Purification and characterization of Clostridium perfringens iota toxin

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Virginia Polytechnic Institute and State University

Clostridium perfringens type E iota toxin is implicated in some cases of fatal diarrhea in calves, lambs, and guinea pigs. A crossreacting "iota-like" toxin, produced by Clostridium spiroforme, is responsible for antibiotic-associated and weaning related enterotoxemias of rabbits. Antisera developed against culture supernatant of either organism neutralized the biological activity of iota or iota-like toxin. By using C. spiroforme antiserum and crossed immunoelectrophoresis (crossed IEP), we found two cross-reacting antigens in C. perfringens type E supernatants. C. perfringens types A, B, C, and D, which do not produce iota toxin, did not cross-react with C. spiroforme antiserum.

To determine if either antigen had iota toxin activity, we separated the cross-reacting antigens of C. perfringens by preparative isoelectric focusing (IEF) and tested all IEF fractions for biological activity in guinea pigs and mice. The fraction containing the faster-migrating antigen seen in crossed IEP, designated iota b (ib), had some guinea pig dermonecrotic and mouse lethal activity. Other fractions, including the one containing the slower migrating iota a (ia) antigen, had little to no biological activity. When fractions containing ia and ib were mixed, there was an 8 and 25 fold increase in mouse lethal and dermonecrotic titers, respectively. Activity was neutralized by C. perfringens type E or C. spiroforme antisera and other fractions, when mixed with ia or ib, did not have a synergistic effect.

Both components of C. perfringens iota toxin were purified using ammonium sulfate precipitation, DEAE anion exchange chromatography, preparative IEF, Sephadex G-100 gel filtration, and flatbed electrophoresis to yield a 12 and 5% final recovery of ia and ib, respectively. Each protein was homogeneous by SDS PAGE, gradient PAGE, and crossed IEP using homologous antiserum. There was at least an 8 fold increase in mouse lethal titer and 64 fold increase in dermonecrotic titer when equimolar amounts of ia and ib were mixed. Monospecific antisera against purified ia and ib neutralizd the iota or iota-like activity of crude supernatants. A sensitive and specific ELISA was developed using monospecific and C. spiroforme antisera.

The ia and ib proteins have a pI of 5.2 and 4.2 and molecular weights of 48,000 and 71,000 (SDS PAGE), respectively. The ia protein is heat stable (85° C/15 min) while ib lost its activity at 55°C. Amino terminus sequencing revealed that both proteins were blocked by an unknown functional group(s). Purified ia, but not ib, has ADP-ribosylating activity specific poly-L-arginine in vitro. Recent evidence suggests that nonmuscle actin, involved in the cytoskeletal structure of eucaryotic cells, may act as the in situ acceptor.