The Bordetella adenylate cyclase toxin-hemolysin (CyaA, ACT, or AC-Hly) forms cation-selective membrane channels and delivers into the cytosol of target cells an adenylate cyclase domain (AC) that catalyzes uncontrolled conversion of cellular ATP to cAMP. Both toxin activities were previously shown to depend on post-translational activation of proCyaA to CyaA by covalent palmitoylation of the internal Lys983 residue (K983). CyaA, however, harbors a second RTX acylation site at residue Lys860 (K860), and the role of K860 acylation in toxin activity is unclear. We produced in E. coli the CyaA-K860R and CyaA-K983R toxin variants having the Lys860 and Lys983 acylation sites individually ablated by arginine substitutions. When examined for capacity to form membrane channels and to penetrate sheep erythrocytes, the CyaA-K860R acylated on Lys983 was about 1 order of magnitude more active than CyaA-K983R acylated on Lys860, although, in comparison to intact CyaA, both monoacylated constructs exhibited markedly reduced activities in erythrocytes. Channels formed in lipid bilayers by CyaA-K983R were importantly less selective for cations than channels formed by CyaA-K860R, intact CyaA, or proCyaA, showing that, independent of its acylation status, the Lys983 residue may play a role in toxin structures that determine the distribution of charged residues at the entry or inside of the CyaA channel. While necessary for activity on erythrocytes, acylation of Lys983 was also sufficient for the full activity of CyaA on CD11b+ J774A.1 monocytes. In turn, acylation of Lys860 alone did not permit toxin activity on erythrocytes, while it fully supported the high-affinity binding of CyaA-K983R to the toxin receptor CD11b/CD18 and conferred on CyaA-K983R a reduced but substantial capacity to penetrate and kill the CD11b+ cells. This is the first evidence that acylation of Lys860 may play a role in the biological activity of CyaA, even if redundant to the acylation of Lys983.

Adenylate cyclase toxin (ACT) is secreted by Bordetella pertussis, the bacterium causing whooping cough. ACT is a member of the RTX (repeats in toxin) family of toxins, and like other members in the family, it may bind cell membranes and cause disruption of the permeability barrier, leading to efflux of cell contents. The present paper summarizes studies performed on cell and model membranes with the aim of understanding the mechanism of toxin insertion and membrane restructuring leading to release of contents. ACT does not necessarily require a protein receptor to bind the membrane bilayer, and this may explain its broad range of host cell types. Adenylate cyclase toxin (ACT) is secreted by Bordetella pertussis, the bacterium responsible for whooping cough. The 1,706-residue protein can enter eukaryotic cells, where, upon activation by endogenous calmodulin, it increases the intracellular levels of cyclic AMP, leading to severe alterations in cellular physiology, often referred to as intoxication (see reference 28 for a review). ACT belongs to the so-called RTX (repeats in toxin) family of proteins, characterized by a Ca 2ϩ -binding nonapeptide repeated in tandem several times, up to 30 to 38 repeats in the case of ACT, depending on the stringency of repeat definition. This toxin represents the most evolutionarily divergent example of the family (for reviews of RTX proteins, see references 40 and 41). Unlike most other members of the family, ACT remains associated with the bacterial surface after secretion, apparently associated with filamentous hemagglutinin (42).In common with other members of the RTX family, and apart from its unique adenylate cyclase activity, ACT has a capacity to induce cell lysis, usually demonstrated as hemolysis. ACT-induced hemolysis requires higher toxin concentrations (by more than 1 order of magnitude) and occurs more slowly than intoxication (17). Active ACT is acylated at two positions inside the chain, and the acylation pattern appears to affect hemolysis, rather than intoxication (19). Moreover, dose-response experiments suggest that intoxication can be triggered by ACT monomers, while hemolysis is a more cooperative event, mediated by at least trimers (5, 17, 32). These and other observations have led to the conclusion that hemolysis and intoxication occur through separate mechanisms (17,28,32,34).Unlike intoxication, ACT-induced cell lysis has received relatively little attention. Benz et al. (4) and Szabo et al. (39), using planar lipid bilayers, demonstrated that ACT increased membrane conductance, giving rise to small, transient, cationselective channels. These authors also found that ACT was less active in this respect than ␣-hemolysin (HlyA), another member of the RTX family, secreted by Escherichia coli (4). In general, the mechanism of HlyA-induced hemolysis has been studied in more detail (see references 16 and 40 for reviews). In particular, studies in one of our laboratories have examined the capacity of HlyA to destroy the permeability barrier of pure-lipid vesicl...