Abstract
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.
Highlights
By instantaneously disrupting bactericidal functions of host phagocytes, the adenylate cyclase toxin-hemolysin (CyaA, ACT, or adenylate cyclase enzyme (AC)-Hly) plays a major role in virulence of pathogenic Bordetellae [1]
The adenylate cyclase toxin (CyaA) of pathogenic Bordetellae eliminates the first line of host innate immune defense by inhibiting the oxidative burst and complementmediated opsonophagocytic killing of bacteria
We show here that the capacity of CyaA to permeabilize phagocytes depends on its ability to mediate influx of extracellular calcium ions into cells
Summary
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 multidomain ,1300 residues-long RTX (Repeat in ToXin) cytolysin moiety of CyaA acts independently as a pore-forming leukotoxin and hemolysin [1]. CyaA can oligomerize into small cation-selective pores that mediate efflux of cytosolic potassium ions from cells [3,4,5,6], eventually provoking colloid-osmotic cell lysis [7,8,9]. This activity synergizes with cytotoxic signaling of the translocated AC enzyme in bringing about the final cytotoxic action of CyaA [8,9]
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