Staphylococcus aureus isolates encode variant staphylococcal enterotoxin B proteins that are diverse in superantigenicity and lethality

Staphylococcus aureus produces superantigens (SAgs) that bind and cross-link T cells and APCs, leading to activation and proliferation of immune cells. SAgs bind to variable regions of the β-chains of T cell receptors (Vβ-TCRs), and each SAg binds a unique subset of Vβ-TCRs. This binding leads to massive cytokine production and can result in toxic shock syndrome (TSS). The most abundantly produced staphylococcal SAgs and the most common causes of staphylococcal TSS are TSS toxin-1 (TSST-1), and staphylococcal enterotoxins B and C (SEB and SEC, respectively). There are several characterized variants of humans SECs, designated SEC1-4, but only one variant of SEB has been described. Sequencing the seb genes from over 20 S. aureus isolates show there are at least five different alleles of seb, encoding forms of SEB with predicted amino acid substitutions outside of the predicted immune-cell binding regions of the SAgs. Examination of purified, variant SEBs indicates that these amino acid substitutions cause differences in proliferation of rabbit splenocytes in vitro. Additionally, the SEBs varied in lethality in a rabbit model of TSS. The SEBs were diverse in their abilities to cause proliferation of human peripheral blood mononuclear cells, and differed in their activation of subsets of T cells. A soluble, high-affinity Vβ-TCR, designed to neutralize the previously characterized variant of SEB (SEB1), was able to neutralize the variant SEBs, indicating that this high-affinity peptide may be useful in treating a variety of SEB-mediated illnesses.     

Characterization of a novel staphylococcal enterotoxin-like superantigen,a member of the group V subfamily of pyrogenic toxins

Staphylococcus aureus is an important human pathogen, causing a variety of diseases. Major virulence factors of this organism include staphylococcal enterotoxins (SEs) that cause food poisoning and toxic shock syndrome. Our study identified a novel enterotoxin-like protein that is a member of the new subfamily (group V) of pyrogenic toxin superantigens (PTSAgs) and examined its biochemical and immunobiological properties. The gene encoding the SE-like protein is directly 5' of another recently identified PTSAg, SEK. The SE-like protein had a molecular weight of 26000 and an experimentally determined isoelectric point between 7.5 and 8.0. We demonstrated that the PTSAg had many of the biological activities associated with SEs, including superantigenicity, pyrogenicity, and ability to enhance endotoxin shock, but lacked both lethality in rabbits when administered in subcutaneous miniosmotic pumps and emetic activity in monkeys. Recombinant protein stimulated human CD4 and CD8 T cells in a T cell receptor variable region, beta chain (TCRVbeta) specific manner. T cells bearing TCRVbeta 2, 5.1, and 21.3 were significantly stimulated.     

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.     

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.     

HLA transgenic mice provide evidence for a direct and dominant role of HLA class II variation in modulating the severity of streptococcal sepsis

Our epidemiologic studies on invasive Group A Streptococci (GAS) infections identified specific HLA class II haplotypes/alleles conferring high-risk or protection from streptococcal toxic shock syndrome with a strong protection conferred by the DRB1*15/DQB1*06 haplotype. We used HLA-transgenic mice to provide an in vitro and in vivo validation for the direct role of HLA class II allelic variation in streptococcal toxic shock syndrome. When splenocytes from mice expressing the protective HLA-DQB1*06 (DQ6) allele were stimulated with a mixture of streptococcal superantigens (SAgs), secreted by the prevalent M1T1 strain, both proliferative and cytokine responses were significantly lower than those of splenocytes from mice expressing the neutral DRB1*0402/DQB1*0302 (DR4/DQ8) alleles (p < 0.001). In crisscross experiments, the presentation of SAgs to pure T cells from either the DQ6 or the DR4/DQ8 mice resulted in significantly different levels of response depending on the HLA type expressed on the APCs. Presentation by HLA-DQ6 APCs elicited significantly lower responses than the presentation by HLA-DR4/DQ8 APCs. Our in vitro data were supported by in vivo findings, as the DQ6 mice showed significantly longer survival post-i.v. infection with live M1T1 GAS (p < 0.001) and lower inflammatory cytokine responses as compared with the DR4/DQ8 mice (p < 0.01). The data presented here provide evidence for a direct role of HLA class II molecules in modulating responses to GAS SAgs and underscore the dominant role of HLA class II allelic variation in potentiating the severity of GAS systemic infections.     

