Biochimie des Interactions Macromoléculaires - Projet Alexandre Chenal
Biophysical and biochemical investigations of the Bordetella pertussis CyaA toxin
This project is performed by Ana Cristina Sotomayor Pérez, Johanna C. Karst, Orso Subrini, Audrey Hessel, Véronique Yvette Ntsogo, Sara Elisabetta Cannella, Darragh O'Brien, Maryline Davi, Daniel Ladant and Alexandre Chenal.
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Our research interests are mainly focused on the study of the molecular mechanisms that underlying protein folding and protein-membrane interactions, using a bacterial toxin, the adenylate cyclase (CyaA) produced by Bordetella pertussis, the causative agent of whooping cough.
CyaA, a 1706 residue-long protein, is one of the major virulence factors produced by B. pertussis and plays an important role in the early stages of respiratory tract colonization. This toxin uses an original intoxication mechanism: secreted by the virulent bacteria, CyaA is able to invade eukaryotic target cells through a unique but poorly understood mechanism that involves a calcium-dependent direct translocation of its N-terminal catalytic domain across the plasma membrane. Then, upon activation by the endogenous cytosolic calmodulin (CaM), CyaA catalyzes massive production of cAMP that in turn alters cellular physiology. Our main objective is to unravel the molecular mechanisms of this unique entry pathway.
One challenging aspect of the structural and biophysical studies of CyaA arises from the complexity of this toxin, a large (1706 amino-acids) multi-domain protein that is post-translationally acylated and exhibits a pronounced hydrophobic character limiting its solubility. The only structural data available thus far on the protein is the 3D structure of the catalytic domain solved by the group of Wei-Jen Tang. In the last times, our work has been focused on the characterization of individual domains of the toxin, mainly the AC catalytic domain (AC) and Repeat-in-ToXin (RTX) Domains (RD and part of it, the so-called C-terminal Block V), using a combination of biochemical and biophysical approaches as summarized below.
In the future, we will pursue the study of the physico-chemical properties of CyaA in solution and upon its insertion into membranes. Biophysical techniques will be developed to follow the translocation process both in vitro on lipid membranes and in vivo on eukaryotic cells. These studies should provide a better understanding of the mechanisms of toxin translocation across biological membranes, and in addition, will be instrumental for further developments of CyaA-based vaccines (two of them are currently in phase I/II clinical trials). Indeed, Daniel Ladant, in collaboration with C. Leclerc’s team at Institut Pasteur, previously showed that CyaA is a potent vaccine vehicle able to deliver antigens into dendritic cells to trigger specific cell-mediated immune responses.
Besides investigating the biophysics of CyaA, I have pursued several projects initiated during my previous post-doctoral positions or within new collaborations established with various groups inside or outside Institut Pasteur. The main goal is to improve the understanding of the behavior of amphitropic proteins, i.e., to describe how soluble proteins are able to partition from the solution to the membrane and translocate across the lipid bilayer. This objective is pursued using mainly biochemical and spectroscopic approaches to characterize the biophysics of amphitropic proteins.