Crystal structure of the superantigen staphylococcal enterotoxin type A.

Staphylococcal enterotoxins are prototype superantigens characterized by their ability to bind to major histocompatibility complex (MHC) class II molecules and subsequently activate a large fraction of T-lymphocytes. The crystal structure of staphylococcal enterotoxin type A (SEA), a 27 kDa monomeric protein, was determined to 1.9 A resolution with an R-factor of 19.9% by multiple isomorphous replacement. SEA is a two domain protein composed of a beta-barrel and a beta-grasp motif demonstrating the same general structure as staphylococcal enterotoxins SEB and TSST-1. Unique for SEA, however, is a Zn2+ coordination site involved in MHC class II binding. Four amino acids including Ser1, His187, His225 and Asp227 were found to be involved in direct coordination of the metal ion. SEA is the first Zn2+ binding enterotoxin that has been structurally determined.     

Staphylococcal enterotoxin superantigens

Staphylococcal enterotoxins (SE) are a family of structurally related proteins that are produced by Staphylococcus aureus. They play a role in the pathogenesis of food poisoning and are the most potent activators of T lymphocytes known. The receptors for SE on antigen-presenting cells are major histocompatibility complex class II molecules. Recent studies have shown that a complex of SE and major histocompatibility complex class II molecules is required for binding to the variable region of the T cell antigen receptor beta-chain. SE mitogenic activity is dependent on induction of interleukin 2, which may be intimately involved in the mechanism of SE toxicity. The minor lymphocyte-stimulating "endogenous" self-superantigen has recently been shown to be a retroviral gene product, so that this too is apparently a microbial superantigen. An understanding of the mechanism of action of these microbial superantigens has implications for normal and pathological immune functions.     

Effect of isotypic and allotypic variations of MHC class II molecules on staphylococcal enterotoxin presentation to murine T cells.

Staphylococcal enterotoxins (SE) are known to be potent T cell activators, stimulating +/- proliferation and lymphokine production. These toxins have recently have been termed "superantigens" because of their ability to bind directly to class II molecules forming a ligand that interacts with particular V beta gene elements within the TCR complex. This interaction between SE and MHC class II molecules plays a central role in toxin-induced mitogenesis. In the present study we have examined the effect of polymorphism on the ability of MHC class II molecules to bind and present SE. Through the use of H-2 congenic mouse strains, it was possible to look directly at haplotype differences within the MHC and their effect on SE presentation to a panel of responsive V beta-bearing T cells. The results demonstrate that toxin presentation by class II-bearing accessory cells to murine T cells is greatly affected by polymorphisms within the H-2 complex. Toxin-pulsed accessory cells obtained from mice of an H-2k and H-2u haplotype were found to be less efficient in activating a variety of T cell clones and hybridomas. However, one T cell clone responded similarly to the enterotoxins presented on all H-2 haplotypes, suggesting that differences in responses of T cells are not simply a function of the degree of binding of these toxins to various class II molecules. Neutralization analysis with monoclonal anti-class II antibodies demonstrates that both I-A and I-E molecules play a significant role in SEA and SEB presentation to murine T cells. These results suggest that the differential activation of T cells by a particular enterotoxin may reflect a difference in recognition of an SE:class II ligand by a surface T cell receptor complex.     

Clonal heterogeneity of superantigen reactivity in human V beta 6+ T cell clones. Limited contributions of V beta sequence polymorphisms.

