Identification of the factors that govern the ability of therapeutic antibodies to provide postchallenge protection against botulinum toxin: a model for assessing postchallenge efficacy of medical countermeasures against agents of bioterrorism and biological warfare.

Therapeutic antibodies are one of the major classes of medical countermeasures that can provide protection against potential bioweapons such as botulinum toxin. Although a broad array of antibodies are being evaluated for their ability to neutralize the toxin, there is little information that defines the circumstances under which these antibodies can be used. In the present study, an effort was made to quantify the temporal factors that govern therapeutic antibody use in a postchallenge scenario. Experiments were done involving inhalation administration of toxin to mice, intravenous administration to mice, and direct application to murine phrenic nerve-hemidiaphragm preparations. As part of this study, several pharmacokinetic characteristics of botulinum toxin and neutralizing antibodies were measured. The core observation that emerged from the work was that the window of opportunity within which postchallenge administration of antibodies exerted a beneficial effect increased as the challenge dose of toxin decreased. The critical factor in establishing the window of opportunity was the amount of time needed for fractional redistribution of a neuroparalytic quantum of toxin from the extraneuronal space to the intraneuronal space. This redistribution event was a dose-dependent phenomenon. It is likely that the approach used to identify the factors that govern postchallenge efficacy of antibodies against botulinum toxin can be used to assess the factors that govern postchallenge efficacy of medical countermeasures against any agent of bioterrorism or biological warfare.

Functional and structural characterization of chimeras of a bacterial genotoxin and human type I DNAse.

Chimeras composed of the cdtB gene of a novel bacterial genotoxin and the human type I DNAse I gene were constructed and their products characterized relative to the biochemical and enzymatic properties of the native proteins. The product of a cdtB/DNAse I chimera formed a heterotrimer with the CdtA and CdtC subunits of the genotoxin, and targeted mutations increased the specific activity of the hybrid protein. Expression of active chimeric gene products established that the CdtB protein is an atypical divalent cation-dependent endonuclease and demonstrated the potential for genetically engineering a new class of therapeutic agent for inhibiting the proliferation of cancer cells.

Role of aromatic amino acids in receptor binding activity and subunit assembly of the cytolethal distending toxin of Aggregatibacter actinomycetemcomitans.

The periodontal pathogen Aggregatibacter actinomycetemcomitans produces a cytolethal distending toxin (Cdt) that inhibits the proliferation of oral epithelial cells. Structural models suggest that the CdtA and CdtC subunits of the Cdt heterotrimer form two putative lectin domains with a central groove. A region of CdtA rich in heterocyclic amino acids (aromatic patch) appears to play an important role in receptor recognition. In this study site-specific mutagenesis was used to assess the contributions of aromatic amino acids (tyrosine and phenylalanine) to receptor binding and CdtA-CdtC assembly. Predominant surface-exposed aromatic residues that are adjacent to the aromatic patch region in CdtA or are near the groove located at the junction of CdtA and CdtC were studied. Separately replacing residues Y105, Y140, Y188, and Y189 with alanine in CdtA resulted in differential effects on binding related to residue position within the aromatic region. The data indicate that an extensive receptor binding domain extends from the groove across the entire face of CdtA that is oriented 180 degrees from the CdtB subunit. Replacement of residue Y105 in CdtA and residues Y61 and F141 in CdtC, which are located in or at the periphery of the groove, inhibited toxin assembly. Taken together, these results, along with the lack of an aromatic amino acid-rich region in CdtC similar to that in CdtA, suggest that binding of the heterotoxin to its cell surface receptor is mediated predominantly by the CdtA subunit. These findings are important for developing strategies designed to block the activity of this prominent virulence factor.

Role of intrachain disulfides in the activities of the CdtA and CdtC subunits of the cytolethal distending toxin of Actinobacillus actinomycetemcomitans.

The cytolethal distending toxin (Cdt) of Actinobacillus actinomycetemcomitans is an atypical A-B-type toxin consisting of a heterotrimer composed of the cdtA, cdtB, and cdtC gene products. The CdtA and CdtC subunits form two heterogeneous ricin-like lectin domains which bind the holotoxin to the target cell. Point mutations were used to study CdtC structure and function. One (mutC216(F97C)) of eight single-amino-acid replacement mutants identified yielded a gene product that failed to form biologically active holotoxin. Based on the possibility that the F97C mutation destabilized a predicted disulfide, targeted mutagenesis was used to examine the contribution of each of four cysteine residues, in two predicted disulfides (C96/C107 and C135/C149), to CdtC activities. Cysteine replacement mutations in two predicted disulfides (C136/C149 and C178/C197) in CdtA were also characterized. Flow cytometry and CHO cell proliferation assays showed that changing either C96 or C149 in CdtC to alanine abolished the biological activity of holotoxin complexes. However, replacing C107 or C135 in CdtC and any of the four cysteines in CdtA with alanine or serine resulted in only partial or no loss of holotoxin activity. Changes in the biological activities of the mutant holotoxins correlated with altered subunit binding. In contrast to elimination of the B chain of ricin, the elimination of intrachain disulfides in CdtC and CdtA by genetic replacement of cysteines destabilizes these subunit proteins but not to the extent that cytotoxicity is lost. Reduction of the wild-type holotoxin did not affect cytotoxicity, and the reduced form of wild-type CdtA exhibited a statistically significant increase in binding to ligand. A diminished role for intrachain disulfides in stabilizing CdtA and CdtC may have clinical relevance for the A. actinomycetemcomitans Cdt. The cdt gene products secreted by this pathogen assemble and bind to target cells in periodontally involved sites, which are decidedly reduced environments in the human oral cavity.

Characterization of point mutations in the cdtA gene of the cytolethal distending toxin of Actinobacillus actinomycetemcomitans.

The Cdt is a family of gram-negative bacterial toxins that typically arrest eukaryotic cells in the G0/G1 or G2/M phase of the cell cycle. The toxin is a heterotrimer composed of the cdtA, cdtB and cdtC gene products. Although it has been shown that the CdtA protein subunit binds to cells in culture and in an enzyme-linked immunosorbent assay (CELISA) the precise mechanisms by which CdtA interacts with CdtB and CdtC has not yet been clarified. In this study we employed a random mutagenesis strategy to construct a library of point mutations in cdtA to assess the contribution of individual amino acids to binding activity and to the ability of the subunit to form biologically active holotoxin. Single unique amino acid substitutions in seven CdtA mutants resulted in reduced binding of the purified recombinant protein to Chinese hamster ovary cells and loss of binding to the fucose-containing glycoprotein, thyroglobulin. These mutations clustered at the 5'- and 3'-ends of the cdtA gene resulting in amino acid substitutions that resided outside of the aromatic patch region and a conserved region in CdtA homologues. Three of the amino acid substitutions, at positions S165N (mutA81), T41A (mutA121) and C178W (mutA221) resulted in gene products that formed holotoxin complexes that exhibited a 60% reduction (mutA81) or loss (mutA121, mutA221) of proliferation inhibition. A similar pattern was observed when these mutant holotoxins were tested for their ability to induce cell cycle arrest and to convert supercoiled DNA to relaxed and linear forms in vitro. The mutations in mutA81 and mutA221 disrupted holotoxin formation. The positions of the amino acid substitutions were mapped in the Haemophilus ducreyi Cdt crystal structure providing some insight into structure and function.