Identification of a region that assists membrane insertion and translocation of the catalytic domain of Bordetella pertussis CyaA toxin

Abstract

The adenylate cyclase (CyaA) toxin, one of the virulence factors secreted by Bordetella pertussis, the pathogenic bacteria responsible for whooping cough, plays a critical role in the early stages of respiratory tract colonization by this bacterium. The CyaA toxin is able to invade eukaryotic cells by translocating its N-terminal catalytic domain directly across the plasma membrane of the target cells, where, activated by endogenous calmodulin, it produces supraphysiological levels of cAMP. How the catalytic domain is transferred from the hydrophilic extracellular medium into the hydrophobic environment of the membrane and then to the cell cytoplasm remains an unsolved question. In this report, we have characterized the membrane-interacting properties of the CyaA catalytic domain. We showed that a protein covering the catalytic domain (AC384, encompassing residues 1-384 of CyaA) displayed no membrane association propensity. However, a longer polypeptide (AC489), encompassing residues 1-489 of CyaA, exhibited the intrinsic property to bind to membranes and to induce lipid bilayer destabilization. We further showed that deletion of residues 375-485 within CyaA totally abrogated the toxin's ability to increase intracellular cAMP in target cells. These results indicate that, whereas the calmodulin dependent enzymatic domain is restricted to the amino-terminal residues 1-384 of CyaA, the membrane-interacting, translocation-competent domain extends up to residue 489. This thus suggests an important role of the region adjacent to the catalytic domain of CyaA in promoting its interaction with and its translocation across the plasma membrane of target cells.

FIGURE 1.

Protein interaction with planar lipid bilayers followed by dual polarization interferometry. A and B, real-time transverse magnetic phase changes upon injections of 0.006, 0.019, 0.055, 0.16, and 0.5 μm AC384 (A) and AC489 (B) on a supported soy PC/DOPE/cholesterol (6:3:1) lipid bilayer. C and D, real-time mass density changes of the proteins, ρ_P, upon injections of 0.006 (dotted lines), 0.019 (dashed lines), 0.055 (continuous lines), 0.16 (heavy dashed lines), and 0.5 μm (heavy continuous lines) of AC384 (C) and AC489 (D). E, partition coefficient (Kx) as a function of the ratio of total protein versus total lipid concentrations (P/L). Inset, free energy (ΔG) of protein partition from the buffer to the lipid phase as a function of P/L. F, observed binding constant rates (k_obs), deduced from fitting the real-time mass density changes ρ_P to a single exponential function, are reported as a function of AC489 protein concentrations.

FIGURE 2.

Specular neutron reflectometry of AC proteins. A, NR profiles of ¹H phospholipid bilayers in D₂O buffer in the presence (■) or absence (□) of AC384. B and C, NR profiles of phospholipid bilayers in SMW (B) or in D₂O (C) contrast conditions, in the presence (■) or absence (□) of AC489. D, fitted scattering length density of phospholipid bilayers in the absence of AC489 (dashed lines) or in the presence of AC489 (continuous lines) in D₂O (heavy lines) or in SMW (light lines) buffers. The spatial organization of the different layers is schematized at the bottom of the plot.

FIGURE 3.

Protein-induced membrane permeabilization. Time course of ANTS and DPX efflux from LUVs composed of POPC/DOPE/cholesterol at a molar ratio of 6:3:1 (A) or 6:3:0 (B) in the presence of CyaA, AC489, or AC384. Lipid and protein concentrations were 0.2 mm and 0.02 μg ml⁻¹, respectively (CyaA, 0.11 μm; AC489, 0.4 μm; AC384, 0.5 μm). A buffer injection was performed as a control (excitation, 360 nm; emission, 510 nm; bandwidths, 5 nm). The maximal fluorescence signal (in arbitrary units (a.u.)) obtained after the addition of Triton X-100 (2 mm) was 795 in the case of the POPC/DOPE/cholesterol preparation (in A) and 980 in the case of the POPC/DOPE preparation (B).

FIGURE 4.

Effect of increasing AC489 concentrations on LUVs permeabilization. A, time course of ANTS and DPX efflux from LUVs composed of POPC/DOPE/cholesterol (6:3:1) in the presence of the indicated concentrations of AC489. For each concentration, the time dependence of dye release was fitted to a single exponential function. B, amplitude and observed constant rates (k_obs) (inset) obtained from the fitting procedure.

FIGURE 5.

Effect of NaCl concentrations on AC489-induced LUVs permeabilization. A, time course of ANTS and DPX efflux from LUVs composed of POPC/DOPE/cholesterol (6:3:1) prepared in 20 mm Hepes, pH 7.4, containing either 150 mm NaCl (A) or 300 mm NaCl (B); for the permeabilization assays, LUVs were diluted in 20 mm Hepes, pH 7.4, supplemented with the indicated NaCl concentrations (from top to bottom): 0, 25, 50, 75, 100, 115, 130, 150, and 300 mm NaCl (A) and 50, 100, 150, 200, 250, 260, 275, 300, 400, and 500 mm NaCl (B). C, amplitudes of fluorescence intensities of the above experiments are plotted as a function of the ratio of extravesicular versus intravesicular NaCl concentrations. 150 mm NaCl inside LUVs (open circles) or 300 mm NaCl inside LUVs (closed circles). a.u., arbitrary units.

FIGURE 6.

Integrity of AC489 is required for LUV-permeabilizing activity. A, proteins (10 μg) were separated by 4–12% SDS-PAGE and detected with Coomassie Blue staining. Lane 1, molecular mass marker (in kDa); lane 2, AC489; lane 3, AC489_TEV; lane 4, AC489_TEV cleaved by TEV protease. Fragments, corresponding to AC(1–373) (41 kDa) and AC(374–489) (12 kDa), are indicated by asterisks. Protein bands around 30 kDa correspond to the TEV protease. Lane 5, AC384. B, time course of ANTS and DPX efflux from LUVs composed of POPC/DOPE/cholesterol (6:3:1) in the presence of the cleaved protein solution. From top to bottom are shown AC489_TEV, AC489, AC489_TEV incubated with TEV protease for 2 h at room temperature, AC384, and TEV protease alone. Note that when AC489 was incubated with TEV protease in the same conditions, no proteolysis occurred (data not shown).