Streptococcal Superantigens Research Articles

Peripheral blood lymphocytes from psoriatic patients are hyporesponsive to beta-streptococcal superantigens.

The strong association of acute guttate psoriasis and streptococcal throat infection, together with the preferential use of T cells expressing a particular T-cell receptor, has suggested a role for bacterial superantigens in the pathogenesis of psoriasis. We examined the proliferative responses of peripheral blood lymphocytes (PBLs), obtained from patients with psoriasis and from healthy controls, to streptococcal superantigens, cytoplasmic membrane-associated protein (CAP) and secretion-type CAP (SCAP), isolated from group A, beta-haemolytic streptococci. PBLs from patients with psoriasis showed significantly less response to SCAP and CAP than those from healthy controls. Because there was no difference between psoriatic patients and controls in the proliferative response of PBLs to staphylococcal enterotoxin A or E (SEA, SEE) or the mitogen phytohaemagglutinin (PHA), these findings strongly suggest that the reduced reactivity to the streptococcal superantigens seems to reflect anergy of a population of PBLs to the superantigens. As the CAP used in the present study stimulates V beta 8 T cells selectively, we further examined the proliferation of V beta 8 T cells after such stimulation using flow cytometry. V beta 8 T cells obtained from three of four psoriatic patients failed to proliferate in the presence of CAP, whereas they proliferated vigorously in the presence of SEE, which activates V beta 8 T cells, confirming the specific hyporesponsiveness of PBLs from psoriatic patients to streptococcal superantigens. We then determined the effects of serum factors on the suppressed response of PBLs to the streptococcal superantigens with SCAP or CAP. It was partially restored when PBLs were cultured with sera obtained from healthy subjects, although the responses were still significantly lower than those of the healthy controls. In contrast, psoriatic sera markedly suppressed the proliferative response of PBLs from healthy controls to CAP or SCAP, but showed no suppression of the proliferative response of PBLs to SEA. Because these findings suggest the presence of specific inhibitory factors in psoriatic sera, we examined whether the inhibitory effect was caused by antisuperantigen antibody. However, no significant increase was detected in antibody titre to CAP in psoriatic sera, as has been noted in sera from patients with poststreptococcal glomerulonephritis. The present results show for the first time the hyporesponsiveness of PBLs to streptococcal superantigens and the presence of serum inhibitors that specifically inhibit T-cell response to the superantigens in psoriatic patients. These findings suggest a pathological role for streptococcal infections in the pathogenesis of psoriasis.

Mechanisms of pathogenesis of staphylococcal and streptococcal superantigens.

In order to infect a host successfully, to access nutrients, and to promote the progression of disease, many pathogenic bacteria, such as the staphylococci and streptococci, produce exoproteins which enhance microbial virulence. Among these proteins is the family of toxins known today as the superantigens (SAg). This family includes the pyrogenic toxin SAg (PTSAg), such as the staphylococcal enterotoxins (SE, serotypes A-E, G, H), group A streptococcal pyrogenic exotoxins (SPE, serotypes A-C and possibly F), streptococcal SAg (SSA), and staphylococcal toxic shock syndrome toxin (TSST)-1. The following is a review of the biochemistry, structure, and mechanisms of pathogenicity of the PTSAg and the shared and unique properties of each. The properties of other relevant superantigenic proteins such as the staphylococcal exfoliative toxins (ETA, ETB) will also be discussed.

Serologic evidence that streptococcal superantigens are not involved in the pathogenesis of Kawasaki disease.

Kawasaki disease (KD) is an acute multisystem vasculitis of unknown etiology and is associated with marked activation of T cells and monocyte macrophages, leading to the assumption that superantigens are involved in its pathogenesis. To determine if an association exists between streptococcal superantigens and KD, we examined serum antibody responses to superantigens in sera from 50 paired acute and convalescent KD patients using purified recombinant streptococcal superantigens, such as SPEA, SPEC, SSA and MF. We found a very low frequency of detection of anti-superantigen antibodies by ELISA and no marked IgG seroconversion to each superantigen, indicating the absence of a serological relationship between toxin-producing streptococcal infection and the onset of KD.

