Simplified procedure for purification of Bordetella bronchiseptica dermonecrotic toxin.

Research Center for Veterinary Science, Kitasato Institute, Chiba, Japan.
FEMS Microbiology Letters (Impact Factor: 2.72). 02/1990; 54(1-3):39-43. DOI: 10.1016/0378-1097(90)90255-O
Source: PubMed

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: 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: Bordetella bronchiseptica is a pathogenic bacterium causing respiratory infections in a broad range of mammals. Recently, we determined the whole genome sequence of B. bronchiseptica S798 strain isolated from a pig infected with atrophic rhinitis and found four single-nucleotide polymorphisms (SNPs) at positions -129, -72, +22, and +38 in the region upstream of dnt encoding dermonecrotic toxin (DNT), when compared with a rabbit isolate, RB50. DNT is known to be involved in turbinate atrophy observed in atrophic rhinitis. Immunoblotting, quantitative real-time PCR, and β-galactosidase reporter assay revealed that these SNPs resulted in the increased promoter activity of dnt and conferred the increased ability to produce DNT on the bacteria. Similar or identical SNPs were also found in other pig isolates kept in our laboratory, all of which produce a larger amount of DNT than RB50. Our analysis revealed that substitution of at least two of the four bases, at positions -72 and +22, influenced the promoter activity for dnt. These results imply that these SNPs are involved in the pathogenicity of bordetellae specific to pig diseases.
    PLoS ONE 02/2015; 10(2):e0116604. DOI:10.1371/journal.pone.0116604 · 3.53 Impact Factor
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    ABSTRACT: Bordetella dermonecrotic toxin (DNT) is a virulence factor produced by bacteria belonging to the genus Bordetella including B. pertussis, B. parapertussis, and B. bronchiseptica. Intensive studies have clarified the structure and function of DNT in the last decade long after its discovery by Bordet and Gengou (Bordet, 1909). DNT is a single-chain polypeptide composed of 1464 amino acids and possesses novel transglutaminase activity that catalyzes polyamination or deamidation of the small GTPases of the Rho family. The modified GTPases lose their GTP hydrolyzing activity, function as a constitutive active molecule, and continuously transduce signals to downstream effectors, which mediate various abnormal phenotypes in intoxicated cells.
    Toxin Reviews 10/2008; 25(1):125-135. DOI:10.1080/15569540500321019 · 0.84 Impact Factor