Basic streptococcal superantigens (SPEX/SMEZ or SPEC) are responsible for the mitogenic activity of the so-called mitogenic factor (MF)

The mitogenic factor (MF) of group A streptococci has been reported to be a superantigen stimulating human T cells carrying Vbeta2, 4 and 8 and has been designated streptococcal pyrogenic exotoxin F (SPEF). MF was also shown to possess DNase activity. Here we have purified MF from culture supernatants of different Streptococcus pyogenes strains. Surprisingly, the MF preparations from different strains showed different Vbeta specificities depending on the expression of SPEC or SMEZ3 by the producing strain. Their mitogenic activity decreased upon further purification. In addition, the mitogenic activity could be only neutralized by antibodies against the basic streptococcal superantigens SPEC or SPEX (SMEZ3) but not by antibodies against MF itself although the latter were able to neutralize completely the DNase activity of MF. We found that streptodornase type B (SDB) was expressed in two molecular forms (SDBI and SDBII), differing only by one additional N-terminal arginine at SDBI. MF was found identical to the enzyme SDBII but is devoid of superantigenic properties and should no longer be called a superantigen or a pyrogenic exotoxin.     

Streptococcus dysgalactiae-derived mitogen (SDM), a novel bacterial superantigen: characterization of its biological activity and predicted tertiary structure

A mitogenic substance, designated Streptococcus dysgalactiae-derived mitogen (SDM), was purified from S. dysgalactiae culture supernatant, and the gene encoding the mitogen was cloned. Both native and recombinant SDM expressed in Escherichia coli significantly activated human V beta 1+ and V beta 23+ T cells in association with major histocompatibility complex (MHC) class II molecules on accessory cells, indicating that SDM possesses superantigenic properties. The sdm gene consists of two segments encoding a signal peptide and a mature 25 kDa protein composed of 212 amino acids. Three of 34 S. dysgalactiae strains but none of 28 Streptococcus pyogenes strains examined carried sdm. Phylogenetic analysis indicated that SDM belongs to a family distinct from established bacterial superantigens. SDM showed around 30% homology with other superantigens at the amino acid sequence level. The tertiary structure of SDM was predicted by modelling onto streptococcal pyrogenic exotoxin C and streptococcal mitogenic exotoxin Z-2, both of which share highly homologous structure-determining regions. SDM showed overall structural similarity to both these superantigens. This is the first study to characterize fully a bacterial superantigen from S. dysgalactiae.     

HLA class II polymorphisms determine responses to bacterial superantigens

The excessive immunological response triggered by microbial superantigens has been implicated in the etiology of a wide range of human diseases but has been most clearly defined for the staphylococcal and streptococcal toxic shock syndromes. Because MHC class II presentation of superantigens to T cells is not MHC-restricted, the possibility that HLA polymorphisms could influence superantigenicity, and thus clinical susceptibility to the toxicity of individual superantigens, has received little attention. In this study, we demonstrate that binding of streptococcal and staphylococcal superantigens to HLA class II is influenced by allelic differences in class II. For the superantigen streptococcal pyrogenic exotoxin A, class II binding is dependent on DQ alpha-chain polymorphisms such that HLA-DQA1*01 alpha-chains show greater binding than DQA1*03/05 alpha-chains. The functional implications of differential binding on T cell activation were investigated in various experimental systems using human T cells and murine Vbeta8.2 transgenic cells as responders. These studies showed quantitative and qualitative differences resulting from differential HLA-DQ binding. We observed changes in T cell proliferation and cytokine production, and in the Vbeta specific changes in T cell repertoire that have hitherto been regarded as a defining feature of an individual superantigen. Our observations reveal a mechanism for the different outcomes seen following infection by toxigenic bacteria.     

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

Abstract 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.     

Streptococcus pyogenes: Insight into the function of the streptococcal superantigens

The group A streptococcus produces a number of highly potent exoproteins that act as superantigens. The cascade of pro-inflammatory events that follow invasive streptococcal infection is greatly enhanced by production of such toxins, leading to profound hypotension and multi-organ failure in some cases. Superantigens such as streptococcal mitogenic exotoxin Z (SMEZ) interact with host MHC class II and the T cell receptor, leading to activation events in both cells. In vitro, these interactions lead to expansion and cytokine production by specified T cell subsets. Studies using humanized HLA class II transgenic mice and isogenic streptococcal strains have characterised the in vivo responses to superantigens produced in the context of live infection. Notwithstanding the obvious deleterious role of superantigens in toxic shock, the evolutionary advantage conferred by these toxins remains a subject of speculation.     

