Translocation-Specific Conformation of Adenylate Cyclase Toxin from Bordetella pertussis Inhibits Toxin-Mediated Hemolysis

Abstract

Bordetella pertussis adenylate cyclase (AC) toxin belongs to the RTX family of toxins but is the only member with a known catalytic domain. The principal pathophysiologic function of AC toxin appears to be rapid production of intracellular cyclic AMP (cAMP) by insertion of its catalytic domain into target cells (referred to as intoxication). Relative to other RTX toxins, AC toxin is weakly hemolytic via a process thought to involve oligomerization of toxin molecules. Monoclonal antibody (MAb) 3D1, which binds to an epitope (amino acids 373 to 399) at the distal end of the catalytic domain of AC toxin, does not affect the enzymatic activity of the toxin (conversion of ATP into cAMP in a cell-free system) but does prevent delivery of the catalytic domain to the cytosol of target erythrocytes. Under these conditions, however, the ability of AC toxin to cause hemolysis is increased three-to fourfold. To determine the mechanism by which the hemolytic potency of AC toxin is altered, we used a series of deletion mutants. A mutant toxin, ⌬AC, missing amino acids 1 to 373 of the catalytic domain, has hemolytic activity comparable to that of wild-type toxin. However, binding of MAb 3D1 to ⌬AC enhances its hemolytic activity three-to fourfold similar to the enhancement of hemolysis observed with 3D1 addition to wild-type toxin. Two additional mutants, ⌬N489 (missing amino acids 6 to 489) and ⌬N518 (missing amino acids 6 to 518), exhibit more rapid hemolysis with quicker onset than wild-type toxin does, while ⌬N549 (missing amino acids 6 to 549) has reduced hemolytic activity compared to wild-type AC toxin. These data suggest that prevention of delivery of the catalytic domain or deletion of the catalytic domain, along with additional amino acids distal to it, elicits a conformation of the toxin molecule that is more favorable for hemolysis.

Bordetella pertussis adenylate cyclase toxin translocation across a tethered lipid bilayer

“…The overall efficiency of translocation (approximately 80% of total tBLM-bound CyaA) observed in vitro compares favorably with that measured previously on sheep erythrocytes (32,43). Furthermore, no translocation was observed with the nonacylated proCyaA protein or after preincubation of CyaA with mAb 3D1, which had been previously shown to block the entry of CyaA into eukaryotic cells (30,33,38). This finding further supports our contention that our in vitro assay captures the essential features of the authentic physiological processes occurring with living cells.…”

“…mAb 3D1 Blocks CyaA Translocation Across the tBLM. Gray et al (33) previously reported that a monoclonal antibody, mAb 3D1, which recognizes an epitope located between residues 373 and 399 of CyaA at the C-terminal end of the catalytic domain, does not affect the adenylate cyclase activity of the toxin or its capacity to bind to target cells, but does inhibit delivery of the catalytic domain into the cytosol of target cells (erythrocytes or J774A.1 cells). Thus, we tested the effect of mAb 3D1 in our in vitro translocation assay.…”

“…Then CyaA is secreted across the bacterial envelope by a dedicated type I secretion machinery and binds to the CD11b/CD18 integrin expressed by a subset of leukocytes including neutrophils, macrophages, and dendritic cells (22,(28)(29)(30). However, CyaA can also invade a wide variety of cells that do not express this receptor, albeit with a lower efficiency (19,(31)(32)(33)(34)(35).…”

The Bordetella Adenylate Cyclase Repeat-in-Toxin (RTX) Domain Is Immunodominant and Elicits Neutralizing Antibodies

“…6C). In contrast, 3D1, a neutralizing IgG that blocks translocation of the catalytic domain (57), and 2A12, a neutralizing antibody with unclear mode-ofaction, had no effect. Minimal nonspecific binding was observed under these assay conditions.…”

Role of CD11b/CD18 in the Process of Intoxication by the Adenylate Cyclase Toxin of Bordetella pertussis

“…Escherichia coli XL-1 Blue cells (Stratagene, La Jolla, CA) containing the appropriate plasmid construct (wild-type [WT] ACT or inactive ACT) were used for toxin production as previously described (23,29). Cultured bacteria were centrifuged, and the resulting pellet was resuspended in 50 mM Tris (pH 7.5), sonicated, and extracted with 8 M urea.…”