Investigating the Importance of the Beta-octyl Glucoside Binding Site for the Toxicity of C. Perfringens Epsilon Toxin

Abstract

C. perfringens epsilon toxin (ε-toxin) is a potent heptameric β-pore forming toxin secreted by C. perfringens toxinotypes B and D. ε-toxin causes fatal disease in livestock populations that is characterised by severe neurological disruption. Numerous formaldehyde based veterinary vaccines against ε-toxin are available. However, these suffer from poor immunogenicity and side effects and are unsuitable for human use. Recently, ε-toxin has also been proposed as an aetiological agent for multiple sclerosis following the isolation of C. perfringens Type B in a multiple sclerosis (MS) patient. Further research has identified ten times greater ε-toxin immunoreactivity in MS patients compared to healthy controls. The aim of this study is to investigate two areas. Firstly, ε-toxin’s betaoctyl glucoside (BOG) binding site was analysed for cytotoxicity against Chinese hamster ovary cells (CHO) expressing the putative myelin and lymphocyte (MAL) receptor using seven site directed mutants (V72A, V72F, F92A, T93A, V166A, V166F, A168F). The V72F, V166F and the A168F mutants demonstrate strongly reduced toxicity in CHO-hMAL cells and show promise as potential recombinant vaccine candidates against ε-toxin. Secondly, sera from control, CIS and MS patients were screened for ε-toxin immunoreactivity by investigating if sera conferred cytotoxic protection to CHO-hMAL cells incubated with ε-toxin. However, high sera lactate dehydrogenase (LDH) enzyme activity distorted experimental results. Consequently, it remains unclear if the sera exhibit immunoreactivity towards WT ε-toxin. The results suggest that fresh standardised sera samples should be used in future to mitigate this issue by ensuring greater experimental control.