Purification and characterization of Clostridium perfringens iota toxin
| dc.contributor.author | Stiles, Bradley G. | en |
| dc.contributor.committeechair | Wilkins, Tracy D. | en |
| dc.contributor.committeemember | Johnson, J.L. | en |
| dc.contributor.committeemember | Smibert, R.M. | en |
| dc.contributor.committeemember | Gregory, Eugene "Mick" | en |
| dc.contributor.committeemember | Chen, Jiann - Shin | en |
| dc.contributor.department | Microbiology | en |
| dc.date.accessioned | 2017-03-10T21:55:00Z | en |
| dc.date.available | 2017-03-10T21:55:00Z | en |
| dc.date.issued | 1987 | en |
| dc.description.abstract | 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 (i<sub>b</sub>), had some guinea pig dermonecrotic and mouse lethal activity. Other fractions, including the one containing the slower migrating iota a (i<sub>a</sub>) antigen, had little to no biological activity. When fractions containing i<sub>a</sub> and i<sub>b</sub> 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 i<sub>a</sub> or i<sub>b</sub>, 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 i<sub>a</sub> and i<sub>b</sub>, 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 i<sub>a</sub> and i<sub>b</sub> were mixed. Monospecific antisera against purified i<sub>a</sub> and i<sub>b</sub> neutralizd the iota or iota-like activity of crude supernatants. A sensitive and specific ELISA was developed using monospecific and C. spiroforme antisera. The i<sub>a</sub> and i<sub>b</sub> proteins have a pI of 5.2 and 4.2 and molecular weights of 48,000 and 71,000 (SDS PAGE), respectively. The i<sub>a</sub> protein is heat stable (85° C/15 min) while i<sub>b</sub> lost its activity at 55°C. Amino terminus sequencing revealed that both proteins were blocked by an unknown functional group(s). Purified i<sub>a</sub>, but not i<sub>b</sub>, 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. | en |
| dc.description.degree | Ph. D. | en |
| dc.format.extent | vi, 121 leaves | en |
| dc.format.mimetype | application/pdf | en |
| dc.identifier.uri | http://hdl.handle.net/10919/76516 | en |
| dc.language.iso | en_US | en |
| dc.publisher | Virginia Polytechnic Institute and State University | en |
| dc.relation.isformatof | OCLC# 16882929 | en |
| dc.rights | In Copyright | en |
| dc.rights.uri | http://rightsstatements.org/vocab/InC/1.0/ | en |
| dc.subject.lcc | LD5655.V856 1987.S844 | en |
| dc.subject.lcsh | Clostridial enteritis | en |
| dc.subject.lcsh | Clostridium diseases | en |
| dc.subject.lcsh | Microbial toxins | en |
| dc.title | Purification and characterization of Clostridium perfringens iota toxin | en |
| dc.type | Dissertation | en |
| dc.type.dcmitype | Text | en |
| thesis.degree.discipline | Microbiology | en |
| thesis.degree.grantor | Virginia Polytechnic Institute and State University | en |
| thesis.degree.level | doctoral | en |
| thesis.degree.name | Ph. D. | en |
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