Structural Studies on the Epsilon Toxin from Clostridium perfringens.
Epsilon toxin is a potent, 32kDa toxin produced by the bacterium C. perfringens. It is third only to the Botulinum and tetanus neurotoxins in its potency. Of the five types of C. perfringens known (A-E) two, B and D produce this toxin. These strains have a limited host range mainly being isolated from sheep and lambs, though occasionally from goats and cattle and rarely from man. The two strains are responsible for producing a very severe and often fatal form of enterotoxaemia. Epsilon toxin is produced as a relatively inactive prototoxin that is activated upon removal of a 13 residue N-terminal peptide and 22 C-terminal residues by trypsin in the gut.
A large dose of the toxin causes an increased intestinal permeability facilitating entry to the blood. The toxin then accumulates in the brain and to a lesser extent in the kidney causing widespread osmotic alterations within the brain and kidney by disrupting vascular endothelia. These degenerative changes eventually cause serum proteins and red blood cells to leak out of the vasculature and cause massive oedema. The accumulation of the toxin in specific organs, the brain and kidney, is thought to be because of specific binding to a cell surface protein, thought to be a 26 kDa sialoglycoprotein.
Experiments on a Madine-Darby canine kidney cells (MDCK cells), a susceptable cell line, have shown that the application of epsilon toxin causes cellular disruption that is consistant with pore formation, Petit et al . Evidence of this has increased with the observation of pore formation and ion passage through lipid bi-layers. A seperate group, Miyata et al , have shown the importance of cleavage at the C-terminus in pore formation. This same group have also shown that the pore is likely to be heptameric.
Here at Birkbeck we have been carrying out structural studies on both the prototoxin and active forms of Epsilon toxin. We have recently solved and published the structure of the prototoxin and have shown that it is indeed likely to be a pore forming toxin in the aeroloysin family of beta-pore forming toxins. We have now began work to try to improve on this knowledge by investigating the steps leading to pore formation including the binding and heptamerisation processes.
A crystal of the Epsilon prototoxin.
Diffraction from these crystals
A sample of the resulting density.
The finished structure