| Summary: | Pneumolysin is a virulence factor produced by the human pathogen Streptococcus pneumoniae. It acts in disease to damage cell membranes via pore formation and to activate the complement system directly. Pore formation is accompanied by the transition of the protein from an aqueous, monomeric conformation to a lipid-inserted, ring-shaped oligomeric state. This thesis describes the behaviour of pneumolysin in solution, investigations concerning its mechanism of pore formation, and the structure of an oligomeric form of the toxin. Pneumolysin self-interacts in solution through one of its four domains. The self-interaction leads to the formation of dimeric toxin. Consequent upon dimerization, pneumolysin oligomerizes in solution into structures apparently the same as those associated with pore formation in membranes. In addition it forms helical oligomers. Small-angle neutron scattering (SANS) suggests the existence of inter-domain flexibility in pneumolysin. The kinetics of pore formation by pneumolysin are dependent on binding and oligomerization of the toxin. SANS allows the observation of a model membrane under attack by pneumolysin, indicating changes in bilayer structure. The structure of a helical pneumolysin oligomer is described determined by electron cryomicroscopy. This thesis demonstrates that oligomerization is an innate property of pneumolysin, although it was previously thought that the monomer-oligomer transition required interaction between toxin and cholesterol. It furthermore describes a novel approach to observing the interaction between a protein and a membrane in seeking to understand the biochemistry of this important and widespread process. The determination of an oligomeric structure for pneumolysin indicates the orientation of the toxin subunit in the pore for the first time, which is very different from that previously proposed. It is also possible to understand on the basis of the oligomeric structure the relationship between self-association and pore formation by pneumolysin and related toxins.
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