Superantigens encoded in the genome or released by bacteria have been identified as potent modulators of the murine immune system. High frequencies of mature or immature T cells are activated or intrathymically deleted when superantigens cross-link MHC class II molecules and the V beta element of the TCR. The V beta specificity discriminates superantigens from polyclonal T cell stimulators as well as specific Ag and determines the immunomodulatory role in shaping the T cell repertoire. A similar regulatory function of superantigens in the human immune system is less well established. Here, we have studied a series of human T cell clones sharing the TCR V beta 6 element and describe a surprising heterogeneity in their responsiveness to staphylococcal exotoxins. The V beta 6 gene segment had the ability to respond to all staphylococcal enterotoxins (SE); however, for individual T cell clones, there was a clear predominance of SE C3 reactivity compared to SE B and SE C2. The clonal heterogeneity of SE responsiveness did not correlate to sequence polymorphisms in the fourth hypervariable region of the V beta 6 segment, the presumptive binding site for superantigens. Superantigen reactivity was crucially influenced by the presenting HLA-DR molecule, especially when the superantigen served as a coligand, enhancing or suppressing the Ag-specific activation of the TCR. These data suggest that the correlation between human TCR V beta gene segments and superantigen responses is not stringent. Potential intrathymic deletion mechanisms controlled by superantigens may be less selective in humans and may result in a leakiness influenced by the host HLA-DR molecules.     

Control of T cell responses to staphylococcal enterotoxins by stimulator cell MHC class II polymorphism

The bacterial toxic mitogens or superantigens are a family of related proteins that elicit potent T cell proliferative responses. These responses require APC that express MHC class II proteins, but they are not MHC restricted and they do not depend on a processing step, presumably because these mitogens bind directly to MHC class II molecules. These mitogens stimulate T cells by interacting in an unknown way with the portion of the TCR encoded by certain V beta gene segments. In this paper, we explore the importance of MHC class II polymorphism in T cell responses to staphylococcal enterotoxins. We find that certain MHC molecules present SEB to V beta 8-bearing T cells far better than others. These data suggest that one route of host defence against bacterial toxic mitogens may be to alter MHC class II molecules so that stimulation is inhibited.     

Selective stimulation of human T cells with streptococcal erythrogenic toxins A and B

Streptococcal exotoxins have been implicated in the pathogenesis of a toxic shock-like syndrome and scarlet fever. Previous studies have demonstrated that these toxins are potent stimulators of human T cells and have structural homology to staphylococcal enterotoxins. In the current study, we investigated the mechanism by which streptococcal erythrogenic toxins type A (SPEA) and B (SPEB) activate T cells and compared it with anti-CD3 and the known "superantigen" staphylococcal enterotoxin B. SPEA was found to selectively activate T cells bearing V beta 8, V beta 12, and V beta 14, whereas SPEB selectively activated T cells bearing V beta 2 and V beta 8. Furthermore, fibroblasts transfected with MHC class II molecules were capable of presenting SPEA and SPEB to purified T cells. The T cell response to these toxins, however, was not MHC-restricted. Although the streptococcal exotoxins stimulated both CD4+ and CD8+ T cells, SPEA but not SPEB stimulated the CD4+ T cell subset proportionately more than the CD8+ T cell subset. Our results indicate that SPEA and SPEB, like the staphylococcal enterotoxins, are superantigens and suggest a mechanism by which they may mediate particular systemic syndromes associated with streptococcal infections.     

The crystal structure of staphylococcal enterotoxin type D reveals Zn2+-mediated homodimerization.

Bacterial superantigens, including the staphylococcal enterotoxins, are the most potent activators of T cells known and have been suggested as a causative factor in Gram-positive shock in humans. Staphylococcal enterotoxin D (SED) is dependent upon Zn2+ for high affinity interactions with MHC class II molecules and thus SED was co-crystallized with Zn2+. The crystal structure of SED has been determined in two different space groups, at 2.3 and 3.0 A resolution respectively. The three-dimensional structure of SED is similar to structures of other bacterial superantigens, although this study has revealed that SED has the unique capability of forming dimers in the presence of Zn2+. The high affinity Zn2+ site used in dimer formation is located on the surface of the beta-sheet in the C-terminal domain. Two bound metal ions are coordinated by residues from both molecules in the dimer interface and thus contribute directly to formation of the dimer. A second Zn2+ site is located on the surface close to the domain interface of the molecule. The unique feature of SED in forming a Zn2+-dependent homodimer seems to facilitate novel and biologically relevant multimeric interactions with MHC class II molecules, as shown by the induction of cytokine mRNA in human monocytes when exposed to SED and SED mutants.     

