Binding of ɛ-toxin from Clostridium perfringens in the nervous system

Abstract

Epsilon-toxin (epsilon-toxin), produced by Clostridium perfringens type D, is the main agent responsible for enterotoxaemia in livestock. Neurological disorders are a characteristic of the onset of toxin poisoning. Epsilon-Toxin accumulates specifically in the central nervous system, where it produces a glutamatergic-mediated excitotoxic effect. However, no detailed study of putative binding structures in the nervous tissue has been carried out to date. Here we attempt to identify specific acceptor moieties and cell targets for epsilon-toxin, not only in the mouse nervous system but also in the brains of sheep and cattle. An epsilon-toxin-GFP fusion protein was produced and used to incubate brain sections, which were then analyzed by confocal microscopy. The results clearly show specific binding of epsilon-toxin to myelin structures. epsilon-Prototoxin-GFP and epsilon-toxin-GFP, the inactive and active forms of the toxin, respectively, showed identical results. By means of pronase E treatment, we found that the binding was mainly associated to a protein component of the myelin. Myelinated peripheral nerve fibres were also stained by epsilon-toxin. Moreover, the binding to myelin was not only restricted to rodents, but was also found in humans, sheep and cattle. Curiously, in the brains of both sheep and cattle, the toxin strongly stained the vascular endothelium, a result that may explain the differences in potency and effect between species. Although the binding of epsilon-toxin to myelin does not directly explain its neurotoxic effect, this feature opens up a new line of enquiry into its mechanism of toxicity and establishes the usefulness of this toxin for the study of the mammalian nervous system.

Dominant-negative Inhibitors of the Clostridium perfringens ϵ-Toxin

“…To overcome this obstacle, we expressed, purified, and trypsin-treated a GFPtagged form of the wild-type ⑀-toxin (5,33,34). Remarkably, this toxin retains cytotoxic activity, and the GFP tag is not lost following trypsin treatment.…”

“…This placed the etxB gene under the regulation of the bacteriophage T7 RNA polymerase and fused the N-terminal end of the prototoxin to the pelB leader peptide and the C-terminal end of the prototoxin to a His 6 affinity tag (to aid in purification of the protein). A derivative plasmid that expressed a GFP-⑀-toxin fusion protein was also constructed (5,33,34).…”

“…pelB leader peptide and the C-terminal end of the prototoxin to a His 6 affinity tag (to aid in purification of the protein). A derivative plasmid that expressed a GFP-⑀-toxin fusion protein was also constructed (5,33,34). The ⑀-prototoxin-expressing plasmid was transformed into an E. coli K12 expression strain, NovaBlue (DE3) (Novagen), along with the plasmid pLysE (encoding bacteriophage T7 lysozyme) and transformants were grown in broth supplemented with antibiotics to an optical density at 600 nm of 0.7.…”

Molecular cloning and expression of epsilon toxin from Clostridium perfringens type D and tests of animal immunization

“…The toxin's potential targets include vascular endothelia, synaptosomal membrane fractions, astrocytes, microglial cells, and myelin (Soler-Jover et al, 2004Dorca-Arévalo et al, 2008). The major manifestations of epsilon neurotoxicity are microvascular endothelial injury and vasogenic edema (Ghabriel et al, 2000;Finnie et al, 1999Finnie et al, , 2008.…”

Role of Pore-Forming Toxins in Bacterial Infectious Diseases

“…When mice were injected intravenously with labeled, functional ε-toxin, it was found to accumulate on endothelia in various organs, especially the kidneys and brain (401,402). In the nervous system, ε-toxin associates with myelin structures (403). Although the neurotoxic effects were initially hypothesized to be caused by damage to brain blood vessels, later work showed that ε-toxin is also able to directly attack brain oligodendrocytes (82).…”