Attenuated Clostridium perfringens Epsilon Toxin Mutants and Insights Into the Mechanism of Attenuation.

Abstract

Clostridium perfringens epsilon toxin (ETX) is a potent pore-forming exotoxin responsible for severe enterotoxemia and necrotizing enterocolitis in ruminants. To elucidate the molecular mechanisms underlying ETX pathogenicity and attenuation, several site-directed mutants (R25A, F92A, Y133A, F206A, D210A, and G221A) were constructed based on structural analysis. Cytotoxicity assays revealed reduced virulence in ETX-Y133A (hereafter referred to as Y133A), F92A, and F206A, with Y133A exhibiting the most significant attenuation. To further investigate the role of residue Y133, additional mutants (Y133E, Y133F, Y133S, Y133W, and Y133G) were generated. Selected mutants were evaluated for cytotoxicity, pathogenicity in BALB/c mice, and in vivo safety through histopathological analysis. Furthermore, their pore-forming ability, binding affinity to MDCK cells, and oligomerization properties were assessed. Results demonstrated that residue Y133 is critical for ETX activity, likely due to the necessity of its aromatic side chain for pore formation. In contrast, F92 and F206 appear to be involved in host-cell interactions via distinct mechanisms. These findings provide insights into ETX structure-function relationships and offer potential strategies for rational attenuation in vaccine development.