Membrane Activity and Channel Formation of the Adenylate Cyclase Toxin (CyaA) of Bordetella pertussis in Lipid Bilayer Membranes

Abstract

The Gram-negative bacterium Bordetella pertussis is the cause of whooping cough. One of its pathogenicity factors is the adenylate cyclase toxin (CyaA) secreted by a Type I export system. The 1706 amino acid long CyaA (177 kDa) belongs to the continuously increasing family of repeat in toxin (RTX) toxins because it contains in its C-terminal half a high number of nine-residue tandem repeats. The protein exhibits cytotoxic and hemolytic activities that target primarily myeloid phagocytic cells expressing the αMβ2 integrin receptor (CD11b/CD18). CyaA represents an exception among RTX cytolysins because the first 400 amino acids from its N-terminal end possess a calmodulin-activated adenylate cyclase (AC) activity. The entry of the AC into target cells is not dependent on the receptor-mediated endocytosis pathway and penetrates directly across the cytoplasmic membrane of a variety of epithelial and immune effector cells. The hemolytic activity of CyaA is rather low, which may have to do with its rather low induced permeability change of target cells and its low conductance in lipid bilayer membranes. CyaA forms highly cation-selective channels in lipid bilayers that show a strong dependence on aqueous pH. The pore-forming activity of CyaA but not its single channel conductance is highly dependent on Ca²⁺ concentration with a half saturation constant of about 2 to 4 mM.

Keywords: Bordetella pertussis; CyaA; adenylate cyclase toxin; lipid bilayer; membrane interaction; pore formation.

Figure 1

Schematic representation of the different domains of cyclase toxin (CyaA) of Bordetella pertussis.

Figure 2

(a) Current–voltage relationships of cyclase toxin (CyaA) pores initiated by positive potential (+ 50 mV) applied to the cis side. The membranes were formed from asolectin/n-decane. The aqueous phase contained 150 mM KCl and 10 mM 2-(4-(2-Hydroxyethyl)-1-piperazinyl)-ethanesulfonic acid-potassium hydroxide (HEPES-KOH) pH 7; the temperature was 20 °C. The cis side contained in addition 320 ng/mL wild-type CyaA (full squares), 320 ng/mL ΔAC-CyaA (open squares), or 320 ng/mL CyaA 1008 (full circles) (adapted from [78]). (b) Current–voltage relationships of CyaA pores initiated by negative potential (−50 mV) applied to the cis side. The membranes were formed from asolectin/n-decane. The aqueous phase contained 150 mM KCl and 10 mM HEPES-KOH pH 7; the temperature was 20 °C. The cis side contained either 20 ng/mL wild-type CyaA (full circles), 160 ng/mL wild-type CyaA (full squares), or 320 ng/mL wild-type CyaA (full triangles) (adapted from [78]). (c) Increase of membrane conductance after the addition of 300 ng/mL CyaA to the cis side of a black asolectin/n-decane membrane (left side arrow) and of 0.8 mM Ca²⁺ first to the trans side of the membrane (middle arrow) and then to the cis side of the membrane (right side arrow). The aqueous phase contained 1 M KCl, pH 7 on both sides of the membrane. The addition of 300 ng/mL and CyaA added to the cis side of the membrane (left-hand side arrow) resulted in an increase of the membrane conductance (full squares) by a factor of about 100 over control in the absence of CyaA (full points). The addition of 0.8 mM CaCl₂ to the trans side (middle arrow) had no impact on membrane conductance, whereas the addition of 0.8 mM CaCl₂ to the cis side (right-hand side arrow) led to a dramatic increase of membrane conductance by many orders of magnitude (full squares). The temperature was 20 °C and 50 mV were applied to the cis side (adapted from [18]). (d) Single-pore recordings of asolectin/n-decane membranes in the presence of a 13 ng/mL concentration of the purified CyaA-E581K protein (lower line) or intact recombinant CyaA (upper line). Mutant E581K showed an increased pore lifetime, which seems to be linked to an increased hemolytic activity. The applied membrane potential was 50 mV, and the temperature was 20 °C (adapted from [72]).

Figure 3

Schematic model of CyaA action on target membranes. Adapted from Osickova et al. [71].