Superantigenicity of helper T-cell mitogen (SPM-2) isolated from culture supernatants of Streptococcus pyogenes.

A superantigen (Streptococcus pyogenes mitogen-2; SPM-2) that stimulates human helper T cells bearing unique types of variable domains of T-cell receptor beta-chain (TCR V beta) was isolated from the culture supernatant of S. pyogenes strain T12. The active molecule isolated by diethylaminoethyl (DEAE)-cellulose chromatography and isoelectric focusing was a protein with a molecular weight (MW) of 29,000 and isoelectric point (pl) of 6.0. This new superantigen was found to activate preferentially V beta 4+, 7+, and 8+ T cells, whereas recombinant streptococcal pyrogenic exotoxin A and C activated V beta 12+ and V beta 2+ T cells, respectively, as determined by flow cytometry and reverse transcriptase-polymerase chain reaction (RT-PCR) methods. This proliferative response was significantly inhibited by anti-HLA-DR monoclonal antibody, and required paraformaldehyde-fixed antigen-presenting cells (APC), indicating that this action is dependent on major histocompatibility complex (MHC) class II molecules without processing. Analysis of the amino-terminal amino acid sequence of the molecule failed to find any identical or significantly homologous proteins. We have previously reported that cytoplasmic membrane-associated protein (CAP), a streptococcal superantigen isolated from the cell membranes of S. pyogenes T12 strain, stimulated mainly V beta 8+ T cells. Both SPM-2 and CAP preferentially stimulated helper T cells, and rabbit antiserum against SPM-2 completely neutralized the T-cell-stimulating activities of CAP, suggesting that SPM-2 and CAP belong to a family of streptococcal mitogenic proteins. The SPM-2 activity with stimulation of V beta 8+ T cells was detected extensively in the culture fluids of group A streptococci, but not in those of other streptococcal species, including groups B and D streptococci, and most of the activities detected were completely inhibited by anti-SPM-2 serum. These results indicate that SPM-2 may be a newly discovered superantigen molecule, which can be commonly synthesized by group A streptococci.

Streptococcal Superantigen, Mitogenic Factor, and Pyrogenic Exotoxin B Expressed by Streptococcus pyogenes

Streptococcus pyogenes is a Gram-positive human bacterial pathogen that causes pharyngitis, tonsillitis, skin infections (impetigo, erysipelis, and other forms of pyoderma), acute rheumatic fever (ARF), scarlet fever (SF), poststreptococcal glomerulonephritis (PSGN), a streptococcal toxic shock syndrome (STSS), and necrotizing fasciitis. These infections are some of the most economically and medically important conditions that affect humans. For example, globally, ARF is the most common cause of pediatric heart disease. It is estimated that in India more than six million school-aged children suffer from rheumatic heart disease (1). In the United States, “sore throat” is the third most common reason for physician office visits and S. pyogenes is recovered from about 30% of children with this complaint (2). It has been estimated that there are 25–35 million cases of streptococcal pharyngitis per year in the United States, and these infections cause 1–2 billion dollars per year in direct health care costs (3,4). Although the continued great morbidity and mortality caused by S. pyogenes in developing nations, the significant health care financial burden attributable to group A streptococci in the United States, and increasing levels of antibiotic resistance (5), have highlighted the need for a fuller understanding of the molecular pathogenesis of streptococcal infection, it has been the relatively recent intercontinental increase in streptococcal disease frequency and severity (6,7) that has resulted in renewed interest in S. pyogenes virulence factors and host-parasite interactions.

Cytokine profile of human peripheral blood mononucleated cells stimulated with a novel streptococcal superantigen, SPEA, SPEC and group A streptococcal cells.

