The structure and interactions of pneumolysin

Pneumolysin is a major virulence factor of Streptococcus pneumoniae. It is a 53-kDa protein with four domains which form pores in host cells and depletes serum-complement resulting in a diminished host immune response. Despite its importance, the high-resolution structure of pneumolysin is yet to be solved. This thesis seeks to address the lack of high-resolution data, enabling the identification of residues important for pore formation and binding to its cellular target ligand, cholesterol. The interactions between complement and pneumolysin were investigated, to further understand the role of pneumolysin in serum complement depletion. The structure of pneumolysin was solved using X-ray crystallography at 2Å resolution. The crystal packing mimics the pre-pore arrangement of pneumolysin, enabling the identification of intermolecular interactions that stabilise the pre-pore. Mutation experiments revealed interacting residues were not structurally important but resulted in a substantial reduction in the pore forming capability of pneumolysin, showing that they are functionally important and most likely inhibit pre-pore oligomerisation. Residues involved in the interactions are 69% conserved across five proteins of the same family implying a common mechanism. The crystal structure revealed other residues that may be functionally important with respect to domain orientation and cholesterol binding in the pre-pore, and these could be investigated in the future. Complement assays showed that pneumolysin diminished complement in the fluidphase and when immobilized on a surface. However pneumolysin did not interact with IgG and a mutation previously identified as being important (D385N) had no effect on the activity observed. Activation occurred via the alternative pathway, with contributions from the classical and/or the lectin pathway.