Insights into pore-formation from studies of Clostridium perfringens Epsilon-toxin (360G-Wellcome-089618_B_09_Z)

Clostridium perfringens epsilon-toxin is uniquely lethal and exquisitely specific. It causes pulpy kidney disease which in unvaccinated livestock can devastate herds in a few days. Pathological changes associated with epsilon-toxin occur mainly in the brain, and it binds MDCK and rat brain synaptosomal cells. Our structure of the monomeric proto-toxin revealed that it is a beta-pore-forming toxin. This family generally shows activity against a broad range of cell types with much larger lethal doses than epsilon-toxin. Understanding why epsilon-toxin shows this atypical behaviour could help the design of a specific, efficient cytolytic agent. We will study oligomerization and receptor interaction. Micro-array analysis has revealed candidate genes for epsilon-toxin sensitivity. We will use siRNA to suppress expression of these genes in MDCK cells and assess their contribution to toxin recognition. We will mutate residues previously identified by structure comparison and conser vation analysis to assess their involvement in receptor interaction. We will determine the heptamer structure by cryo-electron microscopy and crystallography. We already have promising negative stain EM images. Residues revealed to be in the heptamer interface will be mutated and any changes in oligomerization investigated. Non-oligomerizing variants will be used in models of disease to assess their suitability as molecular vaccines.