Production of Highly Active Recombinant Dermonecrotic Toxin of Bordetella Pertussis

Abstract

Pathogenic Bordetella bacteria release a neurotropic dermonecrotic toxin (DNT) that is endocytosed into animal cells and permanently activates the Rho family GTPases by polyamination or deamidation of the glutamine residues in their switch II regions (e.g., Gln63 of RhoA). DNT was found to enable high level colonization of the nasal cavity of pigs by B. bronchiseptica and the capacity of DNT to inhibit differentiation of nasal turbinate bone osteoblasts causes atrophic rhinitis in infected pigs. However, it remains unknown whether DNT plays any role also in virulence of the human pathogen B. pertussis and in pathogenesis of the whooping cough disease. We report a procedure for purification of large amounts of LPS-free recombinant DNT that exhibits a high biological activity on cells expressing the DNT receptors Cav3.1 and Cav3.2Electron microscopy and single particle image analysis of negatively stained preparations revealed that the DNT molecule adopts a V-shaped structure with well-resolved protein domains. These results open the way to structure–function studies on DNT and its interactions with airway epithelial layers.

Design of a Quantitative LC-MS Method for Residual Toxins Adenylate Cyclase Toxin (ACT), Dermonecrotic Toxin (DNT) and Tracheal Cytotoxin (TCT) in Bordetella pertussis Vaccines

“…. No certified analytical reference standard is currently available for DNT; however, a recently published protocol described production of recombinant DNT that could facilitate screening of alternative cell or immunochemical-based approaches, in addition to quantitative accuracy/precision in MS-based workflows [42]. The detection limit for LC-MS analysis of DNT (Table 2) would need to be improved by an estimated 10 to 100-fold before MRM workflows can approach the sensitivity of current animal methods.…”

Vacunación en neonatos contra la enfermedad resurgente pertussis: un desafío, una estrategia

“…Presenta probada actividad necrotizante 299 . Se secreta durante la infección y activa de manera permanente GTPasas de las células blanco (la familia Rho) 300 .…”