Structural basis for the recognition of superantigen streptococcal pyrogenic exotoxin A (SpeA1) by MHC class II molecules and T-cell receptors.

Streptococcal pyrogenic exotoxin A (SpeA) is a superantigen produced by Streptococcus pyogenes and is associated with severe infections characterized by rash, hypotension, multiorgan failure and a high mortality rate. In this study, an allelic form of this toxin, SpeA1, was crystallized with four molecules in the crystallographic asymmetric unit and its crystal structure was determined at 2.6 A resolution. The crystallographic R-factor was 19.4% (33 497 reflections) for 7031 protein atoms and 88 water molecules. The overall structure of SpeA1 is considerably similar to that of other prototype microbial superantigens, either of staphylococcal or streptococcal origin, but has greatest similarity to staphylococcal enterotoxin C (SEC). Based on structural and mutagenesis data, we have mapped several important residues on the toxin molecule, which are involved in the recognition of major histocompatibility complex (MHC) class II molecules and T-cell receptors. Also, the toxin appears to possess a potential zinc-binding site which may have implications in binding to particular MHC class II molecules. Finally, we propose models for SpeA1-MHC class II and SpeA1-T-cell receptor association and the relevance of this phenomenon to the superantigenic action of this toxin is considered.     

Structural and functional role of threonine 112 in a superantigen Staphylococcus aureus enterotoxin B.

Bacterial superantigens are potent T-cell stimulatory protein molecules produced by Staphylococcus aureus and Streptococcus pyogenes. Their superantigenic activity can be attributed to their ability to cross-link major histocompatibility complex class II molecules with T-cell receptors (TCRs) to form a tri-molecular complex. Each superantigen is known to interact with a specific V(beta) element of TCR. Staphylococcal enterotoxin B (SEB, a superantigen), a primary cause of food poisoning, is also responsible for a significant percentage of non-menstrual associated toxic shock syndrome in patients with a variety of staphylococcal infections. Structural studies have elucidated a binding cavity on the toxin molecule essential for TCR binding. To understand the crucial residues involved in binding, mutagenesis analysis was performed. Our analysis suggest that mutation of a conserved residue Thr(112) to Ser (T112S) in the binding cavity induces a selective reduction in the affinity for binding one TCR V(beta) family and can be attributed to the structural differences in the native and mutant toxins. We present a detailed comparison of the mutant structure determined at 2.0 A with the previously reported native SEB and SEB-TCR V(beta) complex structures.     

Generation of biologically active interleukin-1beta by meprin B

Interleukin-1beta (IL-1beta) is a proinflammatory cytokine that is synthesized as an inactive precursor molecule that must be proteolytically processed to generate the biologically active form. Maturation of the precursor is primarily performed by caspase-1, an intracellular cysteine protease; however, processing by other proteases has been described. Meprins are cell surface and secreted metalloproteases expressed by renal and intestinal brush-border membranes, leukocytes, and cancer cells. In this study we show that purified recombinant meprin B can process the interleukin-1beta precursor to a biologically active form. Amino-terminal sequencing and mass spectrometry analysis of the product of digestion by activated meprin B determined that proteolytic cleavage resulted in an additional six amino acids relative to the site utilized by caspase-1. The biological activity of the meprin B-cleaved cytokine was confirmed by measuring the proliferative response of helper T-cells. These results suggest that meprin may play an important role in activation of this proinflammatory cytokine in various pathophysiological conditions.     

<Emphasis Type="Italic">Staphylococcus aureus</Emphasis> enterotoxin C2 mutants: biological activity assay in vitro

Staphylococcal enterotoxin C2 (SEC2) is one member of bacterial superantigens produced by Staphylococcus aureus. It can be attributed to its superantigenic activity to cross-link major histocompatibility complex class II molecules with T-cell receptors and activate a large number of resting T cells resulting in release of massive cytokines, which will produce significant tumor inhibition in vivo and in vitro. However, it could be not broadly applied to cure malignant tumors in clinic because of emetic activity of SEC2. The aim of this study was to inactivate emetic activity of SEC2 through site-directed mutagenesis. Cys93, Cys110 and His118 were selected as substitutional sites based on the functional sites responsible for emesis. The mutated proteins were used to determine Peripheral blood mononuclear cell proliferation activity and anti-tumor activity in vitro. Results showed that these mutated proteins efficiently stimulated T cell and exhibited the same tumor-inhibition effect as SEC2. It is possible to inactivate emetic activity of SEC2 through site-directed mutagenesis and provide satisfying agents for tumor treatment in clinic.     

