Bordetella bronchiseptica dermonecrotic toxin, purification and characterisation
thesisposted on 15.12.2014, 10:32 by Toni Elizabeth. Adams
Dermonecrotic toxin (DNT) is produced by all the Bordetella species, and DNT from B. bronchiseptica is considered to be an important virulence factor in turbinate atrophy of pigs.;Recombinant DNT (rDNT) was purified by sonication, ion-exchange and hydroxylapatite chromatography. Other methods for the purification of wild-type DNT and rDNT, including preparative isoelectric focusing and hydrophobic chromatography, were investigated in detail.;Partially pure preparations of rDNT contained a 145 kDa protein band and were cytotoxic to embryonic bovine lung (EBL) cells. Partially pure rDNT induced the formation of actin stress fibres and focal adhesions in Swiss 3T3 cells. In addition, rDNT stimulated DNA synthesis in quiescent Swiss 3T3 cells but prevented cell proliferation, resulting in binucleated cells. Recombinant DNT has been shown to directly modify the small GTP-binding protein, Rho, (Pullinger, unpublished), which regulates the cell cytoskeleton. Results from this thesis indicate that rDNT causes the assembly of actin stress fibres and focal adhesion possibly by direct activation of the Rho protein.;Partially purified rDNT with a site-directed mutation in a putative nucleotide-binding motif did not induce cytoskeletal rearrangements and did not stimulate DNA synthesis in Swiss 3T3 cells. This suggests that the nucleotide-binding motif is essential for activity.;Two lines of evidence indicate that the toxin is internalised in the endosomal/lysosomal compartment: i) stimulation of DNA synthesis by transient exposure of Swiss 3T3 cells to rDNT, and ii) blocking of rDNT-induced DNA synthesis with methylamine.;Three monoclonal antibodies (mAbs) were produced against B. bronchiseptica DNT. These mAbs recognised rDNT and B. pertussis DNT, but none neutralised the cytotoxic activity of DNT on EBL cells.;The partial purification of rDNT and characterisation of its biological effects provide valuable information for further studies of the toxin, including analysis of its enzymatic mode of action and its role in infection. Also, DNT may prove to be a useful tool for analysis of cell responses involving the important signalling molecule, Rho.