Segments Crucial for Membrane Translocation and Pore-forming Activity of Bordetella Adenylate Cyclase Toxin

Abstract

570 and Glu 581 by helix-breaking proline or positively charged lysine residue reduced (E570K, E581P) or ablated (E570P, E581K) AC membrane translocation. Moreover, E570P, E570K, and E581P substitutions down-modulated also the specific hemolytic activity of CyaA. In contrast, the E581K substitution enhanced the hemolytic activity of CyaA 4 times, increasing both the frequency of formation and lifetime of toxin pores. Negative charge at position 570, but not at position 581, was found to be essential for cation selectivity of the pore, suggesting a role of Glu 570 in ion filtering inside or close to pore mouth. The pairs of glutamate residues in the predicted transmembrane segments of CyaA thus appear to play a key functional role in membrane translocation and pore-forming activities of CyaA.

Phospholipase A activity of adenylate cyclase toxin mediates translocation of its adenylate cyclase domain

“…The location of the catalytic Ser-606 within one of the predicted α-helices (helix IV) of the so-called hydrophobic pore-forming domain of ACT, leads us to plausibly hypothesize, that such a hydrophilic lipidic pore might be of proteolipidic nature, constituted likely by both lipids and one or more helical segments of the pore-forming region. Consistent with this idea, others have previously noted that mutations in specific amino acids of several α-helices from the hydrophobic domain impair not only the hemolytic activity, but also the translocation capacity of ACT (21).…”

“…How this is accomplished remains poorly understood. It is believed that after binding to the CD11b/ CD18 receptor, ACT inserts its amphipathic helices into the plasma membrane and then delivers its catalytic domain directly across the lipid bilayer into the cytosol (20,21) without receptormediated endocytosis (22). Temperatures above 15°C (20), calcium at millimolar range, negative membrane potential, and unfolding of the AC domain (23) have been noted to be necessary for AC delivery.…”

“…Several studies have highlighted the importance of various amino acids and the contribution of distinct domains of the ACT polypeptide in the translocation process (21,24). However, a full description at the molecular level of the individual steps that the Significance Numerous bacterial toxins can cross cell membranes, penetrating the cytosol of their target cells, but to do so exploits cellular endocytosis or intracellular sorting machineries.…”

Calcium, Acylation, and Molecular Confinement Favor Folding of Bordetella pertussis Adenylate Cyclase CyaA Toxin into a Monomeric and Cytotoxic Form

“…Once secreted, CyaA binds in a calcium-dependent manner to the CD11b/CD18 integrin expressed on myeloid cells, such as macrophages, neutrophils, dendritic cells, and natural killer cells, that are the primary targets of this toxin in vivo (31). However, CyaA can also efficiently intoxicate a variety of cell types lacking this receptor (16,(32)(33)(34)(35).…”

“…Moreover, CyaA, after insertion into the membrane, can also form cation-selective pores, which impair membrane impermeability and ultimately cause cell lysis (6,40,45). This membranedamaging activity is thought to synergize with the cAMP intoxication ability, thus increasing the overall cytotoxicity of the toxin (33,40,46,47).…”