The SpeB virulence factor of Streptococcus pyogenes, a multifunctional secreted and cell surface molecule with strepadhesin, laminin-binding and cysteine protease activity

The interactions between pathogenic bacteria and the host need to be resolved at the molecular level in order to develop novel vaccines and drugs. We have previously identified strepadhesin, a novel glycoprotein-binding activity in Streptococcus pyogenes, which is regulated by Mga, a regulator of streptococcal virulence factors. We have now identified the protein responsible for the strepadhesin activity and find that (i) strepadhesin activity is carried by SpeB, streptococcal pyrogenic exotoxin with cysteine protease activity; (ii) SpeB carries laminin-binding activity of the bacteria; and (iii) SpeB is not only a secreted molecule but also occurs unexpectedly tightly bound to the bacterial cell surface. Thus, in contrast to the previous view of SpeB as mainly an extracellular protease, it is also present as a streptococcal surface molecule with binding activity to laminin and other glycoproteins.     

Dissociation between murine spleen cell mitogenic activity of enterotoxin contaminants and anti-tumor activity of staphylococcal protein A

Soluble staphylococcal protein A (SpA) in the form of high m.w. complexes with IgG has been shown to significantly inhibit the growth of Meth A fibrosarcomas in BALB/c mice. Although SpA reportedly is a potent T cell mitogen that can induce immune cell proliferation and production of humoral factors with anti-tumor activity, it has been suggested that mitogenic enterotoxin contaminants might be responsible for these effects. The purpose of the present study was to investigate the nature of SpA-induced cell proliferation and the relationship between mitogenicity and the anti-tumor effect that we observed in our mouse model. SpA stimulated the proliferation of a mixed population of splenic B and T cells from BALB/c mice, but activity did not require the presence of IgG in the culture medium. Furthermore, mitogenic activity could be inhibited completely by anti-SEA plus anti-SEB, but was unaffected by anti-SpA. HPLC-purified SpA was inactive while the mitogenic factor(s) had the same retention time as authentic enterotoxin and its activity was inhibited by anti-SEA and anti-SEB, but not by anti-SpA. Enterotoxin-free rSpA produced in Escherichia coli had the same IgG binding capacity as the staphylococcal product but was not mitogenic. These data indicate that SEA and SEB completely account for mitogenicity in SpA preparations. In contrast, we found that optimal concentrations of rSpA as well as crude and HPLC purified staphylococcal SpA were equally effective in inhibiting the growth of established Meth A fibrosarcomas demonstrating that SpA is responsible for antitumor activity without any apparent role for enterotoxins.     

Inhibition of interleukin-6 release and T-cell proliferation by synthetic mirror pseudo cord factor analogues in human peripheral blood mononuclear cells

Abstract The effects of synthetic alkyl ((alkyl 6-deoxy-a- d - gluco -heptopyranosyluronate) 6-deoxy-a- D -gluco-heptopyranoside) uronates, a novel type of mirror pseudo cord factor, on the in vitro modulation of interleukin-6 production and T-cell proliferation in human peripheral blood mononuclear cells, were investigated. Synthetic mirror pseudo cord factors with alkyl chains ranging from C₁₆ to C₁₈ have very weak interleukin-6-inducing capacities and lack mitogenic activities for T-cell proliferation. However, they could inhibit IL-6 release induced by sonicated Bacillus Calmette-Guérin (S-BCG), bacterial endotoxin, and phytohaemagglutinin in a dose-dependent manner. Inhibition was observed not only with mononuclear cells but also with purified monocytes. Furthermore, these synthetic compounds could suppress T-lymphocyte proliferation stimulated by sonicated Mycobacterium tuberculosis H37Rv (S-H37Rv) antigens, S-BCG antigens, as well as by recombinant 65 kDa mycobacterial heat-shock protein. In contrast, these compounds failed to inhibit the phytohaemagglutinin-induced T-cell proliferation. We conclude that the inhibition of cytokine release and T-cell proliferation by synthetic mirror pseudo cord factors was due to direct blocking of the function and/or activity of monocytes or antigen-presenting cells.     

