Simplified procedure for purification of Bordetella bronchiseptica dermonecrotic toxin
ABSTRACT Bordetella bronchiseptica dermonecrotic toxin was purified by a simplified method. The method consisted of SP Toyopearl 650M chromatography and high performance liquid chromatography on a TSK gel G3000SW column. 47.5% of the activity of the crude cell extract was recovered. The purified toxin behaved as a homogeneous protein in sodium dodecyl sulfate polyacrylamide gel electrophoresis, high performance liquid chromatography, and agar gel double diffusion tests.
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ABSTRACT: We studied the biochemical mechanism of morphological changes in cells treated with Bordetella dermonecrotizing toxin (DNT). DNT caused the morphological changes of serum-starved MC3T3-E1 cells from flat shapes to reflactile ones. These changes were accompanied by the assembly of actin stress fibers and focal adhesions, which is known to be regulated by the small GTP-binding protein rho. Clostridium botulinum C3 exoenzyme, which ADP-ribosylates and inactivates rho protein, 'rounded' the cells within 2 hours after addition to the extracellular fluid and their rounded shapes were maintained for at least 10 hours. However, when the cells were co-treated with C3 exoenzyme and DNT, they were rounded at 2 hours but recovered an apparently intact morphology after 3-8 hours of incubation. rho proteins in lysates from DNT-treated cells and untreated cells were radiolabeled by [32P]ADP-ribosylation with C3 exoenzyme and analyzed by SDS-polyacrylamide gel electrophoresis. Whereas the lysate from untreated cells showed a single band of [32P]ADP-ribosylated rho protein, the lysate from DNT-treated cells showed an additional two bands as well as the band identical to that of the lysate from untreated cells. Recombinant rhoA protein treated with DNT in vitro also showed a mobility shift in SDS-polyacrylamide gel electrophoresis. These results indicate that DNT causes the assembly of actin stress fibers and focal adhesions by directly modifying rho protein.Journal of Cell Science 11/1995; 108 ( Pt 10)(10):3243-51. · 5.33 Impact Factor
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ABSTRACT: incorporation increased inthetoxin-treated cells more than24hafter addition ofthetoxinundertheserum-starved conditions. Thiseffect was dependent on thetoxinconcentration ranging from0.3to3ng/ml andwas eliminated byaphidicolin andhydroxyurea, inhibitors forDNA replication. Inthetoxin-treated culture, thenumberofcellsdidnotincrease butpolynucleated cells appeared andtheir numberincreased toca.50%ofthetotal numberofcells6days afterthetoxin addition. Fromthese results, we concluded that thetoxinstimulates DNAreplication inMC3T3-E1cells without cellproliferation. Bordetella bronchiseptica dermonecrotic toxin (DNT)is considered tobeavinilence factor causing turbinate atrophy in swine atrophic rhinitis (1, 3,4,8,14). Several reports suggested that DNT caused degenerative effects onbonetissue (4,8,14). However, theexactrole ofthetoxin inproduction ofturbinate atrophy hasnotbeenclarified because ofthelackofahighly purified toxin preparation andanappropriate invitro modelfor analysis ofitsaction on bonetissue. In1989, Horiguchi etal.succeeded inpurifying DNT asa single-chain polypeptide withamolecular weight of145,000 (11). Recently, Horiguchi etal.(13) foundthat DNT induced morphological changes inclonal osteoblast-like MC3T3-E1 cells andreducedaccumulation oftypeI collagen and elevation ofalkaline phosphatase activity, bothofwhichare closely linked toosteoblastic differentiation ofMC3T3-E1 cells. Thissuggested thatDNT causesturbinate atrophy by impairing theability ofosteoprogenitor cells todifferentiate, resulting inthereduction ofboneformation. Whileinvestigating themorphological changes inMC3T3-E1 cells induced byDNT,we observed many polynucleated cells inthetoxin-treated cells. TheamountofDNA inMC3T3-E1 cells seemed tobeincreased byDNT because thecellnumbers intoxin-treated cultures anduntreated cultures were similar. Previous reports showedthatDNA synthesis inMC3T3-E1 cells was stimulated byprostaglandin F2 andepidermal growth factor, whichinhibit expression ofosteoblastic pheno- types(5, 6, 10). Inaddition, Pasteurella multocida toxin, which issimilar inbiological activities toDNT butserologically distinct (17), was reported tobea potent mitogen toseveral kindsofcultured fibroblasts (21). Therefore, we examined DNA synthesis inMC3T3-E1cells treated withDNT.Here, we report that DNT stimulates DNA synthesis intheserum- starved MC3T3-E1 cellsandthatDNT leads thecellstoform polynucleated cellsinstead ofstimulating cellproliferation. The results presented hereshowthattheeffect ofDNT on DNA synthesis isobviously different fromthatofP.multocida toxin. MATERIALSAND METHODS DNT andother reagents. DNT was purified fromthecell extract ofB.bronchiseptica S798(provided byN.Terakado,
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ABSTRACT: The effects of Bordetella bronchiseptica dermonecrotic toxin on the structure and function of a clonal osteoblastic cell line, MC3T3-E1, were investigated. The toxin induced a morphological change in the cells from a spindle shape to a spherical form with many blebs. The toxin-treated cells were viable and grew to form confluent cell layers composed of irregularly shaped cells and multinuclear cells. The toxin inhibited elevation of alkaline phosphatase activity in the cells in a dose-dependent manner at concentrations from 10 pg to 10 ng/ml. The accumulation of type I collagen in the cells was also reduced by the toxin. Since high alkaline phosphatase activity and accumulation of collagen are closely linked to differentiation of the cells into osteoblasts, it is considered likely that B. bronchiseptica dermonecrotic toxin impairs the ability of the cells to differentiate.Infection and Immunity 04/1991; 59(3):1112-6. · 4.16 Impact Factor