Binding of ɛ-toxin from Clostridium perfringens in the 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
References (20)
- et al.
Proteolysis of SNAP-25 by types E and A botulinal neurotoxins
J. Biol. Chem.
(1994) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding
Anal. Biochem.
(1976)- et al.
Lethal effects of Clostridium perfringens epsilon toxin are potentiated by alpha and perfringolysin-O toxins in a mouse model
Vet. Microbiol.
(2008) Histopathological changes in the brain of mice given Clostridium perfringens type D epsilon toxin
J. Comp. Pathol.
(1984)Ultrastructural changes in the brain of mice given Clostridium perfringens type D epsilon toxin
J. Comp. Pathol.
(1984)Neurological disorders produced by Clostridium perfringens type D epsilon toxin
Anaerobe
(2004)- et al.
Clostridium perfringens epsilon toxin causes excessive release of glutamate in the mouse hippocampus
FEMS Microbiol. Lett.
(2000) - et al.
Cleavage of a C-terminal peptide is essential for heptamerization of Clostridium perfringens epsilon-toxin in the synaptosomal membrane
J. Biol. Chem.
(2001) - et al.
Clostridium perfringens epsilon-toxin forms a heptameric pore within the detergent-insoluble microdomains of Madin-Darby canine kidney cells and rat synaptosomes
J. Biol. Chem.
(2002) - et al.
Distribution of labeled Clostridium perfringens epsilon toxin in mice
Toxicon.
(1991)
Cited by (74)
1.21 - Gut Microbiota in Brain diseases
2022, Comprehensive Gut MicrobiotaCentral residues of the amphipathic β-hairpin loop control the properties of Clostridium perfringens epsilon-toxin channel
2020, Biochimica et Biophysica Acta - BiomembranesMicrobiome and motor neuron diseases
2020, Progress in Molecular Biology and Translational ScienceImmune-mediated genesis of multiple sclerosis
2020, Journal of Translational AutoimmunityClostridium perfringens epsilon toxin: Toxic effects and mechanisms of action
2019, Biosafety and HealthCitation 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].
- 1
Present address: Department of Medicine, University of California, San Diego, 9500 Gilman Drive 0838, La Jolla, CA 92093-0838, United States.