Among bacterial derived proteins, the superantigens possess the unique property to interact polyclonally with a high proportion of T lymphocytes bearing appropriate Vß motifs of the T cell receptor. In the presence of antigen presenting cells expressing MHC class II molecules the superantigens stimulate in a cascade of activation signals the production of a broad array of cytokines. Clinical and experimental observations strongly suggest that the pathophysiological features of streptococcal toxic shock syndrome (STSS) are mainly due to massive release of proinflammatory cytokines. Furthermore, the production of Th1 and Th2 derived cytokines by superantigen stimulated immune cells in vitro strongly indicates that these effectors have the capacity to play an important role in pathogenic disorders other than acute inflammatory processes by interfering with the regulation of Th1 and Th2 cytokine network in direction of either cell mediated, allergic or autoimmune responses. The precise role of bacterial superantigen involvement in the pathogenesis of chronic sequelae remains to be clarified.KeywordsCytokine ProfileHuman Peripheral Blood Mononuclear CellToxic Shock SyndromePathophysiological FeatureStreptococcal Toxic Shock SyndromeThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

An outbreak of invasive group A streptococcal disease associated with high carriage rates of the invasive clone among school-aged children

To determine if a common strain of group A streptococcus was responsible for an outbreak of invasive streptococcal disease in southeastern Minnesota and to determine whether this strain was prevalent among residents of this area during the outbreak who either had streptococcal pharyngitis or were asymptomatic streptococcal carriers. Pharyngeal culture survey and case-contact evaluation. Three adjacent counties in southeastern Minnesota defined as the outbreak area. Outbreak period, January 1 through March 31, 1995. Seven patients with invasive streptococcal infection, 1249 patients (adults and children) with sore throat who resided in the outbreak area, children from an elementary school located in 1 community where the majority of invasive cases occurred, and 896 students from 3 schools located in Minnesota counties outside the outbreak area. Pulsed-field gel electrophoresis (DNA fingerprinting) of group A streptococcal isolates obtained from patients with invasive disease, throat swabs of patients with sore throat, and throat swabs of asymptomatic school-aged children. All patients with outbreak-associated invasive disease had group A streptococcal isolates that were indistinguishable by pulsed-field gel electrophoresis. Additional testing showed that these isolates carried significant virulence factors including pyrogenic exotoxin A and streptococcal superantigen. Five of these 7 patients with invasive disease had underlying medical conditions; 4 developed toxic shock syndrome and died (case fatality, 57%). The outbreak-associated group A streptococcal clone was found in 69 (26.5%) of the 260 patients with sore throat from whom group A streptococcus was isolated. The frequency of the outbreak clone among pharyngeal carriers from the 3 schools outside the outbreak area was significantly less (range, 0%-10%) than in children from the school in the outbreak area (78%; relative risk, 29; 95% confidence interval, 11.1-78.1; P<.001). Four of the 7 patients with outbreak-associated disease had contact with children who attended the school in the outbreak area. A single clone of group A streptococcus was responsible for 7 cases of invasive streptococcal disease during an outbreak in Minnesota and for a significant number of pharyngitis cases that also occurred during the outbreak. Invasive disease occurred most frequently in persons with underlying medical conditions. This outbreak was also associated with increased carriage rates of the invasive streptococcal clone among community school-aged children. Cases of invasive group A streptococcal infection may therefore reflect the tip of the iceberg with regard to the burden of colonization of a specific invasive streptococcal clone in a community.

Cases of streptococcal toxic shock syndrome in Germany since 1989, toxin (mitogen) expression, content of toxin genes and relation to M-serotypes.

