Bruce D. Wines

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.

The IgG Fc Contains Distinct Fc Receptor (FcR) Binding Sites: The Leukocyte Receptors FcγRI and FcγRIIa Bind to a Region in the Fc Distinct from That Recognized by Neonatal FcR and Protein A

The CH2-CH3 interface of the IgG Fc domain contains the binding sites for a number of Fc receptors including Staphylococcal protein A and the neonatal Fc receptor (FcRn). It has recently been proposed that the CH2-CH3 interface also contains the principal binding site for an isoform of the low affinity IgG Fc receptor II (FcγRIIb). The FcγRI and FcγRII binding sites have previously been mapped to the lower hinge and the adjacent surface of the CH2 domain although contributions of the CH2-CH3 interface to binding have been suggested. This study addresses the question whether the CH2-CH3 interface plays a role in the interaction of IgG with FcγRI and FcγRIIa. We demonstrate that recombinant soluble murine FcγRI and human FcγRIIa did not compete with protein A and FcRn for binding to IgG, and that the CH2-CH3 interface therefore appears not to be involved in FcγRI and FcγRIIa binding. The importance of the lower hinge was confirmed by introducing mutations in the proposed binding site (LL234,235AA) which abrogated binding of recombinant soluble FcγRIIa to human IgG1. We conclude that the lower hinge and the adjacent region of the CH2 domain of IgG Fc is critical for the interaction between FcγRIIa and human IgG, whereas contributions of the CH2-CH3 interface appear to be insignificant.

Structural Basis for FcγRIIa Recognition of Human IgG and Formation of Inflammatory Signaling Complexes

The interaction of Abs with their specific FcRs is of primary importance in host immune effector systems involved in infection and inflammation, and are the target for immune evasion by pathogens. FcγRIIa is a unique and the most widespread activating FcR in humans that through avid binding of immune complexes potently triggers inflammation. Polymorphisms of FcγRIIa (high responder/low responder [HR/LR]) are linked to susceptibility to infections, autoimmune diseases, and the efficacy of therapeutic Abs. In this article, we define the three-dimensional structure of the complex between the HR (arginine, R134) allele of FcγRIIa (FcγRIIa-HR) and the Fc region of a humanized IgG1 Ab, hu3S193. The structure suggests how the HR/LR polymorphism may influence FcγRIIa interactions with different IgG subclasses and glycoforms. In addition, mutagenesis defined the basis of the epitopes detected by FcR blocking mAbs specific for FcγRIIa (IV.3), FcγRIIb (X63-21), and a pan FcγRII Ab (8.7). The epitopes detected by these Abs are distinct, but all overlap with residues defined by crystallography to contact IgG. Finally, crystal structures of LR (histidine, H134) allele of FcγRIIa and FcγRIIa-HR reveal two distinct receptor dimers that may represent quaternary states on the cell surface. A model is presented whereby a dimer of FcγRIIa-HR binds Ag–Ab complexes in an arrangement that possibly occurs on the cell membrane as part of a larger signaling assembly.

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.

The Interaction of FcαRI with IgA and Its Implications for Ligand Binding by Immunoreceptors of the Leukocyte Receptor Cluster

This study defines the molecular basis of the FcαRI (CD89):IgA interaction, which is distinct from that of the other leukocyte Fc receptors and their Ig ligands. A comprehensive analysis using both cell-free (biosensor) and cell-based assays was used to define and characterize the IgA binding region of FcαRI. Biosensor analysis of mutant FcαRI proteins showed that residues Y35, Y81, and R82 were essential for IgA binding, and R52 also contributed. The role of the essential residues (Y35 and R82) was confirmed by analysis of mutant receptors expressed on the surface of mammalian cells. These receptors failed to bind IgA, but were detected by the mAb MY43, which blocks IgA binding to FcαRI, indicating that its epitope does not coincide with these IgA binding residues. A homology model of the ectodomains of FcαRI was generated based on the structures of killer Ig-like receptors, which share 30–34% identity with FcαRI. Key structural features of killer Ig-like receptors are appropriately reproduced in the model, including the structural conservation of the interdomain linker and hydrophobic core (residues V17, V97, and W183). In this FcαRI model the residues forming the IgA binding site identified by mutagenesis form a single face near the N-terminus of the receptor, distinct from other leukocyte Fc receptors where ligand binding is in the second domain. This taken together with major differences in kinetics and affinity for IgA:FcαRI interaction that were observed depending on whether FcαRI was immobilized or in solution suggest a mode of interaction unique among the leukocyte receptors.

