Molecular and Structural Characterisation of Epsilon Toxin and Necrotic Enteritis Toxin B: Two Pore-forming Toxins from Clostridium Perfringens
Fernandes da Costa, Sérgio P.
Thesis or dissertation
University of Exeter
Reason for embargo
To enable publication elsewehere
Epsilon toxin (Etx) and necrotic enteritis toxin B (NetB) are two pore-forming toxins produced by C. perfringens. While Etx has been shown to be the key virulence factor for enterotoxemia in goats and sheep, NetB has been associated with the pathogenesis of avian necrotic enteritis (NE), a gastro-intestinal disease causing economic damage to the poultry industry worldwide. The crystal structure of Etx H149A (an Etx variant with 6x reduced toxicity relative to wild type toxin) was solved to 2.4 Å and showed that the H149A mutation in domain III does not affect organization of the receptor binding region in domain I. The Etx H149A structure also revealed a second putative glycan binding site in domain III. In addition, site-directed mutagenesis in domain I of Etx H149A affirmed the important role of tyrosine residues for toxin binding and demonstrated the capability of Etx H149A to be used as a platform for further receptor binding studies in the future. The crystal structure of the pore-form of NetB was solved to 3.9 Å and revealed high similarities to the Staphylococcus aureus α-hemolysin heptameric structure. However, in particular the region thought to interact with the target cell membrane showed some interesting divergence in amino acid composition. Site-directed mutagenesis within this domain significantly affected binding and toxicity of NetB to target cells. Mutagenesis within the β-sandwich domain of NetB revealed important amino acid residues for toxin oligomerisation and pore-formation. In order to test NetB toxoids as candidate vaccines, a NetB genetic toxoid and a formaldehyde NetB toxoid were used to immunise poultry in an in vivo NE disease model. Vaccination with any of the two antigens resulted in the induction of specific antibody responses against NetB and provided significant protection against disease.
PHD in Biological Sciences