A worldwide increase of severe invasive Streptococcus pyogenes infections beginning from the skin or soft tissue infections (cellulitis, fasciitis, myositis) has been reported during the last decade. This so-called Streptococcal Toxic Shock Syndrome (STSS) is considered as a resurgence of the historically known fulminant or toxic scarlet fever. It is characterized by fever, multiorgan failure, skin desquamation and occurs mainly in young/middle-aged, otherwise healthy adults within hours of the onset of symptoms. The onset of STSS seems to be related to the efficacy of bacterial products and host factors (particularly the immune state) of the patients. Bacterial products which are believed to be causally involved in these infections are the M-protein, different enzymes and the various erythrogenic toxins (ET or SPE), the streptococcal mitogenicity factor MF, the streptococcal superantigen (SSA), low molecular weight proteins (LMP) and the cell bound streptococcal membrane protein (CAP) (1). Our interest in this investigation has been focused on the examination of STSS strains isolated in Germany since 1989, when the first case was reported. More than 70 S. pyogenes strains isolated from cases of STSS in Germany and a group of control strains isolated from healthy people or from patients suffering from harmless streptococcal diseases were typed and investigated with regard to content of speA, speB, speC, and MF genes, toxin expression (ETA and ETC) and for mitogenic activity in culture supernatants. The results of this investigation are presented and discussed.KeywordsBacterial ProductSoft Tissue InfectionMitogenic ActivityToxin GeneGene DetectionThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Streptococcal Superantigens and Their Role in the Pathogenesis of Severe Infections

AbstractGroup A streptococci (GAS), produce a large number of extracellular proteins which contribute to virulence, among these are the streptococcal pyrogenic exotoxins (spe), SpeA, SpeB, SpeC, and the recently identified SpeF and streptococcal superantigen SSA. The streptococcal as well as the staphylococcal exotoxins have all been shown to belong to the rapidly growing group of microbial proteins designated superantigens because of their potency to stimulate massive T-cell proliferation and subsequent release of large amounts of cytokines. Production of cytokines constitute an important part of the host defense against infectious agents; however, at high concentrations these immunomodulatory substances can be harmful. The superantigens have been implied in the pathogenesis of staphylococcal and streptococcal induced toxic shock and the host humoral response against the Spe's have been shown to be of importance for the clinical manifestation of streptococcal infection. Patients who developed severe stre...

An Outbreak of Invasive Group A Streptococcal Disease Associated With High Carriage Rates of the Invasive Clone Among School-aged Children

To determine if a common strain of group A streptococcus was responsible for an outbreak of invasive streptococcal disease in southeastern Minnesota and to determine whether this strain was prevalent among residents of this area during the outbreak who either had streptococcal pharyngitis or were asymptomatic streptococcal carriers.Pharyngeal culture survey and case-contact evaluation.Three adjacent counties in southeastern Minnesota defined as the outbreak area. Outbreak period, January 1 through March 31, 1995.Seven patients with invasive streptococcal infection, 1249 patients (adults and children) with sore throat who resided in the outbreak area, children from an elementary school located in 1 community where the majority of invasive cases occurred, and 896 students from 3 schools located in Minnesota counties outside the outbreak area.Pulsed-field gel electrophoresis (DNA fingerprinting) of group A streptococcal isolates obtained from patients with invasive disease, throat swabs of patients with sore throat, and throat swabs of asymptomatic school-aged children.All patients with outbreak-associated invasive disease had group A streptococcal isolates that were indistinguishable by pulsed-field gel electrophoresis. Additional testing showed that these isolates carried significant virulence factors including pyrogenic exotoxin A and streptococcal superantigen. Five of these 7 patients with invasive disease had underlying medical conditions; 4 developed toxic shock syndrome and died (case fatality, 57%). The outbreak-associated group A streptococcal clone was found in 69 (26.5%) of the 260 patients with sore throat from whom group A streptococcus was isolated. The frequency of the outbreak clone among pharyngeal carriers from the 3 schools outside the outbreak area was significantly less (range, 0%-10%) than in children from the school in the outbreak area (78%; relative risk, 29; 95% confidence interval, 11.1-78.1; P<.001). Four of the 7 patients with outbreak-associated disease had contact with children who attended the school in the outbreak area.A single clone of group A streptococcus was responsible for 7 cases of invasive streptococcal disease during an outbreak in Minnesota and for a significant number of pharyngitis cases that also occurred during the outbreak. Invasive disease occurred most frequently in persons with underlying medical conditions. This outbreak was also associated with increased carriage rates of the invasive streptococcal clone among community school-aged children. Cases of invasive group A streptococcal infection may therefore reflect the tip of the iceberg with regard to the burden of colonization of a specific invasive streptococcal clone in a community.

