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.