus mirabilis

To circumvent problems encountered in the synthesis of active chymosin in a number of bacteria and fungi, a recombinant DNA L-form expression system that directed the complete secretion of fully activable prochymosin into the extracellular culture medium was developed. The expression plasmid constructions involved the in-frame fusion of prochymosin cDNA minus codons 1 to 4 to streptococcal pyrogenic exotoxin type A gene (speA') sequences, including the speA promoter, ribosomal binding site, and signal sequence and five codons of mature SpeA. Secretion of fusion prochymosin enzymatically and immunologically indistinguishable from bovine prochymosin was achieved after transformation of two stable protoplast type L-form strains derived from Proteus mirabilis. The secreted proenzyme was converted by autocatalytic processing to chymosin showing milk-clotting activity. In controlled laboratory fermentation processes, a maximum specific rate of activable prochymosin synthesis of 0.57 x 10(-3)/h was determined from the time courses of biomass dry weight and product formation. Yields as high as 40 +/- 10 micrograms/ml were obtained in the cell-free culture fluid of strain L99 carrying a naturally altered expression plasmid of increased segregational stability. The expression-secretion system described may be generally useful for production of recombinant mammalian proteins synthesized intracellularly as aberrantly folded insoluble aggregates.

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.

Cloning and characterization of the gene, speC, for pyrogenic exotoxin type C from Streptococcus pyogenes.

The structural gene of streptococcal pyrogenic exotoxin type C (SPE C) was cloned from the chromosome of Streptococcus pyogenes strain T18P into Escherichia coli using pBR328 as the vector plasmid. Subcloning enabled the localization of the gene (speC) to a 1.7 kb fragment. Partially purified E. coli-derived SPE C and purified streptococcal-derived SPE C, were shown to have the same molecular weight (23,800) and biological activities. A DNA probe, prepared from cloned speC, cross-hybridized with the structural genes of SPE A and SPE B indicating relatedness at the nucleotide level. The speC-derived probe also hybridized to a fragment of CS112 bacteriophage DNA containing the phage attachment site.

Cloning and expression of streptococcal pyrogenic exotoxin A and staphylococcal toxic shock syndrome toxin-1 in Bacillus subtilis.

The genes encoding streptococcal pyrogenic exotoxin type A (SPE A) and staphylococcal toxic shock syndrome toxin-1 (TSST-1) were stably cloned and expressed in Bacillus subtilis. In the non-pathogenic Bacillus background, the recombinant speA clone expressed 32-fold more SPE A than the native streptococcus, and similarly, the recombinant plasmid harboring tst expressed 4-fold more TSST-1 in Bacillus than in the native Staphylococcus aureus. The Bacillus-derived products were secreted into the culture fluid, were resistant to proteolytic degradation and their biological activities mimicked native preparations.

Streptococcal pyrogenic exotoxin type A (scarlet fever toxin) is related to Staphylococcus aureus enterotoxin B.

The nucleotide sequence of the gene encoding group A streptococcal pyrogenic exotoxin type A (SPE A) was determined by the dideoxy chain termination method. The first 30 residues of the translation product represented a hydrophobic signal peptide. The mature protein was 220 amino acids in length and had a molecular weight of 25,805. It has significant protein sequence homology with Staphylococcus aureus enterotoxin B but not with other proteins in the Dayhoff library.

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.