Epitope Mapping of Monoclonal Antibodies againstBordetella pertussisAdenylate Cyclase Toxin

Abstract

Adenylate cyclase (AC) toxin from Bordetella pertussisis a 177-kDa repeats-in-toxin (RTX) family protein that consists of four principal domains; the catalytic domain, the hydrophobic domain, the glycine/aspartate-rich repeat domain, and the secretion signal domain. Epitope mapping of 12 monoclonal antibodies (MAbs) directed against AC toxin was conducted to identify regions important for the functional activities of this toxin. A previously developed panel of in-frame deletion mutants of AC toxin was used to localize MAb-specific epitopes on the toxin. The epitopes of these 12 MAbs were located throughout the toxin molecule, recognizing all major domains. Two MAbs recognized a single epitope on the distal portion of the catalytic domain, two reacted with the C-terminal 217 amino acids, one bound to the hydrophobic domain, and one bound to either the hydrophobic domain or the functionally unidentified region adjacent to it. The remaining six MAbs recognized the glycine/aspartate-rich repeat region. To localize these six MAbs, different peptides derived from the repeat region were constructed. Two of the six MAbs appeared to react with the repetitive motif and exhibited cross-reactivity with Escherichia coli hemolysin. The remaining four MAbs appeared to interact with unique epitopes within the repeat region. To evaluate the roles of these epitopes on toxin function, each MAb was screened for its effect on intoxication (cyclic AMP accumulation) and hemolytic activity. The two MAbs recognizing the distal portion of the catalytic domain blocked intoxication of Jurkat cells by AC toxin but had no effect on hemolysis. On the other hand, a MAb directed against a portion of the repeat region caused partial inhibition of AC toxin-induced hemolysis without affecting intoxication. In addition, the MAb recognizing either the hydrophobic domain or the unidentified region adjacent to it inhibited both intoxication and hemolytic activity of AC toxin. These findings extend our understanding of the regions necessary for the complex events required for the biological activities of AC toxin and provide a set of reagents for further study of this novel virulence factor.

Oligomerization is involved in pore formation byBordetellaadenylate cyclase toxin

“…The top CyaA species corresponded well to the full-length CyaA form, which is known to migrate in SDS-PAGE slower than expected ϳ205 kDa, presumably because of lower relative binding of SDS to the acidic CyaA protein (55). This ϳ205-kDa CyaA species was recognized in immunoblots by both the 9D4 mAb binding to Cterminal RTX repeats of CyaA, as well as by the 3D1 mAb that specifically recognizes the segment of the AC domain located between residues 373 and 400 (38). In contrast, the 3D1 antibody failed to detect the bottom ϳ170-kDa CyaA species, while the same protein was detected unambiguously by the 9D4 antibody.…”

“…Mouse monoclonal anti-CyaA antibodies 9D4 and 10A8 (38) were obtained from Erik L. Hewlett (University of Virginia School of Medicine, Charlottesville, VA, USA) and protein A-gold 5-nm particles from Jan Slot (University Medical Center, Utrecht, The Netherlands).…”

“…Cells were treated with 25 g/ml CyaA for 30 min at 37°C in the presence of 5 mM EDTA or 2 mM Ca 2ϩ . A) Extracts of toxin-treated cells were separated by SDS-PAGE and subjected to Western blotting using the 9D4 mAb and anti-mouse IgG-peroxidase conjugate to detect total CyaA or by the 3D1 antibody, recognizing a CyaA segment between residues 373 and 400 (38). B) CyaA species were extracted from erythrocyte membranes and separated by BN-PAGE.…”

Bordetella adenylate cyclase toxin interacts with filamentous haemagglutinin to inhibit biofilm formation in vitro

“…1C is a schematic representation of modifications). These variants, which are partial deletions from full-length DcyaA (Sebo and Ladant, 1993;Sadilkova et al, 2008), were previously used to characterize monoclonal antibodies (Lee et al, 1999) and have been used previously in functional assays (Sakamoto et al, 1992;Iwaki et al, 1995;Macdonald-Fyall et al, 2004;Eby et al, 2014).…”

Back to the future: recombinant polyclonal antibody therapeutics

“…There are also several examples in the literature of synergistic antibody combinations with potencies surpassing that of polyclonal antisera in mice [49]. Since the key antigens are wellcharacterized with, in most cases, neutralizing antibodies and protective epitopes already identified [49][50][51], antibodies could be rationally combined to identify a potently protective cocktail. The antibodies could be individually engineered for traits likely to correlate with protection, including high affinity, recognition of all circulating clinical strains with characterized mechanisms of protection [50,52].…”