Structural and functional characterisation of staphylococcal superantigen-like protein 11 (SSL11)

Abstract

Staphylococcus aureus is a human pathogen with significant socioeconomic impact. It is the causative agent of many life-threatening disease states, including sepsis, staphylococcal toxic shock syndrome and necrotizing pneumonia. S. aureus is associated with 1% of all hospital stays and estimated costs of US$9.5 billion / year (for 2000 and 2001) in the USA alone (Noskin et al., 2005). Recently, a novel family of virulence factors produced by S. aureus was identified, which share sequence and structural homology to the infamous superantigens (Arcus et al., 2002; Williams et al., 2000) – proteins that cause a range of symptoms, including food poisoning and toxic shock. This family of staphylococcal superantigen-like proteins (SSLs) includes 14 members, of which 11 (SSLs 1-11) are closely linked within a mobile genetic element, called a pathogenicity island (Kuroda et al., 2001). To date, the literature suggests that the SSLs exhibit important host immune-evasion functions that are distinct from the superantigens. SSL5 has been reported to inhibit leukocyte rolling, a key process in their recruitment to sites of infection (Bestebroer et al., 2006). SSL7 inhibits serum-mediated killing of bacteria (Langley et al., 2005). This thesis describes the investigation into the structure and function of the related protein, SSL11. The binding specificity of SSL11 has been identified as the trisaccharide Neu5Acα2-3Galβ1- 4GlcNAc, where Neu5Ac is essential for interaction, whereas the NAc sidechain of GlcNAc is not, but is highly favoured. The interaction between the related carbohydrate, sialyl Lewis X (sLex, or Neu5Acα2-3Galβ1-4[Fucα1-3]GlcNAc), and SSL11 has been characterised at an atomic level by X-ray crystallography. The dissociation constant of SSL11 binding to sLex was determined by surface plasmon resonance to be in the micromolar to sub-micromolar range. Investigation into the effects of SSL11 on neutrophils reveals that nanomolar concentrations of SSL11 inhibited neutrophil rolling on a P-selectin surface in vitro. Additionally, SSL11 was rapidly internalized, exhibited a cytoplasmic distribution and caused changes in actin, visible by 2-D gel analysis. The characterization of the interaction between SSL11 and the previously-identified ligand, FcαRI (the myeloid immunoglobulin A receptor), is also described. The dissociation constant - II - of SSL11 binding to FcαRI was determined by surface plasmon resonance to be in the micromolar to sub-micromolar range and was found to be sialic acid-dependent. Binding of SSL11 to FcαRI inhibited the binding of its native ligand, IgA. The sialic acid-dependent glycan-binding site of SSL11 is the sole site of interaction with the neutrophil surface, shown by neuraminidase treatment of cells and complete abrogation of binding by a site-directed functional knockout mutant. Residues involved in glycan binding were found to be conserved in SSLs 2-6, giving rise to a potential related function between this subset of the SSL family.