Superantigen-induced proliferation of human CD4+CD25- T cells is followed by a switch to a functional regulatory phenotype

Bacterial superantigens are potent T cell activators. In humans they cause toxic shock and scarlet fever, and they are implicated in Kawasaki's disease, autoimmunity, atopy, and sepsis. Their function remains unknown, but it may be to impair host immune responses increasing bacterial carriage and transmission. Regulatory (CD25(+)FOXP3(+)) T cells (Tregs) play a role in controlling inflammatory responses to infection. Approximately 2% of circulating T cells are naturally occurring Tregs (nTregs). Conventional Ag stimulation of naive FOXP3(-) T cells induces Ag-specific Tregs. Polyclonal T cell activation has been shown to produce non-Ag-specific Tregs. Because superantigens are unique among microbial virulence factors in their ability to trigger polyclonal T cell activation, we wanted to determine whether superantigen stimulation of T cells could induce non-Ag-specific Tregs. We assessed the effect of superantigen stimulation of human T cells on activation, regulatory markers, and cytokine production by flow cytometry and T cell suppression assays. Stimulation of PBMCs with staphylococcal exotoxin A and streptococcal pyrogenic exotoxins A and K/L resulted in dose-dependent FOXP3 expression. Characterization of this response for streptococcal pyrogenic exotoxin K/L confirmed its Vβ specificity, that CD25(+)FOXP3(+) cells arose from CD25(-) T cells and required APCs. These cells had increased CTLA-4 and CD127 expression, typical of the recently described activated converted Treg-like cells, and exhibited functional suppressor activity comparable to nTregs. Superantigen-stimulated CD25(+)FOXP3(+) T cells expressed IL-10 at lower superantigen concentrations than was required to trigger IFN-γ production. This study provides a mechanism for bacterial evasion of the immune response through the superantigen induction of Tregs.     

Alpha and beta chains of hemoglobin inhibit production of Staphylococcus aureus exotoxins

Prior studies suggest Staphylococcus aureus exotoxins are not produced when the organism is cultured in human blood. Human blood was fractionated into plasma and water-lysed red blood cells, and it was demonstrated that mixtures of alpha and beta globins of hemoglobin (as low as 1 mug/mL) inhibited S. aureus exotoxin production while increasing production of protein A and not affecting bacterial growth. Pepsin but not trypsin digestion destroyed the ability of alpha and beta globin to inhibit exotoxin production. Exotoxin production by both methicillin-resistant and methicillin-susceptible organisms was inhibited. Production of streptococcal pyrogenic exotoxin A by Streptococcus pyogenes was unaffected by alpha and beta globin chains but was inhibited when produced in S. aureus. Use of isogenic S. aureus strains suggested the targets of alpha and beta globin chains, leading to inhibition of staphylococcal exotoxins, included the two-component system SrrA-SrrB. delta hemolysin production was also inhibited, suggesting the two-component (and quorum sensing) system AgrA-AgrC was targeted. The alpha and beta globin chains represent promising molecules to interfere with the pathogenesis of serious staphylococcal diseases.     

The mitogenic activity of human T-cell leukemia virus type I is T-cell associated and requires the CD2/LFA-3 activation pathway.

The presence of a high number of activated T cells in the bloodstream and spontaneous proliferation of peripheral blood mononuclear cells in vitro are striking characteristics of human T-cell leukemia virus type I (HTLV-I) infection. The HTLV-I regulatory protein Tax and the envelope protein gp46 have been implicated in mediating the activation process. In this study, HTLV-I-producing cell lines and purified virus from the cell lines were examined for the ability to activate peripheral blood lymphocytes (PBLs) and Jurkat cells. Antisera and monoclonal antibodies against several cellular adhesion proteins involved in T-cell activation and against viral proteins were used to identify which molecules may be participating in the activation process. First, neither virus from a T-cell line, MT2, nor virus produced from the human osteosarcoma cell line HOS/PL was able to induce PBLs to proliferate. In contrast, both fixed and irradiated HTLV-I-producing T-cell lines induced proliferation of PBLs; HOS/PL cells did not activate PBLs. Second, HTLV-I-positive T-cell lines were capable of activating interleukin-2 mRNA expression in Jurkat cells. Induction of interleukin-2 expression was inhibited by anti-CD2 and anti-lymphocyte function-associated antigen 3 (LFA-3) monoclonal antibodies but not anti-human leukocyte antigen-DR, anti-CD4, anti-LFA-1, or anti-intercellular adhesion molecule 1. Similar results were obtained with PBLs as the responder cells. Furthermore, monoclonal antibodies and antisera against various regions of the HTLV-I envelope proteins gp46 and gp21 as well as p40tax did not block activation. These data indicate that HTLV-I viral particles are not intrinsically mitogenic and that infection of target T cells is not necessary for activation. Instead, the mitogenic activity is restricted to virus-producing T cells, requires cell-to-cell contact, and may be mediated through the LFA-3/CD2 activation pathway.     

