Identification Of Functional Amino Acids Of Clostridium Perfringens Epsilon Toxin Domain ? And Immunoprotection Of Non-toxic Mutants

? toxin(Etx),which is an extremely potent toxin produced by Clostridium perfringens toxinotypes B and D,belongs to the ?-pore-forming toxin family.Etx is the main causative factor causing fatal enterotoxiemia in livestock,and the mortality rate of diseased animals is almost 100%.Generating protective antibodies against Etx through immunization is an effective way to prevent and control diseases.In this study,based on the analysis of the three-dimensional structure of Etx,a series of Etx mutants were constructed by site-directed mutation of the aromatic amino acid cluster of Domain ?.The MDCK cytotoxicity test,calcium efflux test,detection of the binding and oligomerization of toxins on the cell membrane,and mouse lethality test were used to identify the function of the Etx mutant.The results confirmed that the 71st tyrosine(Y71)in Domain ? is a key amino acid that affects the toxicity of Etx,and the side chain aromatic ring group of amino acid residue 71 is the determinant of Etx cell killing activity in vitro and lethality in vivo.In order to further determine the potential of the constructed non-toxic mutant as a subunit vaccine antigen,we selected Etx-Y71A as a representative mutant for safety and immunogenicity studies in vivo.Histopathological observations showed that the mice did not show any pathological damage after inoculation with the mutant Etx-Y71A,and the fluorescence trace results also showed that the mutant lost the ability to cross the blood-brain barrier of the mice.These results suggested that the Etx-Y71A mutant is sufficiently safe in vivo to be a vaccine candidate.Both homology modeling and circular dichroism analysis showed that the structure of Etx-Y71A was not significantly different from that of wild-type Etx,indicating that the antigenic structure of the toxin did not change significantly,providing a structural basis for Etx-Y71A to retain the immunogenicity of the toxin.Mice inoculated with Etx-Y71A were induced to generate high levels of humoral immune activities and were completely protected from a 100 LD50 of trypsin-activated Etx challenge.Sheep can produce high titers of Etx-specific antibodies after inoculation with Etx-Y71A,and these antibodies have high levels of neutralizing activity by in vitro and in vivo tests.Importantly,lambs could receive passive immunity through immunization of pregnant ewes.These results indicate that Etx-Y71A has the potential as a subunit vaccine candidate.In summary,this study for the first time determined that the aromatic ring side chain at residue 71 of Domain ? is the determinant of Etx toxicity,filling the gap in the structure and function of Etx Domain ?;the constructed non-toxic mutant Etx-Y 71A has remarkable immune efficacy in both in model and host animals,providing a theoretical and experimental foundation the development of non-toxic recombinant Etx vaccines.