Uptake and Intracellular Trafficking of Superantigens in Dendritic Cells

Bacterial superantigens (SAgs) are exotoxins produced mainly by Staphylococcus aureus and Streptococcus pyogenes that can cause toxic shock syndrome (TSS). According to current paradigm, SAgs interact directly and simultaneously with T cell receptor (TCR) on the T cell and MHC class II (MHC-II) on the antigen-presenting cell (APC), thereby circumventing intracellular processing to trigger T cell activation. Dendritic cells (DCs) are professional APCs that coat nearly all body surfaces and are the most probable candidate to interact with SAgs. We demonstrate that SAgs are taken up by mouse DCs without triggering DC maturation. SAgs were found in intracellular acidic compartment of DCs as biologically active molecules. Moreover, SAgs co-localized with EEA1, RAB-7 and LAMP-2, at different times, and were then recycled to the cell membrane. DCs loaded with SAgs are capable of triggering in vitro lymphocyte proliferation and, injected into mice, stimulate T cells bearing the proper TCR in draining lymph nodes. Transportation and trafficking of SAgs in DCs might increase the local concentration of these exotoxins where they will produce the highest effect by promoting their encounter with both MHC-II and TCR in lymph nodes, and may explain how just a few SAg molecules can induce the severe pathology associated with TSS.     

Diverse repertoire of the MHC class II-peptide complexes is required for presentation of viral superantigens

Among other features, peptides affect MHC class II molecules, causing changes in the binding of bacterial superantigens (b-Sag). Whether peptides can alter binding of viral superantigens (v-Sag) to MHC class II was not known. Here we addressed the question of whether mutations limiting the diversity of peptides bound by the MHC class II molecules influenced the presentation of v-Sag and, subsequently, the life cycle of the mouse mammary tumor virus (MMTV). T cells reactive to v-Sag were found in mice lacking DM molecules as well as in A(b)Ep-transgenic mice in which MHC class II binding grooves were predominantly occupied by an invariant chain fragment or Ealpha(52-68) peptide, respectively. APCs from the mutant mice failed to present v-Sag, as determined by the lack of Sag-specific T cell activation, Sag-induced T cell deletion, and by the aborted MMTV infection. In contrast, mice that express I-A(b) with a variety of bound peptides presented v-Sag and were susceptible to MMTV infection. Comparison of v-Sag and b-Sag presentation by the same mutant cells suggested that presentation of v-Sag had requirements similar to that for presentation of toxic shock syndrome toxin-1. Thus, MHC class II peptide repertoire is critical for recognition of v-Sag by the T cells and affects the outcome of infection with a retrovirus.     

Invasive infection caused Streptococcus group A and streptococcal toxic shock syndrome

Modern data on the etiology and pathogenesis of invasive streptococcal infection and the syndrome of streptococcal toxic shock are presented. In the course of the last 10-15 years essential changes in the system of interaction of group A streptococci and the macroorganism have been noted. The growth of morbidity in severe invasive forms of streptococcal infection with different clinical manifestations, including the syndrome of toxic shock, is observed. Most often this disease develops in elderly people, making up a group of risk, but sometimes affects healthy young people. Different pathogenicity factors of streptococci, capable of inducing the development of infection, are analyzed. Special attention is given to superantigens: pyrogenic toxins and M-protein. The suggestion that the development of the disease is seemingly linked with the state of specific protective immunity is substantiated. In spite of achievements in the field of the microbiology and immunology of group A streptococci, the causes of the appearance and development of invasive streptococcal infection have not yet been determined.     

