Expression and characterization of recombinant epsilon toxin variants of Clostridium perfringens type D in...

Epsilon toxin (ETX) is the most potent clostridial toxin after tetanus and botulinum neurotoxins. ETX belongs to the heptameric β-pore-forming proteins produced by Clostridium perfringens types B and D, which cause fatal enterotoxemia and significant economic losses in livestock. ETX is synthesized as a protoxin and activated by proteolytic enzymes. Due to limitations in diagnosis and treatment, prevention through vaccination and good management practices is essential. Several commercial vaccines exist for C. perfringens enterotoxemia prevention. However, industrial production is costly and elicits variable immune responses. This has led to the study of recombinant subunit vaccines of ETX with lower toxicity and improved efficacy. The purpose of this study was to produce two versions of the recombinant toxin: truncated epsilon (rETX) and mutated epsilon (rETX^H106P). Both were cloned and expressed in a Pichia pastoris expression system, followed by small-scale cultivation and a cytotoxicity assay. The assays confirmed that both recombinant polypeptides were non-toxic. Large-scale cultivation conditions were subsequently adjusted to facilitate the evaluation of kinetic parameters and potential expression, focusing on rETX^H106P. This work demonstrated that rETX^H106P could potentially be used in a diagnostic kit or as a subunit vaccine candidate against enterotoxemia and other diseases caused by ETX.