Glycerol monolaurate inhibits the effects of Gram-positive select agents on eukaryotic cells

Many exotoxins of Gram-positive bacteria, such as superantigens [staphylococcal enterotoxins, toxic shock syndrome toxin-1 (TSST-1), and streptococcal pyrogenic exotoxins] and anthrax toxin are bioterrorism agents that cause diseases by immunostimulation or cytotoxicity. Glycerol monolaurate (GML), a fatty acid monoester found naturally in humans, has been reported to prevent synthesis of Gram-positive bacterial exotoxins. This study explored the ability of GML to inhibit the effects of exotoxins on mammalian cells and prevent rabbit lethality from TSS. GML (>or=10 microg/mL) inhibited superantigen (5 microg/mL) immunoproliferation, as determined by inhibition of (3)H-thymidine incorporation into DNA of human peripheral blood mononuclear cells (1 x 10(6) cells/mL) as well as phospholipase Cgamma1, suggesting inhibition of signal transduction. The compound (20 microg/mL) prevented superantigen (100 microg/mL) induced cytokine secretion by human vaginal epithelial cells (HVECs) as measured by ELISA. GML (250 microg) inhibited rabbit lethality as a result of TSST-1 administered vaginally. GML (10 microg/mL) inhibited HVEC and macrophage cytotoxicity by anthrax toxin, prevented erythrocyte lysis by purified hemolysins (staphylococcal alpha and beta) and culture fluids containing streptococcal and Bacillus anthracis hemolysins, and was nontoxic to mammalian cells (up to 100 microg/mL) and rabbits (250 microg). GML stabilized mammalian cell membranes, because erythrocyte lysis was reduced in the presence of hypotonic aqueous solutions (0-0.05 M saline) or staphylococcal alpha- and beta-hemolysins when erythrocytes were pretreated with GML. GML may be useful in the management of Gram-positive exotoxin illnesses; its action appears to be membrane stabilization with inhibition of signal transduction.     

Sensitive enzyme-linked immunosorbent assays for the detection of bacterial superantigens and antibodies against them in human plasma

Enzyme-linked immunosorbent assays for the quantitation of bacterial superantigens, staphylococcal enterotoxins A, B and C, toxic-shock syndrome toxin-1 and streptococcal pyrogenic exotoxin A, were developed. The assays had sensitivity to quantitate these toxins to 1.4, 5.9, 16.3, 2.5 and 4.3 pg/ml, respectively, in a buffer including 50% human plasma. It takes only 150 min to complete the assays after plate preparation. Specificity of the assays agreed with those of reverse latex agglutination assay. We also developed enzyme-linked immunosorbent assays to detect antibodies against these five superantigens. The assays are expected to be significant tools for the study of superantigens in several diseases.     

Yersinia enterocolitica produces superantigenic activity.

We have recently observed that antigenic preparations from Yersinia enterocolitica are capable of inducing strong proliferative responses in normal murine spleen cell cultures. As a consequence of this observation, we evaluated whether Yersinia-derived Ag possess superantigenic activity. Stimulatory activity can be found in culture supernatants, as well as membrane and cytoplasmic fractions of Y. enterocolitica. Cell depletion studies indicate that the primary responding cell is a CD4+ T cell, which requires the presence of APC for responsiveness to Y. enterocolitica Ag. Furthermore, these APC must express MHC class II Ag, as evidenced by the fact that either antibody depletion of class II+ APC or addition of anti-class II antibodies (that block class II Ag on the surface of APC) eliminates the proliferative response. Evaluation of TCR usage by BALB/c T cells responsive to Y. enterocolitica revealed that those T cells bearing V beta 3, 6, and 11 and possibly 7 and 9 were expanded after exposure to Y. enterocolitica Ag preparations. By using a panel of T cell hybridomas, we have shown that hybridomas bearing V beta 3, 7, 8.1, 9, and 11 but not 2, 8.2, 8.3, and 13 respond to Yersinia. When cytoplasmic fractions of Y. enterocolitica were subjected to column chromatography, proliferative activity was enriched approximately 27-fold, and the elution characteristics of the active material suggest that it possesses hydrophobic regions and is, therefore, probably membrane associated. These data indicate that Y. enterocolitica produces antigenic material that has properties consistent with those of T cell superantigens.