An immunogenetic and molecular basis for differences in outcomes of invasive group A streptococcal infections

The role of host genetic factors in conferring predisposition or protection in infectious diseases has become evident. Infection with group A streptococci causes a wide spectrum of disease ranging from pharyngitis to streptococcal toxic shock syndrome. The release of inflammatory cytokines triggered by streptococcal superantigens has a pivotal role in invasive streptococcal disease. However, individuals infected with the same strain can develop very different manifestations. We report here that the immunogenetics of the host influence the outcome of invasive streptococcal infection, and demonstrate the underlying mechanism for these genetic associations. Specific human leukocyte antigen class II haplotypes conferred strong protection from severe systemic disease, whereas others increased the risk of severe disease. Patients with the DRB1*1501/DQB1*0602 haplotype mounted significantly reduced responses and were less likely to develop severe systemic disease (P < 0.0001). We propose that human leukocyte antigen class II allelic variation contributes to differences in severity of invasive streptococcal infections through their ability to regulate cytokine responses triggered by streptococcal superantigens.     

Proinflammatory exoprotein characterization of toxic shock syndrome Staphylococcus aureus

Pulsed-field gel electrophoresis (PFGE) clonal type USA200 is the most widely disseminated Staphylococcus aureus colonizer of the nose and is a major cause of toxic shock syndrome (TSS). Exoproteins derived from these organisms have been suggested to contribute to their colonization and causation of human diseases but have not been well-characterized. Two representative S. aureus USA200 isolates, MNPE (α-toxin positive) and CDC587 (α-toxin mutant), isolated from pulmonary post-influenza TSS and menstrual vaginal TSS, respectively, were evaluated. Biochemical, immunobiological, and cell-based assays, including mass spectrometry, were used to identify key exoproteins derived from the strains that are responsible for proinflammatory and cytotoxic activity on human vaginal epithelial cells. Exoproteins associated with virulence were produced by both strains, and cytolysins (α-toxin and γ-toxin), superantigens, and proteases were identified as the major exoproteins, which caused epithelial cell inflammation and cytotoxicity. Exoprotein fractions from MNPE were more proinflammatory and cytotoxic than those from CDC587 due to high concentrations of α-toxin. CDC587 produced a small amount of α-toxin, despite the presence of a stop codon (TAG) at codon 113. Additional exotoxin identification studies of USA200 strain [S. aureus MN8 (α-toxin mutant)] confirmed that MN8 also produced low levels of α-toxin despite the same stop codon. The differences observed in virulence factor profiles of two USA200 strains provide insight into environmental factors that select for specific virulence factors. Cytolysins, superantigens, and proteases were identified as potential targets, where toxin neutralization may prevent or diminish epithelial damage associated with S. aureus.     

The mechanism of superantigen-mediated toxic shock: not a simple Th1 cytokine storm

The profound clinical consequences of Gram-positive toxic shock are hypothesized to stem from excessive Th1 responses to superantigens. We used a new superantigen-sensitive transgenic model to explore the role of TCRalphabeta T cells in responses to staphylococcal enterotoxin B (SEB) in vitro and in two different in vivo models. The proliferative and cytokine responses of HLA-DR1 spleen cells were 100-fold more sensitive than controls and were entirely dependent on TCRalphabeta T cells. HLA-DR1 mice showed greater sensitivity in vivo to two doses of SEB with higher mortality and serum cytokines than controls. When d-galactosamine was used as a sensitizing agent with a single dose of SEB, HLA-DR1 mice died of toxic shock whereas controls did not. In this sensitized model of toxic shock there was a biphasic release of cytokines, including TNF-alpha, at 2 h and before death at 7 h. In both models, mortality and cytokine release at both time points were dependent on TCRalphabeta T cells. Anti-TNF-alpha pretreatment was protective against shock whereas anti-IFN gamma pretreatment and delayed anti-TNF-alpha treatment were not. Importantly, anti-TNF-alpha pretreatment inhibited the early TNF-alpha response but did not inhibit the later TNF-alpha burst, to which mortality has previously been attributed. Splenic T cells were shown definitively to be the major source of TNF-alpha during the acute cytokine response. Our results demonstrate unequivocally that TCRalphabeta T cells are critical for lethality in toxic shock but it is the early TNF-alpha response and not the later cytokine surge that mediates lethal shock.     