Staphylococcal toxins bind to different sites on HLA-DR

Staphylococcal enterotoxins (SE) and toxic shock syndrome toxin 1 (TSST-1) bind to MHC class II molecules and the toxin-class II complexes induce proliferation of T cells bearing specific V beta sequences. We have previously reported that these toxins display varying binding affinities for HLA-DR1. We now investigated whether these differences simply reflected differences in binding affinity for a single class II binding site or, at least in part, the engagement of different binding sites on the HLA-DR complex. Through competitive binding studies we show that SEB and TSST-1, which are not closely related by their amino acid sequences, bind to two different sites on HLA-DR. Both of these sites are also occupied by staphylococcal enterotoxin A (SEA), enterotoxin D (SED), and enterotoxin E (SEE) which exhibit more than 70% amino acid sequence homology. SEB and TSST-1 failed to inhibit SEA binding to HLA-DR. These studies suggest that there may be three distinct, although perhaps overlapping, binding sites on HLA-DR for these toxins. Further, although SED and SEE are similar to SEA in structure, and appear to bind the same sites on HLA-DR as SEA, they displayed significantly lower binding affinities. T cell proliferative responses to SED required a higher concentration of the toxin than SEA, probably reflecting its lower binding affinity. SEE, however, elicited T cell responses at very low concentrations, similar to SEA, despite its much lower binding affinity. Therefore, although the affinities of these toxins to MHC class II molecules appear to significantly influence the T cell responses, the effective recognition of the toxin-class II complex by the TCR may also contribute to such responses.     

Superantigens interact with MHC class II molecules outside of the antigen groove

Superantigens, including the staphylococcal enterotoxins and the minor lymphocyte stimulatory antigens, are highly potent immunostimulatory molecules, capable of activating virtually all T cells that express particular T cell receptor (TCR) variable regions. Superantigen stimulation of T lymphocytes depends on major histocompatibility complex (MHC) class II molecules, so there has been some debate as to whether superantigens interact with the antigen binding "groove" on class II complexes, just like conventional peptide antigens, or whether they bind elsewhere and serve as TCR coligands. We compared the presentation of peptide antigens and superantigens by a panel of mutant-presenting cell lines, each displaying an A kappa alpha chain with a single alanine replacement along the alpha helix proposed to form one face of the groove. The negligible effect of these 30 mutations on superantigen presentation, versus their drastic consequences for peptide presentation, prompts us to conclude that superantigens interact with MHC class II molecules outside the groove.     

Binding of staphylococcal enterotoxin A to purified murine MHC class II molecules in supported lipid bilayers

The staphylococcal enterotoxins are a family of bacterial toxins that are thought to exert their pathogenic effects by the massive activation of T lymphocytes to produce lymphokines. Activation of T cells by these toxins is dependent on MHC class II+ APC. Recent studies from a number of laboratories have implicated MHC class II proteins as the APC surface receptor for a number of the staphylococcal enterotoxins. The present report shows that staphylococcal enterotoxin A, (SEA) binds to the purified murine MHC class II molecule I-Ed reconstituted in supported planar membranes, indicating that no other cell surface proteins are required for SEA binding. The Kd for SEA binding to I-Ed was determined to be 3.5 +/- 1.6 x 10(-6) M. Specific binding of SEA to I-Ad was also observed, but the interaction was of significantly lower affinity. Binding of SEA to purified I-Ed was blocked by antibodies against both the alpha- and the beta-chain of the I-Ed molecule, but not by antibodies specific for an unrelated MHC class II protein. Binding of SEA to I-Ad was blocked by an A beta d but not by an A alpha d-specific antibody. Planar membranes containing only lipid and purified I-Ed molecules were sufficient for activation of a V beta 1 expressing T hybrid by SEA. The T cells responded to as few as 180 toxin molecules per T cell.     

