title: Bordetella pertussis adenylate cyclase toxin translocation across a tethered lipid bilayer.
abstract: Numerous bacterial toxins can cross biological membranes to reach the cytosol of mammalian cells, where they exert their cytotoxic effects. Our model toxin, the adenylate cyclase (CyaA) from Bordetella pertussis, is able to invade eukaryotic cells by translocating its catalytic domain directly across the plasma membrane of target cells. To characterize its original translocation process, we designed an in vitro assay based on a biomimetic membrane model in which a tethered lipid bilayer (tBLM) is assembled on an amine-gold surface derivatized with calmodulin (CaM). The assembled bilayer forms a continuous and protein-impermeable boundary completely separating the underlying calmodulin (trans side) from the medium above (cis side). The binding of CyaA to the tBLM is monitored by surface plasmon resonance (SPR) spectroscopy. CyaA binding to the immobilized CaM, revealed by enzymatic activity, serves as a highly sensitive reporter of toxin translocation across the bilayer. Translocation of the CyaA catalytic domain was found to be strictly dependent on the presence of calcium and also on the application of a negative potential, as shown earlier in eukaryotic cells. Thus, CyaA is able to deliver its catalytic domain across a biological membrane without the need for any eukaryotic components besides CaM. This suggests that the calcium-dependent CyaA translocation may be driven in part by the electrical field across the membrane. This study's in vitro demonstration of toxin translocation across a tBLM provides an opportunity to explore the molecular mechanisms of protein translocation across biological membranes in precisely defined experimental conditions.
authors.affiliation: Equipe Matériaux Avancés pour la Catalyse et la Santé, Unité Mixte de Recherche 5253, Centre National de la Recherche Scientifique-Ecole Nationale Supérieure de Chimie de Montpellier-Université Montpellier 2-Université Montpellier 1, Institut Charles Gerhardt Montpellier, 34296 Montpellier Cedex 5, France.
authors.forename: Rémi
authors.initials: R
authors.lastname: Veneziano
authors.forename: Claire
authors.initials: C
authors.lastname: Rossi
authors.forename: Alexandre
authors.initials: A
authors.lastname: Chenal
authors.forename: Jean-Marie
authors.initials: JM
authors.lastname: Devoisselle
authors.forename: Daniel
authors.initials: D
authors.lastname: Ladant
authors.forename: Joel
authors.initials: J
authors.lastname: Chopineau
mesh_terms: D037361:Adenylate Cyclase Toxin / Q000737:chemistry* / Q000378:metabolism; D001886:Bordetella pertussis / Q000201:enzymology*; D002118:Calcium / Q000737:chemistry* / Q000378:metabolism; D002147:Calmodulin / Q000737:chemistry / Q000378:metabolism; D008051:Lipid Bilayers / Q000737:chemistry* / Q000378:metabolism; D017434:Protein Structure, Tertiary; D021381:Protein Transport / Q000502:physiology; D020349:Surface Plasmon Resonance
chemical_list: D037361:Adenylate Cyclase Toxin; D002147:Calmodulin; D008051:Lipid Bilayers; D002118:Calcium
keywords: adenylate cyclase activity; synthetic biomembrane; toxin internalization