Research Article: Calcium Influx Rescues Adenylate Cyclase-Hemolysin from Rapid Cell Membrane Removal and Enables Phagocyte Permeabilization by Toxin Pores

Date Published: April 5, 2012

Publisher: Public Library of Science

Author(s): Radovan Fiser, Jiri Masin, Ladislav Bumba, Eva Pospisilova, Catherine Fayolle, Marek Basler, Lenka Sadilkova, Irena Adkins, Jana Kamanova, Jan Cerny, Ivo Konopasek, Radim Osicka, Claude Leclerc, Peter Sebo, Steven R. Blanke.


Bordetella adenylate cyclase toxin-hemolysin (CyaA) penetrates the cytoplasmic membrane of phagocytes and employs two distinct conformers to exert its multiple activities. One conformer forms cation-selective pores that permeabilize phagocyte membrane for efflux of cytosolic potassium. The other conformer conducts extracellular calcium ions across cytoplasmic membrane of cells, relocates into lipid rafts, translocates the adenylate cyclase enzyme (AC) domain into cells and converts cytosolic ATP to cAMP. We show that the calcium-conducting activity of CyaA controls the path and kinetics of endocytic removal of toxin pores from phagocyte membrane. The enzymatically inactive but calcium-conducting CyaA-AC− toxoid was endocytosed via a clathrin-dependent pathway. In contrast, a doubly mutated (E570K+E581P) toxoid, unable to conduct Ca2+ into cells, was rapidly internalized by membrane macropinocytosis, unless rescued by Ca2+ influx promoted in trans by ionomycin or intact toxoid. Moreover, a fully pore-forming CyaA-ΔAC hemolysin failed to permeabilize phagocytes, unless endocytic removal of its pores from cell membrane was decelerated through Ca2+ influx promoted by molecules locked in a Ca2+-conducting conformation by the 3D1 antibody. Inhibition of endocytosis also enabled the native B. pertussis-produced CyaA to induce lysis of J774A.1 macrophages at concentrations starting from 100 ng/ml. Hence, by mediating calcium influx into cells, the translocating conformer of CyaA controls the removal of bystander toxin pores from phagocyte membrane. This triggers a positive feedback loop of exacerbated cell permeabilization, where the efflux of cellular potassium yields further decreased toxin pore removal from cell membrane and this further enhances cell permeabilization and potassium efflux.

Partial Text

By instantaneously disrupting bactericidal functions of host phagocytes, the adenylate cyclase toxin-hemolysin (CyaA, ACT, or AC-Hly) plays a major role in virulence of pathogenic Bordetellae[1]. The toxin rapidly paralyzes phagocytes [1], [2] by translocating across their cytoplasmic membrane an N-terminal adenylate cyclase enzyme (AC) domain (∼400 residues) that binds cytosolic calmodulin and converts ATP to a key signaling molecule, cAMP [1]. In parallel, the multidomain ∼1300 residues-long RTX (Repeat in ToXin) cytolysin moiety of CyaA acts independently as a pore-forming leukotoxin and hemolysin [1]. This employs a hydrophobic pore-forming domain (residues 500 to 700), a domain with covalently palmitoylated lysine residues 860 and 983, and a typical calcium-binding RTX repeat domain within the last 700 residues of CyaA that accounts for receptor binding [1]. CyaA can oligomerize into small cation-selective pores that mediate efflux of cytosolic potassium ions from cells [3]–[6], eventually provoking colloid-osmotic cell lysis [7]–[9]. This activity synergizes with cytotoxic signaling of the translocated AC enzyme in bringing about the final cytotoxic action of CyaA [8], [9].

We show here that by conducting Ca2+ ions across target cell membrane, CyaA decelerates its endocytic uptake and escapes from rapid macropinocytic removal from cell membrane and destruction in endosomes. By redirecting the toxin into a decelerated clathrin-dependent uptake pathway, the calcium-conducting activity of toxin translocation intermediates protracts toxin pore persistence within cytoplasmic membrane, thus extending phagocyte permeabilization and maximizing cytotoxic action of CyaA, as summarized in the model proposed in Figure 13.