Elsevier

Veterinary Microbiology

Volume 131, Issues 1–2, 18 September 2008, Pages 14-25
Veterinary Microbiology

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

https://doi.org/10.1016/j.vetmic.2008.02.015Get rights and content

Abstract

Epsilon-toxin (ɛ-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. ɛ-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 ɛ-toxin, not only in the mouse nervous system but also in the brains of sheep and cattle. An ɛ-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 ɛ-toxin to myelin structures. ɛ-Prototoxin-GFP and ɛ-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 ɛ-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 ɛ-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.

Introduction

Epsilon-toxin (ɛ-toxin), a protein synthesized by the B and D strains of the anaerobic bacteria Clostridium perfringens, causes fatal enterotoxaemia in livestock, characterized by acute neurological signs and sudden death (Finnie, 2004). The toxin is produced as a non-toxic precursor molecule that is activated upon proteolytic cleavage of amino and carboxy terminal peptides (Minami et al., 1997). Although non-active, the prototoxin molecule presumably binds to the same surface cell receptors as the full active molecule and can prevent its binding and further toxicity. The active toxin produces diffuse vasogenic oedema in organs such as the lungs, kidneys and brain and causes neurological disorders such as opisthotonus, convulsions and agonal struggling, leading rapidly to death (Finnie, 2004, Smedley et al., 2004).

Mice have been widely used to study the effect of ɛ-toxin and provide a useful model for laboratory controlled intoxication studies (Finnie, 1984a, Finnie, 1984b, Fernandez-Miyakawa et al., 2007a, Fernandez-Miyakawa et al., 2007b, Fernandez-Miyakawa et al., 2008). After i.v. or i.p. injections into mice the toxin accumulates in several organs, above all in the kidneys and in the nervous system: the binding to the nervous system is specific and saturable (Nagahama and Sakurai, 1991). In mice, as in sheep and other naturally sensitive species, the toxin has the capacity to cross the blood brain barrier (BBB) and enter the brain parenchyma (Soler-Jover et al., 2007). However, there is little information about the final location of the toxin in the brain once it crosses the BBB. Previous results have shown the binding of ɛ-toxin to a synaptosomal fraction (Nagahama and Sakurai, 1992) and glial cells (Soler-Jover et al., 2007), suggesting possible targets. Based on the variety of symptoms and on the distribution of the toxin, we decided to examine the toxin's potential targets by studying its binding to the nervous system in mice. We then extended the study to include sheep, one of ɛ-toxin's principal natural targets.

Section snippets

Expression of the recombinant protein ɛ-prototoxin-GFP

ɛ-Prototoxin and ɛ-prototoxin-GFP were produced and purified as previously described (Soler-Jover et al., 2007). Briefly, the expression of either ɛ-prototoxin or ɛ-prototoxin-GFP was induced overnight with 0.4 mM isopropyl beta-d-thiogalactopyranoside (IPTG) at room temperature (RT), in 250 ml LB medium cultures. Cells were pelleted and resuspended in ice cold phosphate buffer (PB) 20 mM pH 7.5 with NaCl 250 mM, sonicated and centrifuged at 15,000 × g for 20 min. The resultant supernatant was

Glutamate release from brain synaptosomes

Isolated nerve terminal preparations (synaptosomes) have been widely used to analyze the effect of numerous substances, including excitatory and inhibitory neurotoxins, on neurotransmitter release. Synaptosomes were isolated from rat or mouse brains and a specific fluorometric assay was performed to detect “on line” glutamate release (Nicholls and Sihra, 1986), to study the possible excitatory effect of ɛ-toxin on glutamate release directly from nerve terminals. No glutamate release from rat or

Discussion

Some of the neurological disorders produced by ɛ-toxin in experimental and naturally poisoned animals are due to the “massive” secretion of glutamate from glutamatergic nerve terminals, triggering an excitotoxic episode characterized by convulsions (seizures) and neuronal cell death. This effect is an established characteristic of ɛ-toxin, observed in laboratory animals when lethal and sublethal doses of the toxin were injected (Miyamoto et al., 1998, Miyamoto et al., 2000). The massive

Conclusions

ɛ-Toxin binds specifically to myelin in both the central and peripheral nerve systems. The binding is mediated, at least in part, by a protein component of myelin. This characteristic of the toxin is not limited to mouse or rat myelin, the animal models used here, but is also evident in ovine, bovine and human myelin, though with different levels of intensity. In both ovine and bovine brains, at least, the toxin binds strongly to vascular endothelia. The functional importance of the ɛ-toxin

Acknowledgements

We are grateful to Serveis Cientificotècnics of the University of Barcelona (Bellvitge Campus) for their assistance with the confocal microscopy tests. We would also like to thank Inmaculada Gómez de Aranda and Benjamín Torrejón for their excellent technical assistance, Xenia Grandes and Ezequiel Mas for their help obtaining isolated nerve fibres, Dr. Isidre Ferrer and the Brain Bank of the Institute of Neuropathology for providing the human nerve fibres and Serveis Lingüístics (University of

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    Citation Excerpt :

    Moreover, the binding to myelin was found not only in rodents, but also in humans, sheep and cattle [36]. However, Dorca-Arevalo et al. reported that myelin structures bound to ETX were probably the contaminants from the synaptosomal preparation, and ETX did not act directly on nerve terminals [36]. Notably, ETX strongly bound to the vascular endothelium in the brains of both sheep and cattle, but not in rodent species, which may explain the differences in potency and effect of ETX among different animal species [36].

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Present address: Department of Medicine, University of California, San Diego, 9500 Gilman Drive 0838, La Jolla, CA 92093-0838, United States.

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