Functional Analysis of Neutralizing Antibodies against Clostridium perfringens Epsilon-Toxin

Abstract

The Clostridium perfringens epsilon-toxin causes a severe, often fatal illness (enterotoxemia) characterized by cardiac, pulmonary, kidney, and brain edema. In this study, we examined the activities of two neutralizing monoclonal antibodies against the C. perfringens epsilon-toxin. Both antibodies inhibited epsilon-toxin cytotoxicity towards cultured MDCK cells and inhibited the ability of the toxin to form pores in the plasma membranes of cells, as shown by staining cells with the membrane-impermeant dye 7-aminoactinomycin D. Using an antibody competition enzyme-linked immunosorbent assay (ELISA), a peptide array, and analysis of mutant toxins, we mapped the epitope recognized by one of the neutralizing monoclonal antibodies to amino acids 134 to 145. The antibody competition ELISA and analysis of mutant toxins suggest that the second neutralizing monoclonal antibody also recognizes an epitope in close proximity to this region. The region comprised of amino acids 134 to 145 overlaps an amphipathic loop corresponding to the putative membrane insertion domain of the toxin. Identifying the epitopes recognized by these neutralizing antibodies constitutes an important first step in the development of therapeutic agents that could be used to counter the effects of the epsilon-toxin.The Clostridium perfringens species is divided into five types, A through E, based on production of the four "major toxins," the alpha-, beta-, epsilon-, and iota-toxins (50). Epsilon-toxin is one of the toxins produced by type B and D strains (44). The epsilon-toxin can lead to a fatal illness (enterotoxemia) in a variety of livestock animals, most frequently in sheep (50). Clinical signs in intoxicated sheep may include colic, diarrhea, and numerous neurological symptoms. Postmortem analysis reveals widespread increases in vascular permeability with cerebral, cardiac, pulmonary, and kidney edema (52, 53). Experimental intoxication of mice and rats with epsilon-toxin causes a rapidly fatal illness and pathological changes similar to those seen in livestock (12,13,28,46). The dose of epsilon-toxin required to kill 50% of mice has been estimated at between 65 and 110 ng per kg (27), which indicates that epsilon-toxin is one of the most potent known bacterial protein toxins (14). Despite reports of epsilon-toxin-producing C. perfringens being isolated from humans, it is unclear whether or not epsilontoxin causes illness in humans (15,22,26,31,34,49). Due to the clear threat posed to livestock and the potential threat to human health, the epsilon-toxin is classified as a category B overlap select agent by the U.S. Department of Health and Human Services and the U.S. Department of Agriculture.To protect livestock from epsilon-toxin, both a vaccine (based on formalin-inactivated epsilon-toxin) and an equinederived antitoxin are available. Due to the rapid progression of the disease among livestock animals, treatment is generally not possible or practical, and the emphasis is placed on prevention either by vaccination or by administration of ant...

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“…The Ni-NTA column was washed with a buffer composed of 20 mM sodium phosphate, 300 mM sodium chloride, and 20 mM imidazole (pH 8.0), and the ⑀-prototoxin was eluted in a buffer composed of 20 mM sodium phosphate, 300 mM sodium chloride, and 250 mM imidazole (pH 8.0). The identification of the ⑀-prototoxin in the purified sample was confirmed by immunoblotting with ⑀-toxinspecific monoclonal antibody 5B7 (35,36). Protein concentrations were determined using micro-BCA (Pierce).…”

“…Activation of ⑀-Toxin by Trypsin-The ⑀-prototoxin can be cleaved with trypsin to remove short peptides from both the Nand C-terminal ends of the protein to yield the active ⑀-toxin (2,28,35). Trypsin-coated agarose beads (Pierce) were washed and resuspended in 5 mM Tris, pH 7.5.…”

“…Following precedent in the literature, the N-terminal amino acid of the mature ⑀-toxin (i.e. following trypsin treatment) is numbered 1 (35,37).…”

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