John D. Fraser

The bacterial superantigen and superantigen-like proteins

Summary: The bacterial superantigens are protein toxins that bind to major histocompatibility complex class II and T‐cell receptor to stimulate large numbers of T cells. The majority are produced by the Gram‐positive organisms Staphylococcus aureus and Streptococcus pyogenes and are the causative agents in toxic shock syndrome, an acute disease caused by the sudden and massive release of T‐cell cytokines into the blood stream. The structure and function of the superantigens has revealed a common architecture that is also shared by another group of staphylococcal virulence factors called the superantigen‐like proteins (SSL). Together, this family of structurally related molecules highlights how a common pathogenic organism has employed a simple but adaptable protein to generate an armamentarium of potent defense molecules designed to target of the innate and adaptive immune response.

The staphylococcal superantigen-like protein 7 binds IgA and complement C5 and inhibits IgA-Fc alpha RI binding and serum killing of bacteria.

The staphylococcal superantigen-like proteins (SSLs) are close relatives of the superantigens but are coded for by a separate gene cluster within a 19-kb region of the pathogenicity island SaPIn2. rSSL7 (formally known as SET1) bound with high affinity (KD, 1.1 nM) to the monomeric form of human IgA1 and IgA2 plus serum IgA from primate, pig, rat, and horse. SSL7 also bound the secretory form of IgA found in milk from human, cow, and sheep, and inhibited IgA binding to cell surface FcαRI (CD89) and to a soluble form of the FcαRI protein. In addition to IgA, SSL7 bound complement factor C5 from human (KD, 18 nM), primate, sheep, pig, and rabbit serum, and inhibited complement-mediated hemolysis and serum killing of a Gram-negative organism Escherichia coli. SSL7 is a superantigen-like protein secreted from Staphylococcus aureus that blocks IgA-FcR interactions and inhibits complement, leading to increased survival of a sensitive bacterium in blood.

Superantigens - powerful modifiers of the immune system

Superantigens are powerful microbial toxins that activate the immune system by binding to class II major histocompatibility complex and T-cell receptor molecules. They cause a number of diseases characterized by fever and shock and are important virulence factors for two human commensal organisms, Staphylococcus aureus and Streptococcus pyogenes, as well as for some viruses. Their mode of action and variation around the common theme of over-stimulating T cells, provides a rich insight into the constant battle between microbes and the immune system.

Crystal structure of the streptococcal superantigen SPE-C: dimerization and zinc binding suggest a novel mode of interaction with MHC class II molecules

Bacterial superantigens are small proteins that have a very potent stimulatory effect on T lymphocytes through their ability to bind to both MHC class II molecules and T-cell receptors. We have determined the three-dimensional structure of a Streptococcal superantigen, SPE-C, at 2.4 Å resolution. The structure shows that SPE-C has the usual superantigen fold, but that the surface that forms a generic, low-affinity MHC-binding site in other superantigens is here used to create a SPE-C dimer. Instead, MHC class II binding occurs through a zinc binding site that is analogous to a similar site in staphylococcal enterotoxin A. Consideration of the SPE-C dimer suggests a novel mechanism for promotion of MHC aggregation and T-cell activation.

Two novel superantigens found in both group A and group C Streptococcus.

ABSTRACT Two novel streptococcal superantigen genes (speLSe and speMSe) were identified from the Streptococcus equi genome database at the Sanger Center. Genotyping of 8 S. equi isolates and 40 Streptococcus pyogenes isolates resulted in the detection of the orthologous genes speL and speM in a restricted number of S. pyogenes isolates (15 and 5%, respectively). Surprisingly, the novel superantigen genes could not be found in any of the analyzed S. equi isolates. The results suggest that both genes are located on a mobile element that enables gene transfer between individual isolates and between streptococci from different Lancefield groups. S. equi pyrogenic exotoxin L (SPE-LSe)/streptococcal pyrogenic exotoxin L (SPE-L) and SPE-MSe/SPE-M are most closely related to SMEZ, SPE-C, SPE-G, and SPE-J, but build a separate branch within this group. Recombinant SPE-L (rSPE-L) and rSPE-M were highly mitogenic for human peripheral blood lymphocytes, with half-maximum responses at 1 and 10 pg/ml, respectively. The results from competitive binding experiments suggest that both proteins bind major histocompatibility complex class II at the β-chain, but not at the α-chain. The most common targets for both toxins were human Vβ1.1 expressing T cells. Seroconversion against SPE-L and SPE-M was observed in healthy blood donors, suggesting that the toxins are expressed in vivo. Interestingly, the speL gene is highly associated with S. pyogenes M89, a serotype that is linked to acute rheumatic fever in New Zealand.

Superantigens and Streptococcal Toxic Shock Syndrome

Superantigens produced by Streptococcus pyogenes have been implicated with streptococcal toxic shock syndrome (STSS). We analyzed 19 acute-phase serum samples for mitogenic activity from patients with severe streptococcal disease. The serum samples from two patients in the acute phase of STSS showed strong proliferative activity. Streptococcal mitogenic exotoxin (SME) Z-1 and streptococcal pyrogenic exotoxin (SPE)-J were identified in one patient with peritonitis who recovered after 2 weeks in intensive care. SMEZ-16 was found in a second patient who died on the day of admission. Sequential serum samples taken on day 3 after admission from patient 1 showed clearance of mitogenic activity but absence of neutralizing anti-SMEZ antibodies. Serum samples taken on day 9 from this patient showed evidence of seroconversion with high levels of anti-SMEZ antibodies that neutralized SMEZ-1 and 12 other SMEZ-variants. These results imply that a high level of SMEZ production by group A streptococcus is a causative event in the onset and subsequent severity of STSS.

