Production of Highly Active Recombinant Dermonecrotic Toxin of Bordetella Pertussis.

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.

Transglutaminase 2 accelerates neuroinflammation in amyotrophic lateral sclerosis through interaction with misfolded superoxide dismutase 1.

Although the aberrant assembly of mutant superoxide dismutase 1 (mSOD1) is implicated in the pathogenesis of familial amyotrophic lateral sclerosis (ALS), the molecular basis of superoxide dismutase 1 (SOD1) oligomerization remains undetermined. We investigated the roles of transglutaminase 2 (TG2), an endogenous cross-linker in mSOD1-linked ALS. TG2 interacted preferentially with mSOD1 and promoted its oligomerization in transfected cells. Purified TG2 directly oligomerized recombinant mutant SOD1 and the apo-form of the wild-type SOD1 proteins in a calcium-dependent manner, indicating that misfolded SOD1 is a substrate of TG2. Moreover, the non-cell-autonomous effect of extracellular TG2 on the neuroinflammation was suggested, since the TG2-mediated soluble SOD1 oligomers induced tumor necrosis factor-α, interleukin-1ß, and nitric oxide in microglial BV2 cells. TG2 was up-regulated in the spinal cord of pre-symptomatic G93A SOD1 transgenic mice and in the hypoglossal nuclei of mice suffering nerve ligation. Furthermore, inhibition of spinal TG2 by cystamine significantly delayed the progression and reduced SOD1 oligomers and microglial activation. These results indicate a novel role of TG2 in SOD1 oligomer-mediated neuroinflammation, as well as in the involvement in the intracellular aggregation of misfolded SOD1 in ALS.

Characterization of a microbial transglutaminase cross-linked type II collagen scaffold.

This study investigated the effect on the mechanical and physicochemical properties of type II collagen scaffolds after cross-linking with microbial transglutaminase (mTGase). It is intended to develop a collagen-based scaffold to be used for the treatment of degenerated intervertebral discs. By measuring the amount of epsilon-(gamma-glutamyl)lysine isodipeptide formed after cross-linking, it was determined that the optimal enzyme concentration was 0.005% (w/v). From the production of covalent bonds induced by mTGase cross-linking, the degradation resistance of type II collagen scaffolds can be enhanced. Rheological analysis revealed an almost sixfold increase in storage modulus (G') with 0.005% (w/v) mTGase cross-linked scaffolds (1.31 +/- 0.03 kPa) compared to controls (0.21 +/- 0.01 kPa). There was a significant reduction in the level of cell-mediated contraction of scaffolds with increased mTGase concentrations. Cell proliferation assays showed that mTGase crosslinked scaffolds exhibited similar cytocompatibility properties in comparison to non-cross-linked scaffolds. In summary, cross-linking type II collagen with mTGase imparted more desirable properties, making it more applicable for use as a scaffold in tissue engineering applications.

A single amino acid substitution in the enzymatic domain of cytotoxic necrotizing factor type 1 of Escherichia coli alters the tissue culture phenotype to that of the dermonecrotic toxin of Bordetella spp.

Cytotoxic necrotizing factor type 1 (CNF1) and dermonecrotic toxin (DNT) share homology within their catalytic domains and possess deamidase and transglutaminase activities. Although each toxin has a preferred enzymatic activity (i.e. deamidation for CNF1 and transglutamination for DNT) as well as target substrates, both modify a specific glutamine residue in RhoA, Rac1 and Cdc42, which renders these GTPases constitutively active. Here we show that despite their similar mechanisms of action CNF1 and DNT induced unique phenotypes on HEp-2 and Swiss 3T3 cells. CNF1 induced multinucleation of HEp-2 cells and was cytotoxic for Swiss 3T3 cells (with binucleation of the few surviving cells) while DNT showed no morphological effects on HEp-2 cells but did induce binucleation of Swiss 3T3 cells. To determine if the enzymatic domain of each toxin dictated the induced phenotype, we constructed enzymatically active chimeric toxins and mutant toxins that contained single amino acid substitutions within the catalytic site and tested these molecules in tissue culture and enzymatic assays. Moreover, both site-directed mutant toxins showed reduced time to maximum transglutamination of RhoA compared with the parent toxins. Nevertheless, the substitution of threonine for Lys(1310) in the DNT-based mutant, while affecting transglutamination efficiency of the toxin, did not abrogate that enzymatic activity.