Structural basis for inhibition of complement C5 by the SSL7 protein from Staphylococcus aureus

Staphylococcus aureus secretes the SSL7 protein as part of its immune evasion strategy. The protein binds both complement C5 and IgA, yet it is unclear whether SSL7 cross-links these two proteins and, if so, what purpose this serves the pathogen. We have isolated a stable IgA-SSL7-C5 complex, and our crystal structure of the C5-SSL7 complex confirms that binding to C5 occurs exclusively through the C-terminal β-grasp domain of SSL7 leaving the OB domain free to interact with IgA. SSL7 interacts with C5 >70 Å from the C5a cleavage site without inducing significant conformational changes in C5, and efficient inhibition of convertase cleavage of C5 is shown to be IgA dependent. Inhibition of C5a production and bacteriolysis are all shown to require C5 and IgA binding while inhibition of hemolysis is achieved by the C5 binding SSL7 β-grasp domain alone. These results provide a conceptual and structural basis for the development of a highly specific complement inhibitor preventing only the formation of the lytic membrane attack complex without affecting the important signaling functions of C5a.

Specificity of staphylococcal superantigen-like protein 10 toward the human IgG1 Fc domain.

Staphylococcal superantigen-like protein 10 (SSL10) is a highly conserved member of the SSL family secreted by Staphylococcus aureus that displays structural but not functional similarity to superantigens. SSL10 bound to fibrinogen and fibronectin from plasma and in addition displayed striking specificity toward the γ-1 subclass of human Igs. SSL10 also bound strongly to primate IgG but not to any other species tested, including rabbit, pig, guinea pig, cow, sheep, or mouse. A soluble form of the 12-kDa β-grasp C-terminal domain of SSL10 (SSL1095–197) retained fibrinogen and fibronectin binding but lost the ability to bind IgG1, indicating that SSL10 bound to IgG1 primarily through its N-terminal oligonucleotide binding fold domain. SSL10 blocked the binding of IgG1 to FcγRs on monocytes and neutrophil phagocytosis of IgG1-opsonized bacteria. Mutagenesis of human IgG1 at key sites significantly reduced SSL10 binding including Lys322 that is important for C1q binding, a combination of Leu234 and Leu235 that are important for FcγR binding, and a combination of Lys274 and Asp276 that together are unique to IgG1. These mutations suggest that the most likely site bound by SSL10 is the outer face of the Cγ2 domain in close proximity to both the FcγR and C1q binding sites. SSL10 is a potential virulence factor for S. aureus targeting IgG1-mediated immunity.

A competitive mechanism for staphylococcal toxin SSL7 inhibiting the leukocyte IgA receptor, Fc alphaRI, is revealed by SSL7 binding at the C alpha2/C alpha3 interface of IgA.

Leukocyte recruitment and effector functions like phagocytosis and respiratory burst are key elements of immunity to infection. Pathogen survival is dependent upon the ability to overwhelm, evade or inhibit the immune system. Pathogenic group A and group B streptococci are well known to produce virulence factors that block the binding of IgA to the leukocyte IgA receptor, FcαRI, thereby inhibiting IgA-mediated immunity. Recently we found Staphylococcus aureus also interferes with IgA-mediated effector functions as the putative virulence factor SSL7 also binds IgA and blocks binding to FcαRI. Herein we report that SSL7 and FcαRI bind many of the same key residues in the Fc region of human IgA. Residues Leu-257 and Leu-258 in domain Cα2 and residues 440–443 PLAF in Cα3 of IgA lie at the Cα2/Cα3 interface and make major contributions to the binding of both the leukocyte receptor FcαRI and SSL7. It is remarkable this S. aureus IgA binding factor and unrelated factors from streptococci are functionally convergent, all targeting a number of the same residues in the IgA Fc, which comprise the binding site for the leukocyte IgA receptor, FcαRI.

The Mechanism Underlying Defective Fcγ Receptor-Mediated Phagocytosis by HIV-1-Infected Human Monocyte-Derived Macrophages

Clearance of IgG-opsonized erythrocytes is impaired in HIV-1-infected patients, suggesting defective FcγR-mediated phagocytosis in vivo. We have previously shown defective FcγR-mediated phagocytosis in HIV-1-infected human monocyte-derived macrophages (MDM), establishing an in vitro model for defective tissue macrophages. Inhibition was associated with decreased protein expression of FcR γ-chain, which transduces immune receptor signals via ITAM motifs. FcγRI and FcγRIIIa signal via γ-chain, whereas FcγRIIa does not. In this study, we showed that HIV-1 infection inhibited FcγRI-, but not FcγRIIa-dependent Syk activation in MDM, showing that inhibition was specific for γ-chain-dependent signaling. HIV-1 infection did not impair γ-chain mRNA levels measured by real-time PCR, suggesting a posttranscriptional mechanism of γ-chain depletion. HIV-1 infection did not affect γ-chain degradation (n = 7, p = 0.94) measured in metabolic labeling/chase experiments, whereas γ-chain biosynthesis was inhibited (n = 12, p = 0.0068). Using an enhanced GFP-expressing HIV-1 strain, we showed that FcγR-mediated phagocytosis inhibition is predominantly due to a bystander effect. Experiments in which MDM were infected in the presence of the antiretroviral drug 3TC suggest that active viral replication is required for inhibition of phagocytosis in MDM. These data suggest that HIV-1 infection may affect only γ-chain-dependent FcγR functions, but that this is not restricted to HIV-1-infected cells.