The Bacterial Superantigen Streptococcal Mitogenic Exotoxin Z Is the Major Immunoactive Agent of Streptococcus pyogenes

The gene encoding streptococcal mitogenic exotoxin Z (SMEZ) was disrupted in Streptococcus pyogenes. Despite the presence of other superantigen genes, mitogenic responses in human and murine HLA-DQ transgenic cells were abrogated when cells were stimulated with supernatant from the smez− mutant compared with the parent strain. Remarkably, disruption of smez led to a complete inability to elicit cytokine production (TNF-α, lymphotoxin-α, IFN-γ, IL-1 and -8) from human cells, when cocultured with streptococcal supernatants. The potent effects of SMEZ were apparent even though transcription and expression of SMEZ were barely detectable. Human Vβ8+ T cell proliferation in response to S. pyogenes was SMEZ-dependent. Cells from HLA-DQ8 transgenic mice were 3 logs more sensitive to SMEZ-13 than cells from HLA-DR1 transgenic or wild-type mice. In the mouse, SMEZ targeted the human Vβ8+ TCR homologue, murine Vβ11, at the expense of other TCR T cell subsets. Expression of SMEZ did not affect bacterial clearance or survival from peritoneal streptococcal infection in HLA-DQ8 mice, though effects of SMEZ on pharyngeal infection are unknown. Infection did lead to a rise in Vβ11+ T cells in the spleen which was partly reversed by disruption of the smez gene. Most strikingly, a clear rise in murine Vβ4+ cells was seen in mice infected with the smez− mutant S. pyogenes strain, indicating a potential role for SMEZ as a repressor of cognate anti-streptococcal responses.

The Three-dimensional Structure of a Superantigen-like Protein, SET3, from a Pathogenicity Island of the Staphylococcus aureus Genome

The staphylococcal enterotoxin-like toxins (SETs) are a family of proteins encoded within the Staphylococcus aureus genome that were identified by their similarity to the well described bacterial superantigens. The first crystal structure of a member of the SET family, SET3, has been determined to 1.9 Å (R = 0.205, R free = 0.240) and reveals a fold characteristic of the superantigen family but with significant differences. The SET proteins are secreted at varying levels by staphylococcal isolates, and seroconversion studies of normal individuals indicate that they are strongly antigenic to humans. Recombinant SETs do not exhibit any of the properties expected of superantigens such as major histocompatibility complex class II binding or broad T-cell activation, suggesting they have an entirely different function. The fact that the whole gene family is clustered within the pathogenicity island SaIn2 of theS. aureus genome suggests that they are involved in host/pathogen interactions.

Structural basis for evasion of IgA immunity by Staphylococcus aureus revealed in the complex of SSL7 with Fc of human IgA1

Infection by Staphylococcus aureus can result in severe conditions such as septicemia, toxic shock, pneumonia, and endocarditis with antibiotic resistance and persistent nasal carriage in normal individuals being key drivers of the medical impact of this virulent pathogen. In both virulent infection and nasal colonization, S. aureus encounters the host immune system and produces a wide array of factors that frustrate host immunity. One in particular, the prototypical staphylococcal superantigen-like protein SSL7, potently binds IgA and C5, thereby inhibiting immune responses dependent on these major immune mediators. We report here the three-dimensional structure of the complex of SSL7 with Fc of human IgA1 at 3.2 Å resolution. Two SSL7 molecules interact with the Fc (one per heavy chain) primarily at the junction between the Cα2 and Cα3 domains. The binding site on each IgA chain is extensive, with SSL7 shielding most of the lateral surface of the Cα3 domain. However, the SSL7 molecules are positioned such that they should allow binding to secretory IgA. The key IgA residues interacting with SSL7 are also bound by the leukocyte IgA receptor, FcαRI (CD89), thereby explaining how SSL7 potently inhibits IgA-dependent cellular effector functions mediated by FcαRI, such as phagocytosis, degranulation, and respiratory burst. Thus, the ability of S. aureus to subvert IgA-mediated immunity is likely to facilitate survival in mucosal environments such as the nasal passage and may contribute to systemic infections.

The crystal structure of staphylococcal superantigen-like protein 11 in complex with sialyl Lewis X reveals the mechanism for cell binding and immune inhibition.

Staphylococcus aureus is a major pathogen that produces a family of 14 staphylococcal superantigen‐like (SSL) proteins, which are structurally similar to superantigens but do not stimulate T cells. SSL11 is one member of the family that is found in all staphylococcal strains. Recombinant SSL11 bound to granulocytes and monocytes through a sialic acid‐dependent mechanism and was rapidly internalized. SSL11 also bound to sialic acid‐containing glycoproteins, such as the Fc receptor for IgA (FcαRI) and P‐selectin glycoprotein ligand‐1 (PSGL‐1), and inhibited neutrophil attachment to a P‐selectin‐coated surface. Biosensor analysis of two SSL11 alleles binding to sialyl Lewis X [sLex– Neu5Acα2‐3Galβ1‐4(Fuc1‐3)GlcNAc] coupled to bovine serum albumin gave dissociation constants of 0.7 and 7 μm respectively. Binding of SSL11 to a glycan array revealed specificity for glycans containing the trisaccharide sialyllactosamine (sLacNac – Neu5Acα2‐3Galβ1‐4GlcNAc). A 1.6 Å resolution crystal structure of SSL11 complexed with sLex revealed a discrete binding site in the C‐terminal β‐grasp domain, with predominant interactions with the sialic acid and galactose residues. A single amino acid mutation in the carbohydrate binding site abolished all SSL11 binding. Thus, SSL11 is a staphylococcal protein that targets myeloid cells by binding sialyllactosamine‐containing glycoproteins.