Monocyte chemoattractant protein-1 production by intestinal myofibroblasts in response to staphylococcal enterotoxin a: relevance to staphylococcal enterotoxigenic disease

Food poisoning due to staphylococcal enterotoxins A and B (SEA and SEB) affects hundreds of thousands of people annually. SEA and SEB induce massive intestinal cytokine production, which is believed to be the key factor in staphylococcal enterotoxin enteropathy. MHC class II molecules are the major receptors for staphylococcal enterotoxins. We recently demonstrated that normal human subepithelial intestinal myofibroblasts (IMFs) express MHC class II molecules. We hypothesized that IMFs are among the first cells to respond to staphylococcal enterotoxins and contribute to the cytokine production associated with staphylococcal enterotoxin pathogenesis. We demonstrated here that primary cultured IMFs bind staphylococcal enterotoxins in a MHC class II-dependent fashion in vitro. We also demonstrated that staphylococcal enterotoxins can cross a CaCo-2 epithelial monolayer in coculture with IMFs and bind to the MHC class II on IMFs. IMFs responded to SEA, but not SEB, exposure with 3- to 20-fold increases in the production of proinflammatory chemokines (MCP-1, IL-8), cytokines (IL-6), and growth factors (GM-CSF and G-CSF). The SEA induction of the proinflammatory mediators by IMFs resulted from the efficient cross-linking of MHC class II molecules because cross-linking of class II MHC by biotinylated anti-HLA-DR Abs induced similar cytokine patterns. The studies presented here show that MCP-1 is central to the production of other cytokines elicited by SEA in IMFs because its neutralization with specific Abs prevented the expression of IL-6 and IL-8 by IMFs. Thus, MCP-1 may play a leading role in initiation of inflammatory injury associated with staphylococcal enterotoxigenic disease.     

Crystal structure of staphylococcal enterotoxin I (SEI) in complex with a human major histocompatibility complex class II molecule

Superantigens are bacterial or viral proteins that elicit massive T cell activation through simultaneous binding to major histocompatibility complex (MHC) class II and T cell receptors. This activation results in uncontrolled release of inflammatory cytokines, causing toxic shock. A remarkable property of superantigens, which distinguishes them from T cell receptors, is their ability to interact with multiple MHC class II alleles independently of MHC-bound peptide. Previous crystallographic studies have shown that staphylococcal and streptococcal superantigens belonging to the zinc family bind to a high affinity site on the class II beta-chain. However, the basis for promiscuous MHC recognition by zinc-dependent superantigens is not obvious, because the beta-chain is polymorphic and the MHC-bound peptide forms part of the binding interface. To understand how zinc-dependent superantigens recognize MHC, we determined the crystal structure, at 2.0 A resolution, of staphylococcal enterotoxin I bound to the human class II molecule HLA-DR1 bearing a peptide from influenza hemagglutinin. Interactions between the superantigen and DR1 beta-chain are mediated by a zinc ion, and 22% of the buried surface of peptide.MHC is contributed by the peptide. Comparison of the staphylococcal enterotoxin I.peptide.DR1 structure with ones determined previously revealed that zinc-dependent superantigens achieve promiscuous binding to MHC by targeting conservatively substituted residues of the polymorphic beta-chain. Additionally, these superantigens circumvent peptide specificity by engaging MHC-bound peptides at their conformationally conserved N-terminal regions while minimizing sequence-specific interactions with peptide residues to enhance cross-reactivity.     

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.     

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.     

Apoptosis observed in murine peritoneal macrophages treated with interferon gamma through staphylococcal enterotoxin-dependent cell-mediated cytotoxicity

The concept of superantigens is well-known and widely accepted. In this brief communication, we analyze the behaviour of antigen-presenting cells after T-cell activation by staphylococcal enterotoxin B, a representative superantigen. We tried to activate murine T cells by inflammatory mouse peritoneal macrophage in the presence of staphylococcal enterotoxin B, but no T-cell activation was observed. We, therefore, analyzed surface-specific antigens of the macrophages. They expressed insufficient amounts of MHC class II, CD80 and CD86 molecules on the surface of the cells. On the contrary, increased amounts of MHC class II and CD86 molecules on the cell surfaces were observed after incubation with interferon gamma. Interferon gamma-primed macrophages were found to be competent to activate T cells in the presence of staphylococcal enterotoxin B. To our surprise, these macrophages underwent apoptosis in parallel with T-cell activation.     

