The Structural and Functional Properties of Staphylococcal Superantigen-Like Protein 4 (SSL4)

Abstract

Staphylococcus aureus is a major cause of hospital acquired infections and is capable of causing life threatening illness. S. aureus produces a wide range of both cell surface and secreted virulence factors which enable it to cause disease. Among its repertoire of virulence factors are the 14 staphylococcal superantigen-like (SSL) proteins that have been shown to target components of the immune response. This thesis examined SSL4, which belongs to a subfamily of the SSLs that contain a conserved sialylated-glycan binding site in the Cterminal β-grasp domain. SSL4 binds to, and is actively internalised by, myeloid cells in a sialic-acid dependent manner. SSL4 is endocytosed by neutrophils to a separate intracellular compartment to S. aureus, likely to be the lysosome. Within macrophages SSL4 displays perinuclear cytoskeletal staining of an as yet unidentified intracellular structure. While no specific function has been attributed to SSL4, this protein likely has a role in the modulation of myeloid recruitment and function through sialylated glycan specific interactions. These results suggest that SSL4 may force the internalisation of important cell surface glycoproteins, effect cell-mediated killing of S. aureus by interfering with the lysosome, or disrupt the capacity of macrophages to respond to bacterial infection by affecting the cytoskeleton. While sialyllactosamine has been identified as the core ligand for SSL4, cell binding and quantitative binding data of SSL4 indicate the blood group antigen sialyl Lewis X (sLex) is a preferred ligand and likely is a component of the target receptor(s) of this protein. A 2.5 Å crystal structure of SSL4 binding sLex has been refined and revealed a similar glycan-binding site to SSL5 and SSL11, other members of the sugar-binding subfamily. Despite sharing a remarkably similar binding site to SSL11, glycan arrays, biosensor analysis of ligand binding, and competitive cell binding studies reveal these proteins differ in their affinity and preference for ligands. Together with site directed mutagenesis this shows how subtle differences in the glycan-binding site affect SSL specificities for host receptors and how S. aureus uses multiple related molecules to target myeloid cells through specific sialyllactosamine containing glycoproteins.