Novel superantigens from streptococcal toxic shock syndrome Streptococcus pyogenes isolates.

The incidence of severe invasive group A streptococcal infections (GAS) and streptococcal toxic shock syndrome (STSS) continues to rise (1), but the reason for this impressive change in epidemiology and clinical manifestation of GAS infections remains a mystery. Streptococcal superantigens (SAgs) have been implicated in the pathogenesis of STSS, but no single SAg can be directly associated with disease (reviewed in 2). A recent outbreak of severe GAS infections in Canada since November 1994 has led to the identification of 95 patients with invasive GAS infections or STSS. These invasive cases could be classified into severe and nonsevere invasive disease based on the clinical symptoms. The severe cases included patients with STSS and necrotizing fasciitis whereas the nonsevere cases were those with no sign of hypotension, organ failure, deep soft tissue involvement or shock. In this study we examined the distribution of genes encoding known streptococcal pyrogenic exotoxins (Spes), which belong to the family of microbial superantigens, among isolates from severe and nonsevere disease. In addition, we investigated the pattern of SAg production by GAS strains isolated from these patients. We report that invasive strains of S. pyogenes produce a number of known Spes as well as novel SAgs that remain to be fully characterized.

Altered orientation of streptococcal superantigen (SSA) on HLA-DR1 allows unconventional regions to contribute to SSA Vbeta specificity.

Crystallographic studies reveal even distantly related bacterial superantigens (SAg) to adopt a common structural topology. Mutational analyses confirm that this shared folding pattern often confers a conserved function to analogous residues in different SAg, albeit with specificities for particular TCR or MHC class II molecules. It was thus surprising that the streptococcal SAg SSA differed from related SAg in the location of its Vbeta-determining residues. Because it seemed unlikely that SSA would deviate significantly from an SAg-like topology, we hypothesized that variations in SSA Vbeta-determining regions might result from differences in SSA-MHC class II interactions relative to other SAg during SSA presentation to the TCR. Comparison of the DR1-binding properties of SSA with its closest homologue SEB found different amino acid positions within SAg primary sequences to contribute to SSA-DR1 and SEB-DR1 interactions, and suggested that SSA bound DR1 with an altered orientation relative to SEB. The common involvement of DR1 alpha39K, however, predicted that the two SAg bound overlapping sites on DR1. Nevertheless, SSA and SEB did not effectively cross-compete for DR1 binding and had opposite patterns of DR1-binding affinity in the presence of distinct DR1-expressing cell lines. The data thus suggest that SSA and SEB bind not only with different orientations on DR1, but may bind preferentially to distinct DR1 subsets delineated by cell-specific factors. Differences in orientation of SSA on DR1 and/or interaction of SSA with particular DR1 subsets may explain why unconventional regions influence SSA TCR Vbeta specificity.

Definition of the mitogenic factor (MF) as a novel streptococcal superantigen that is different from streptococcal pyrogenic exotoxins A, B, and C.

Human T cell activation by recombinant mitogenic factor (rMF) was investigated in comparison with that by recombinant streptococcal pyrogenic exotoxins (rSPE) A, B, and C. Recombinant MF, rSPEA, and rSPEC were mitogenic for peripheral blood mononuclear cells (PBMC), whereas rSPEB was not. Recombinant MF required only HLA-DR for the stimulation of PBMC, as determined using monoclonal antibodies (mAb) to HLA class II molecules and the mouse L cells transfected with HLA class II molecules. Recombinant SPEA and rSPEC required HLA-DR or HLA-DQ molecule. Recombinant MF selectively stimulated V beta 2, V beta 7, V beta 8, V beta 18 and V beta 21-bearing T cells, whereas rSPEA and rSPEC activated V beta 2 and V beta 6-bearing T cells as evaluated by the quantitative T cell receptor (TCR) analytical method. No clonality was observed in the nucleotide sequences of complementarity determining region 3 of TCR V beta in T cells responding to rMF. The profiles of cytokine production by PBMC in response to rMF, rSPEA, and rSPEC were quite similar. In summary, these results demonstrate that both HLA class II molecules and the TCR V beta required for rMF-mediated T cell activation are distinct from those required for rSPEA or rSPEC-mediated activation. Therefore, the MF is a novel streptococcal super-antigen which is different from SPEA, SPEB, and SPEC.