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.     

Staphylococcal Enterotoxins A,D, and E

Abstract

Staphylococcal enterotoxins (SEs) are a family of structurally related exotoxin molecules produced by certain Gram-positive Staphylococcus aureus bacterial strains. SEs are a major cause of food poisoning and are involved in bacterial Gram-positive shock in humans. SEs bind to major histocompatibility complex (MHC) class II molecules on antigen-presenting cells (APCs) and subsequently activate a large fraction, 5–20%, of T lymphocytes (1). This property has led to their classification of superantigens (SAg). The T cells are activated by SAg to proliferate and produce cytokines such as interleukin-2 (IL-2), interferon-γ (IFN-γ), and tumor necrosis factor-α and β (TNF-α and β) (2,3). Depending on origin, superantigens can be divided in two groups, viral and bacterial. For reviews see Refs. 4–8.     

Superantigen presentation by airway smooth muscle to CD4+ T lymphocytes elicits reciprocal proasthmatic changes in airway function

Microbial products serving as superantigens (SAgs) have been implicated in triggering various T cell-mediated chronic inflammatory disorders, including severe asthma. Given earlier evidence demonstrating that airway smooth muscle (ASM) cells express MHC class II molecules, we investigated whether ASM can present SAg to resting CD4(+) T cells, and further examined whether this action reciprocally elicits proasthmatic changes in ASM responsiveness. Coincubation of CD4(+) T cells with human ASM cells pulsed with the SAg, staphylococcal enterotoxin A (SEA), elicited adherence and clustering of class II and CD3 molecules at the ASM/T cell interface, indicative of immunological synapse formation, in association with T cell activation. This ASM/T cell interaction evoked up-regulated mRNA expression and pronounced release of the Th2-type cytokine, IL-13, into the coculture medium, which was MHC class II dependent. Moreover, when administering the conditioned medium from the SEA-stimulated ASM/T cell cocultures to isolated naive rabbit ASM tissues, the latter exhibited proasthmatic-like changes in their constrictor and relaxation responsiveness that were prevented by pretreating the tissues with an anti-IL-13 neutralizing Ab. Collectively, these observations are the first to demonstrate that ASM can present SAg to CD4(+) T cells, and that this MHC class II-mediated cooperative ASM/T cell interaction elicits release of IL-13 that, in turn, evokes proasthmatic changes in ASM constrictor and relaxant responsiveness. Thus, a new immuno-regulatory role for ASM is identified that potentially contributes to the pathogenesis of nonallergic (intrinsic) asthma and, accordingly, may underlie the reported association between microbial SAg exposure, T cell activation, and severe asthma.     

H2-O inhibits presentation of bacterial superantigens, but not endogenous self antigens

H2-O/HLA-DO are MHC class II accessory molecules that modulate exogenous Ag presentation. Most class II accessory molecules are expressed in all professional APC; however, H2-O is only expressed in B cells and medullary thymic epithelial cells. Because B cells present exogenous Ags and superantigens (SAgs), and medullary thymic epithelial cells are specialized APC for self Ags during negative selection in the thymus, we have hypothesized that H2-O might play a role in MHC class II-restricted SAg and self Ag presentation. In this study, we demonstrate that H2-O expression inhibits presentation of the bacterial SAgs staphylococcal enterotoxins A and B to four SAg-reactive T hybridoma cells. In contrast, H2-O has no effect on presentation of endogenous self Ags, as measured by tumorigenicity in vivo and Ag presentation to three self Ag-specific T hybridoma cells. Additional experiments suggest that H2-O inhibits presentation of exogenous Ags by both newly synthesized and recycling MHC class II molecules. These data suggest H2-O may have a physiological role in tolerance induction and SAg-mediated toxic shock.