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.     

A toxic shock syndrome toxin mutant of Staphylococcus aureus isolated by allelic replacement lacks virulence in a rabbit uterine model

The gene coding for toxic shock syndrome toxin-1 in S. aureus was inactivated by allelic replacement in two TSS-associated strains. One mutant derived from FRI1169 (a non-enterotoxigenic strain) lacked virulence in the rabbit uterine chamber infection model. This suggests that TSST-1 is the only determinant produced by this strain that can induce the symptoms of shock in rabbits. A novel method for allelic replacement involving transduction of plasmid integrants is described.     

链球菌引发宿主中毒性休克综合症相关机制的研究进展

某些链球菌在感染过程中,会引发宿主系统性的细胞因子风暴,病程进展迅速,致死率高,即发生链球菌性中毒性休克综合症(streptococcal toxic shock syndrome,STSS).最初认为STSS由链球菌的超抗原(superantigen,SAg)引发,但近年来也有不依赖超抗原的STSS病例报道,致病机制...     

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.     

The Refined Crystal Structure of Toxic Shock Syndrome Toxin-1 at 2.07 Å Resolution

The pyrogenic toxin toxic shock syndrome toxin-1 fromStaphylococcus aureusis a causative agent of the toxic shock syndrome disease. It belongs to a family of proteins known as superantigens that cross-link major histocompatibility class II molecules and T-cell receptors leading to the activation of a substantial number of T cells. The crystal structure of this protein has been refined to 2.07 Å with anR_crystvalue of 20.4% for 51,240 reflections. The final model contains three molecules in the asymmetric unit with good stereochemistry and a root-mean-square deviation of 0.009 Å and 1.63° from ideality for bond lengths and bond angles, respectively. The overall fold is considerably similar to that of other known microbial superantigens (staphylococcal enterotoxins). However, a detailed structural analysis shows that toxic shock syndrome toxin-1 lacks several structural features that affect its specificity for Vβ elements of the T-cell receptor and also its recognition by major histocompatibility class II molecules.     

IL-3 induces B7.2 (CD86) expression and costimulatory activity in human eosinophils.

Eosinophils in tissues are often present in intimate contact with T cells in allergic and parasitic diseases. Resting eosinophils do not express MHC class II proteins or costimulatory B7 molecules and fail to induce proliferation of T cells to Ags. IL-5 and GM-CSF induce MHC class II and B7 expression on eosinophils and have been reported in some studies to induce eosinophils to present Ag to T cells. The cytokine IL-3, like IL-5 and GM-CSF, is a survival and activating factor for eosinophils and the IL-3 receptor shares with the IL-5 and GM-CSF receptors a common signal transducing beta-chain. IL-3-treated eosinophils expressed HLA-DR and B7.2, but not B7.1 on their surface and supported T cell proliferation in response to the superantigen toxic shock syndrome toxin 1, as well as the proliferation of HLA-DR-restricted tetanus toxoid (TT) and influenza hemagglutinin-specific T cell clones to antigenic peptides. This was inhibited by anti-B7.2 mAb. In contrast, IL-3-treated eosinophils were unable to present native TT Ag to either resting or TT-specific cloned T cells. In parallel experiments, eosinophils treated with IL-5 or GM-CSF were also found to present superantigen and antigenic peptides, but not native Ag, to T cells. These results suggest that eosinophils are deficient in Ag processing and that this deficiency is not overcome by cytokines that signal via the beta-chain. Nevertheless, our findings suggest that eosinophils activated by IL-3 may contribute to T cell activation in allergic and parasitic diseases by presenting superantigens and peptides to T cells.