Species-dependent post-translational modification and position 2 allelism: effects on streptococcal superantigen SSA structure and V beta specificity.

Epidemiologic and molecular population genetic analyses support a role for superantigens (SAg) in the pathogenesis of severe staphylococcal and streptococcal infections. To investigate how variations in SAg structure influence immunomodulatory activity, we examined the biochemical and functional properties of two allelic variants of streptococcal SAg SSA that differ at position 2. Mass spectrometry revealed both recombinant (Escherichia coli) and native (Streptococcus pyogenes) SSA allelic variants to have significantly larger molecular masses than predicted by primary sequence alone and provided evidence that the proteins were modified by the addition of biochemical moieties, a phenomenon that has not been described for related SAg. Furthermore, the molecular masses of native and recombinant SSA were not the same; SSA was differentially post-translationally modified by the two bacterial genera. The substitution of E. coli-dependent processing for that of S. pyogenes altered both protease digestion and V beta specificity, suggesting that recombinant SAg from E. coli may not accurately represent the native toxin. In addition, the observation that SSA allelic variants differed in V beta specificity supports a role for position 2 in SSA-TCR interactions. That SSA position 2 contributes to V beta specificity could not have been predicted from functional or crystallographic studies of other SAg and suggests that SSA may adopt unique interactions with TCR and/or MHC class II molecules. Determining the structural basis for these differences should offer additional clues to the manner in which SAg exert their effects on the immune system during infection and may allow the designing of SAg mutants with specific quantitative and qualitative immunomodulatory properties.

Isolation of a new superantigen with potent mitogenic activity to murine T cells from Streptococcus pyogenes

A mitogenic substance on murine lymphocytes was detected in the culture supernate of Streptococcus pyogenes type 12 strain. This substance had a molecular weight of 28 000 and p I 9.2, and was designated as S. pyogenes mitogen (SPM). The proliferative response of C3H HeN spleen cells began at 1 ng ml −1 and reached a maximal response at 100 ng ml −1 of SPM for 4 days culture. Anti-Thy 1.2 mAb and complement-treated spleen cells abrogated the proliferative response to any dose of SPM. Although the anti-major histocompatibility complex class I mAbs had no blocking effect on proliferation by SPM, this proliferation was substantially inhibited by the addition of either anti-I-A or anti-I-E mAb, and complete inhibition was produced by the addition of both mAbs. Fixed antigen-presenting cells still induced T cell proliferation by SPM. A significant expansion of T cells bearing Vβ13 T-cell receptor was observed up to 73% among the Thy1.2 + cells in cultures stimulated with SPM, indicating expansion in a Vβ-specific manner. Immunoblotting of IEF-separated proteins showed that anti-streptococcal pyrogenic exotoxin (SPE) C reacted with a protein of p I 6.9 and anti-SPEB did not show any reactivity. SPEA was reported to expand Vβ8.1 and 8.2 bearing murine T cells, and SPM did not. SPM also exhibited potent mitogenic activity on human T cells and Vβ21 + T cells were selectively expanded. These results lead to the conclusion that SPM was neither SPEA, B nor C, but a new protein belonging to a group of streptococcal superantigens with activity on not only human but also murine lymphocytes.

Isolation of a new superantigen with potent mitogenic activity to murine T cells from Streptococcus pyogenes.

