Purification and characterization of Clostridium perfringens iota toxin

dc.contributor.authorStiles, Bradley G.en
dc.contributor.committeechairWilkins, Tracy D.en
dc.contributor.committeememberJohnson, J.L.en
dc.contributor.committeememberSmibert, R.M.en
dc.contributor.committeememberGregory, Eugene "Mick"en
dc.contributor.committeememberChen, Jiann - Shinen
dc.contributor.departmentMicrobiologyen
dc.date.accessioned2017-03-10T21:55:00Zen
dc.date.available2017-03-10T21:55:00Zen
dc.date.issued1987en
dc.description.abstractClostridium 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.degreePh. D.en
dc.format.extentvi, 121 leavesen
dc.format.mimetypeapplication/pdfen
dc.identifier.urihttp://hdl.handle.net/10919/76516en
dc.language.isoen_USen
dc.publisherVirginia Polytechnic Institute and State Universityen
dc.relation.isformatofOCLC# 16882929en
dc.rightsIn Copyrighten
dc.rights.urihttp://rightsstatements.org/vocab/InC/1.0/en
dc.subject.lccLD5655.V856 1987.S844en
dc.subject.lcshClostridial enteritisen
dc.subject.lcshClostridium diseasesen
dc.subject.lcshMicrobial toxinsen
dc.titlePurification and characterization of Clostridium perfringens iota toxinen
dc.typeDissertationen
dc.type.dcmitypeTexten
thesis.degree.disciplineMicrobiologyen
thesis.degree.grantorVirginia Polytechnic Institute and State Universityen
thesis.degree.leveldoctoralen
thesis.degree.namePh. D.en

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