Functional characterization of the Clostridium perfringens quadruple point mutant epsilon toxin.

Epsilon toxin (Etx) is one of the exotoxins (∼18) secreted by the spore-forming bacterium Clostridium perfringens, which plays a major role in the pathogenesis of enterotoxaemia (ET) leading to sudden death in affected small ruminants. A chemically inactivated toxoid of Etx has been used as a vaccine to control ET in animals. Non-toxic Etx-mutant proteins could potentially be used in the development of efficient immuno-assays and alternative subunit vaccine formulations for the control of ET in animals. In this study, a codon-optimized synthetic quadruple point mutant (Y30A, H106P, H149A, Y196A) of the etx gene of Clostridium perfringens was expressed in Escherichia coli to produce a recombinant protein (331 aa, ∼36 kDa). The rEtx-mutant protein was purified under both non-denaturing and denaturing-renatured conditions using a single-step affinity chromatography and was functionally characterized in vivo and in vitro. A quadruple point mutant of Etx was structurally similar to the wildtype Etx. The rEtx protein was activated by trypsin, and had no toxicity when tested using in vitro and in vivo models. Furthermore, the protein elicited antigen-specific antibodies in mice, rabbit, and guinea pigs. In an indirect ELISA, the rEtx-mutant protein was able to detect specific antibodies in sera from ET-vaccinated sheep.