A mitogenic substance on murine lymphocytes was detected in the culture supernate of Streptococcus pyogenes type 12 strain. This substance had a molecular weight of 28,000 and pI 9.2, and was designated as S. pyogenes mitogen (SPM). The proliferative response of C3H/HeN spleen cells began at 1 ng ml-1 and reached a maximal response at 100 ng ml-1 of SPM for 4 days culture. Anti-Thy 1.2 mAb and complement-treated spleen cells abrogated the proliferative response to any dose of SPM. Although the anti-major histocompatibility complex class 1 mAbs had no blocking effect on proliferation by SPM, this proliferation was substantially inhibited by the addition of either anti-I-A or anti-I-E mAb, and complete inhibition was produced by the addition of both mAbs. Fixed antigen-presenting cells still induced T cell proliferation by SPM. A significant expansion of T cells bearing V beta 13 T-cell receptor was observed up to 73% among the Thy 1.2+ cells in cultures stimulated with SPM, indicating expansion in a V beta-specific manner. Immunoblotting of IEF-separated proteins showed that anti-streptococcal pyrogenic exotoxin (SPE) C reacted with a protein of pI 6.9 and anti-SPEB did not show any reactivity. SPEA was reported to expand V beta 8.1 and 8.2 bearing murine T cells, and SPM did not. SPM also exhibited potent mitogenic activity on human T cells and V beta 21+ T cells were selectively expanded. These results lead to the conclusion that SPM was neither SPEA, B nor C, but a new protein belonging to a group of streptococcal superantigens with activity on not only human but also murine lymphocytes.

Plasma from patients with severe invasive group A streptococcal infections treated with normal polyspecific IgG inhibits streptococcal superantigen-induced T cell proliferation and cytokine production.

Previous studies have suggested a central role for superantigen-induced immune responses in the pathogenesis of streptococcal toxic shock syndrome. The production of streptococcal superantigens by clinical group A streptococcal (GAS) isolates was studied, and the ability of plasma collected from patients with severe invasive GAS infections to neutralize the proliferative- and cytokine-inducing activities of these superantigens was investigated. Overnight culture supernatants from all GAS isolates obtained from patients with invasive disease were found to contain superantigenic activity, as evident from their ability to drive potent T cell proliferation, induce high production of cytokines, and stimulate T cells in a V beta-specific manner. Twelve patients with severe invasive GAS infections, including 11 streptococcal toxic shock syndrome cases and one necrotizing fasciitis without shock, were treated with i.v. infusions of normal polyspecific Ig (IVIG). Plasma samples collected from each patient before and after IVIG administration were analyzed for their ability to neutralize the activity of streptococcal superantigens produced by the GAS isolate that caused their disease. In all IVIG-treated patients, the capacity to neutralize the superantigenic activity, produced by their respective GAS isolate or by purified streptococcal pyrogenic exotoxins, increased in plasma following IVIG administration. Of particular clinical relevance, post-IVIG plasma from each patient completely blocked cytokine production elicited by their respective GAS culture supernatants or by purified streptococcal pyrogenic exotoxins. This study shows that IVIG treatment confers in vivo inhibitory activity against a large array of streptococcal superantigens and suggests that IVIG may be useful in the treatment of severe invasive streptococcal infections.

Phylogenetic distribution of streptococcal superantigen SSA allelic variants provides evidence for horizontal transfer of ssa within Streptococcus pyogenes.

Phylogenetic analyses recently found the gene encoding the streptococcal superantigen SSA of Streptococcus pyogenes to occur in several well-differentiated clones comprising 10 (12.5%) of 80 clonal lineages examined. To determine if distinct clonal lineages carried the same ssa coding sequence or harbored a group of allelic variants, ssa was sequenced from 23 S. pyogenes strains representing the 10 clones identified by multilocus enzyme electrophoresis. Three alleles of ssa were found in natural populations of S. pyogenes. ssa-1 and ssa-3 differed by a single synonymous substitution in codon 94; both encoded SSA-1. Each of these alleles was present in phylogenetically diverse clones that had not shared a recent common ancestor. ssa-2 was present in a single clonal lineage. It was identical to ssa-3 at codon 94 but had a nonsynonymous substitution at codon 28 that changed the second amino acid of the mature protein from serine to arginine. This substitution altered the predicted isoelectric point and affected the apparent molecular mass during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Despite sequence variation both upstream of and within the ssa locus, all ssa-positive lineages expressed either SSA-1 or SSA-2. The observed patterns of ssa allele-clone distribution provide evidence for individual incidences of horizontal transfer and recombination of ssa among distinct group A streptococcal lineages. Although the extensive homology of SSA to the staphylococcal superantigen SEB raises the possibility of intergeneric gene transfer, a search for ssa in 68 genetically diverse clones of Staphylococcus aureus did not identify the gene. Moreover, the absence of ssa among 119 representative strains of Lancefield group B, C, or G streptococci suggests that ssa is confined to S. pyogenes.

