Simplified procedure for purification of Bordetella bronchiseptica dermonecrotic toxin

Research Center for Veterinary Science, Kitasato Institute, Chiba, Japan.
FEMS Microbiology Letters (Impact Factor: 2.12). 02/1990; 54(1-3):39-43. DOI: 10.1016/0378-1097(90)90255-O
Source: PubMed


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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    • "The anti-mouse nidogen-2 (M-300, sc-33143) and anti-collagen type I (234168) antibodies were from Santa Cruz and Calbiochem, respectively. The anti-DNT monoclonal antibody 2B3 and anti-DNT polyclonal antibody were prepared as reported [4,26]. Alexa 488-conjugated goat anti-rabbit IgG, Alexa 546-conjugated goat anti-mouse IgG, and Alexa 488-conjugated streptavidin were from Molecular Probes/Invitrogen. "
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    ABSTRACT: Bordetella dermonecrotic toxin (DNT) causes the turbinate atrophy in swine atrophic rhinitis, caused by a Bordetella bronchiseptica infection of pigs, by inhibiting osteoblastic differentiation. The toxin is not actively secreted from the bacteria, and is presumed to be present in only small amounts in infected areas. How such small amounts can affect target tissues is unknown. Fluorescence microscopy revealed that DNT associated with a fibrillar structure developed on cultured cells. A cellular component cross-linked with DNT conjugated with a cross-linker was identified as fibronectin by mass spectrometry. Colocalization of the fibronectin network on the cells with DNT was also observed by fluorescence microscope. Several lines of evidence suggested that DNT interacts with fibronectin not directly, but through another cellular component that remains to be identified. The colocalization was observed in not only DNT-sensitive cells but also insensitive cells, indicating that the fibronectin network neither serves as a receptor for the toxin nor is involved in the intoxicating procedures. The fibronectin network-associated toxin was easily liberated when the concentration of toxin in the local environment decreased, and was still active. Components in the extracellular matrix are known to regulate activities of various growth factors by binding and liberating them in response to alterations in the extracellular environment. Similarly, the fibronectin-based extracellular matrix may function as a temporary storage system for DNT, enabling small amounts of the toxin to efficiently affect target tissues or cells.
    Full-text · Article · Sep 2010 · BMC Microbiology
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    • "Bacterial toxins, proteins and chemicals DNT was purified from the cell extracts of B. bronchiseptica S798 by the method reported previously (Horiguchi et al., 1990). C3 and component I of botulinum C2 toxin were provided by S. Kozaki, University of Osaka Prefecture, Osaka, Japan. "
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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.
    Full-text · Article · Nov 1995 · Journal of Cell Science
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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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