Membrane Restructuring by Bordetella pertussis Adenylate Cyclase Toxin, a Member of the RTX Toxin Family

Abstract

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...

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

“…As expected, addition of ACT (10 nM) to DOPC liposomes induced a rapid reduction of the fluorescence signal at 545 nm, in a time scale of minutes (Fig. 7A), consistent with ACT capacity to promote lipid flip-flop, as previously noted by our group (32). In contrast, no changes in the fluorescence signal were detected upon addition of S606A or D1079A mutant proteins to liposomes (Fig.…”

“…With this aim, we determined the capacity of ACT to induce transbilayer lipid motion in DOPC vesicles, using for that a flip-flop assay previously adapted for this purpose in our laboratory (32). This assay is based on the phenomenon of FRET (32).…”

“…In recent years our laboratory has extensively explored ACT interaction with lipid membranes, and we have discovered that toxin insertion into lipid bilayers induces transbilayer lipid motion (lipid "scrambling" or "flip-flop") (32). Transbilayer lipid movement may occur as a result of the insertion of foreign molecules (detergents, lipids, or even proteins) in one of the membrane leaflets, or it may result from the enzymatic generation of lipids (e.g., lysophospholipids, diacylglycerol, or ceramide) at one side of the membrane (33), and it has been involved in enhancing the transmembrane permeability of the lipid membranes (33).…”

Bordetella pertussis Adenylate Cyclase Toxin Modulates Innate and Adaptive Immune Responses: Distinct Roles for Acylation and Enzymatic Activity in Immunomodulation and Cell Death

“…Previous studies have demonstrated that acylation is important for binding of CyaA to RBC, but that interaction with CD11b-expressing cells is via residues 1166 -1281 (2). In addition, CyaA binding to CD11b-expressing cells is saturable, while binding to RBC is not (3,26). These data suggest that CyaA may interact with RBC and CD11b-expressing cells by two different mechanisms.…”

Only two amino acids are essential for cytolytic toxin recognition of cholesterol at the membrane surface

“…hemolysin | intermedilysin | sterol | toxin | pore M embrane cholesterol is important to a variety of pathogenic processes that include virus fusion and budding (1) and the mechanisms of eukaryotic (2,3) and prokaryotic toxins (4)(5)(6)(7). Whether cholesterol is bound directly by these proteins as a receptor or it indirectly influences the binding or activity of the protein at the membrane surface remains unknown.…”