There is no disease-specific role for streptococci-responsive synovial T lymphocytes in the pathogenesis of psoriatic arthritis.

The initiation or exacerbation of psoriasis vulgaris is associated with infections by group A streptococci. T lymphocytes specific for streptococcal antigens or expressing a restricted, for streptococcal superantigens typical T cell receptor Vbeta chain repertoire have been described in psoriatic skin lesions. The aim of our study was, therefore, to clarify whether streptococci-reactive T lymphocytes played a role in the pathogenesis of psoriatic arthritis (PsA), and by which antigens they might be stimulated. Synovial membrane mononuclear cells from patients with PsA and other arthropathies, separated by collagenase digestion, were expanded in interleukin-2-supplemented medium and subsequently cloned in a representative cloning procedure. The T cell lines and about 30% of the T cell clones proliferated in response to preparations of group A streptococci but not to other bacteria as tested by [3H]thymidine incorporation assays. Interestingly, they did not proliferate in response to exotoxin-negative streptococci, but did so in response to the streptococcal pyrogenic exotoxins A and C, which are known to be superantigens. Accordingly, no HLA-DR restriction was seen for the proliferative response. The remaining 70% of the established T cell clones did not react to an antigen of group A streptococci. Our results show that in patients with PsA, osteoarthritis or rheumatoid arthritis a significant number of synovial T lymphocytes were responsive to streptococcal superantigens, but not to conventional streptococcal antigens. A disease-specific role of streptococci-reactive T lymphocytes in the pathogenesis of PsA is, therefore, unlikely.

Reactivity of mouse T-cell hybridomas expressing human Vbeta gene segments with staphylococcal and streptococcal superantigens.

A panel of 15 mouse T-cell hybridomas, each expressing a different human Vbeta gene segment (hVbeta) in an otherwise mouse T-cell receptor (i.e., mouse alpha chain and CD3 complex), was constructed by transfection of hVbeta/mouse Cbeta chimeric T-cell receptor (TCR)-beta genes into a mouse T-cell hybridoma recipient lacking the endogenous TCR-beta chain. Several qualities that are conferred by the hVbeta chain of the TCR are retained in the chimeric human-mouse TCR complex: a large panel of hVbeta-specific antibodies specifically stained the hVbeta expressed by the mouse T-cell hybridomas. Moreover, hVbeta-transfected mouse cells could readily produce interleukin 2 when stimulated by superantigens presented by antigen-presenting cells. These characteristics made it possible to refine the reactivity of 17 superantigen preparations with the available transfected Vbetas. Each superantigen gave a characteristic pattern of reactivity on the transfectants. Positive reactivities with some of these transfectants, which differ only by the expressed hVbeta, demonstrate unambiguously the superantigenic character of a protein or fraction and its potential to react with the corresponding Vbetas. Therefore, these hVbeta-transfected cells constituted a valuable tool for determining "specificity fingerprints" of known or putative superantigens. First, commonly used, commercially available superantigens such as staphylococcal enterotoxin B and toxic shock syndrome toxin-1 (TSST-1) showed additional Vbeta reactivities, compared with those of their recombinant counterparts. This stresses the importance of using defined preparations of superantigens for the definition of Vbeta specificities. Second, the stimulatory pattern of a strain of Streptococcus pyogenes demonstrated that this strain, unlike others, produces a potent Vbeta 8-specific superantigen that is an yet undefined at the molecular level.