The role of four cholesterol-recognition motifs localized between amino acid residues 400-550 in regulating translocation and lytic activity of Adenylate Cyclase Toxin

Adenylate Cyclase Toxin (ACT or CyaA) is an important virulence factor secreted by Bordetella pertussis, the bacterium causative of whooping cough, playing an essential role in the establishment of infection in the respiratory tract. ACT is a pore-forming cytolysin belonging to the RTX (Repeats in ToXin) family of leukotoxins, capable of permeabilizing several cell types and pure lipid vesicles. Besides, the toxin delivers its N-terminal adenylate cyclase domain into the target cytosol, where catalyzes the conversion of ATP into cAMP, which affects cell signalling. In this study we have made two major observations. First, we show that ACT binds free cholesterol, and identify in its sequence 38 potential cholesterol-recognition motifs. Second, we reveal that four of those motifs are real, functional cholesterol-binding sites. Mutations of the central phenylalanine residues in said motifs have an important impact on the ACT lytic and translocation activities, suggesting their direct intervention in cholesterol recognition and toxin functionality. From our data a likely transmembrane topology can be inferred for the ACT helices constituting the translocation and the hydrophobic regions. From this topology a simple and plausible mechanism emerges by which ACT could translocate its AC domain into target cells, challenging previous views in the field. Blocking the ACT-cholesterol interactions might thus be an effective approach for inhibiting ACT toxicity on cells, and this could help in mitigating the severity of pertussis disease in humans.