Research Article: Bordetella Adenylate Cyclase Toxin Mobilizes Its β2 Integrin Receptor into Lipid Rafts to Accomplish Translocation across Target Cell Membrane in Two Steps

Date Published: May 13, 2010

Publisher: Public Library of Science

Author(s): Ladislav Bumba, Jiri Masin, Radovan Fiser, Peter Sebo, Kenneth A. Bradley.


Bordetella adenylate cyclase toxin (CyaA) binds the αMβ2 integrin (CD11b/CD18, Mac-1, or CR3) of myeloid phagocytes and delivers into their cytosol an adenylate cyclase (AC) enzyme that converts ATP into the key signaling molecule cAMP. We show that penetration of the AC domain across cell membrane proceeds in two steps. It starts by membrane insertion of a toxin ‘translocation intermediate’, which can be ‘locked’ in the membrane by the 3D1 antibody blocking AC domain translocation. Insertion of the ‘intermediate’ permeabilizes cells for influx of extracellular calcium ions and thus activates calpain-mediated cleavage of the talin tether. Recruitment of the integrin-CyaA complex into lipid rafts follows and the cholesterol-rich lipid environment promotes translocation of the AC domain across cell membrane. AC translocation into cells was inhibited upon raft disruption by cholesterol depletion, or when CyaA mobilization into rafts was blocked by inhibition of talin processing. Furthermore, CyaA mutants unable to mobilize calcium into cells failed to relocate into lipid rafts, and failed to translocate the AC domain across cell membrane, unless rescued by Ca2+ influx promoted in trans by ionomycin or another CyaA protein. Hence, by mobilizing calcium ions into phagocytes, the ‘translocation intermediate’ promotes toxin piggybacking on integrin into lipid rafts and enables AC enzyme delivery into host cytosol.

Partial Text

The secreted adenylate cyclase toxin-hemolysin (CyaA, ACT, or AC-Hly) plays a key role in virulence of Bordetellae. This multifuctional protein binds the αMβ2 integrin (CD11b/CD18, CR3 or Mac-1) of myeloid phagocytic cells and delivers into their cytosol a calmodulin-activated adenylate cyclase enzyme that ablates bactericidal capacities of phagocytes by uncontrolled conversion of cytosolic ATP to the key signaling molecule cAMP [1]–[5]. In parallel, the hemolysin moiety of CyaA forms oligomeric pores that permeabilize cell membrane for monovalent cations and contribute to overall cytoxicity of CyaA towards phagocytes [6]–[10].

We show here that membrane translocation of the adenylate cyclase domain of CyaA occurs by a two step mechanism and involves toxin piggybacking on the αMβ2 integrin for relocation into lipid rafts. The present results allow us to propose a new model of CyaA mechanism of action, as summarized in Fig. 8. Upon initial binding of CyaA to the CD11b/CD18 receptor distributed in the bulk phase of cell membrane, a ‘translocation intermediate’ of CyaA would insert into the cytoplasmic membrane. It is assumed that in this ‘translocation intermediate’ a part of the AC domain is already inserted within the membrane and is shielded form the lipids by association with the amphipathic α-helical transmembrane segments of the hydrophobic domain of CyaA (residues 502–522, 529–549, 571–591, 607–627 and 678–698 [19], [20]). This ‘translocation intermediate’ then forms a path conducting external Ca2+ ions across cellular membrane into the submembrane compartment of cells. Incoming calcium ions activate the Ca2+-dependent protease calpain, located in the submembrane compartment, which produces cleavage of the talin tether. This liberates the toxin-receptor complex from association with actin cytoskeleton and mobilizes it for recruitment into lipid rafts. Within the specific liquid-ordered environment of cholesterol-rich lipid rafts, translocation of the positively charged AC domain across the cellular membrane is completed, driven by the negative gradient of membrane potential.