A panel of ten site-directed mutants of Clostridium perfringens ε-toxin was generated. All of the mutated proteins expressed in Escherichia coli were recognized in immunoblots by a neutralizing mAb raised against wild-type native ε-toxin. The cytotoxicity of the site-directed mutated toxins was assayed in vitro against MDCK cells. One mutation resulting in loss of activity in the assay was identified. This non-toxic protein was derived by substituting a proline for the histidine at residue 106 of the toxin. Immunization of mice with the non-toxic mutated ε-toxin resulted in the induction of a specific antibody response and immunized mice were protected against 1000 LD50 doses of wild-type recombinant ε-toxin.
BhownA. S.,
HabeebA. F. S. A.1977; Structural studies on ε- prototoxin of Clostridium perfingens type D. Localisation of the site of tryptic scission necessary for activation to ε-toxin.. Biochem Biophys Res Commun 73:889–896
BrooksM. E.,
SterneM.,
WarrackG. H.1957; A reassessment of the criteria used for type differentiation of Clostridium perfringens
. J Pathol Bacteriol 74:185–195
BullenJ. J.1970 Role of toxins in host-parasite relationships.. In Microbial Toxins vol 1 pp 233–276 Edited by
AjlS.,
KadisS.,
MontieT. C.
New York: Academic Press;
BullenJ. J.,
BattyI.1956; The effect of Clostridium welcbii type D culture filtrates on the permeability of the mouse intestine.. J Pathol Bacteriol 71:311–323
CanardB.,
Saint JoanisB.,
ColeS. T.1992; Genomic diversity and organization of virulence genes in the pathogenic anaerobe Clostridium perfringens
. Mol Microbiol 6:1421–1429
GarnierJ.,
OsguthorpeD.,
RobsonB.1978; Analysis of the accuracy and implications of simple methods for predicting the secondary structure of globular proteins.. J Mol Biol 120:97–120
HabeebA. F.1969; Studies on epsilon-prototoxin of Clostridium perfringens type D. I. Purification methods: evidence for multiple forms of epsilon-prototoxin.. Arch Biochem Biophys 130:430–440
HavardH. L.,
HunterS. E.,
TitballR. W.1992; Comparison of the nucleotide sequence and development of a PCR test for the epsilon toxin gene of Clostridium perfringens type B and type D.. FEMS Microbiol Lett 76:77–81
HunterS. E.,
ClarkeI. N.,
KellyD. C.,
TitballR. W.1992; Cloning and nucleotide sequencing of the Clostridium perfringens epsilon-toxin gene and its expression in Escherichia coli
. Infect Immun 60:102–110
LoganA. J.,
WilliamsonE. D.,
TitballR. W.,
PercivalD. A.,
ShuttleworthA. D.,
ConlanJ. W.,
KellyD. C.1991; Epitope mapping of the alpha-toxin of Clostridium perfringens
. Infect Immun 59:4338–4342
NakamayeK. L.,
EcksteinF.1986; Inhibition of restriction endonuclease Neil cleavage by phosphorothioate groups and its application to oligonucleotide-directed mutagenesis.. Nucleic Acids Res 14:9679–9698
PayneD. W.,
WilliamsonE. D.,
HavardH.,
ModiN.,
BrownJ.1994; Evaluation of a new cytotoxicity assay for Clostridium perfringens type D epsilon toxin.. FEMS Microbiol Lett 116:161–167
PercivalD. A.,
ShuttleworthA. D.,
WilliamsonE. D.,
KellyD. C.1990; Anti-idiotypic antibody-induced protection against Clostridium perfringens type D.. Infect Immun 58:2487–2492
SakuraiJ.,
NagahamaM.1985; Role of one tryptophan residue in the lethal activity of Clostridium perfringens epsilon toxin.. Biochem Biophys Res Commun 128:760–766
SakuraiJ.,
NagahamaM.1987b; The inactivation of Clostridium perfringens epsilon toxin by treatment with tetra- nitromethane and N-acetylimidazole.. Toxicon 25:279–284
SayersJ. R.,
SchmidtW.,
EcksteinF.1988; 5ʹ-3ʹ exonucleases in phosphorothioate-based oligonucleotide-directed mutagenesis.. Nucleic Acids Res 16:791–801
StokkaG. L.,
EdwardsA. J.,
SpireM. F.,
BrandtR. T.,
SmithJ. E.1994; Inflammatory response to clostridial vaccines in feedlot cattle.. J Am Vet Med Assoc 204:415–419
TaylorJ. W.,
OttJ.,
EcksteinF.1985a; The rapid generation of oligonucleotide-directed mutations at high frequency using phosphorothioate-modified DNA.. Nucleic Acids Res 13:8765–8785
TaylorJ. W.,
SchmidtW.,
CosstickR.,
OkruszekA.,
EcksteinE.1985b; The use of phosphorothioate-modified DNA in restriction enzyme reactions to prepare nicked DNA.. Nucleic Acids Res 13:8749–8764
TitballR. W.,
HunterS. E.,
MartinK. L.,
MorrisB. C.,
Shuttle- worthA. D.,
RubidgeT.,
AndersonD. W.,
KellyD. C.1989; Molecular cloning and nucleotide sequence of the alpha-toxin (phospholipase) of Clostridium perfringens
. Infect Immun 57:367–376
WorthingtonR. W.,
MuldersM. S.1977; Physical changes in the epsilon prototoxin molecule of Clostridium perfringens during enzymatic activation.. Infect Immun 18:549–551
WorthingtonR. W.,
MuldersM. S.,
van RensburgJ. J.1973; Enzymatic activation of Clostridium perfringens epsilon prototoxin and some biological properties of activated toxin.. Onder- stepoort J Vet Res 40:151–154