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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 Cav3.2. Electron 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.
Highlights
Bordetella pertussis causes the respiratory infectious disease called pertussis, or whooping cough, which used to be the first cause of infant mortality prior to global introduction of efficient whole cell-based pertussis vaccines [1]
High level production of soluble recombinant dermonecrotic toxin (DNT) protein was achieved by expression of the dnt gene at 20 ◦ C for 24 h in Escherichia coli Rosetta 2 cells that harbor tRNAs genes for rare codons and efficiently translate the mRNA transcripts of G:C-rich genes
Sepharose at pH 7.4 followed by ion metal affinity chromatography on Ni-NTA agarose (Figure 1b), where the contaminating E. coli outer membrane lipopolysaccharide (LPS) and other components were removed by extensive washing of the column with 1% (v/v) Triton X-100 [31]
Summary
Bordetella pertussis causes the respiratory infectious disease called pertussis, or whooping cough, which used to be the first cause of infant mortality prior to global introduction of efficient whole cell-based pertussis (wP) vaccines [1]. Still, whooping cough remains the least-controlled vaccine-preventable infectious disease and accounts for more than 24 million cases and 160,000 pertussis-linked deaths annually worldwide [2]. Two decades ago the wP vaccines were replaced in the most developed countries by the less reactogenic acellular pertussis (aP) vaccines that confer a less complete and shorter-lasting protection [3] that led to resurgence of pertussis outbreaks [4,5]. Development of improved pertussis vaccines, capable to control B. pertussis transmission in highly vaccinated populations, is required and calls for better understanding of the pathophysiology of B. pertussis infections. The virulence factors that enable the bacterium to colonize human nasopharynx need to be identified.
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