Biochemistry of Macromolecular Interactions - CNRS URA2185  

  HEADDr LADANT Daniel /

  Annual Report

Our research interests are mainly focused on the study of the molecular mechanisms that underlie protein-protein and protein-membrane interactions, using as a model system a bacterial toxin, the adenylate cyclase (CyaA) produced by Bordetella pertussis, the causative agent of whooping cough. We are currently pursuing two major themes of research:

I Molecular mechanisms of action of CyaA toxin

CyaA is able to invade eukaryotic cells where it is activated by calmodulin to produce supra physiological levels of cAMP. CyaA is a 1706 residue-long protein with a unique mechanism of penetration into eukaryotic cells (see scheme below): after binding through its C-terminal part (red), to a receptor, the αMβ2 integrin, at the surface of target cells, the central region (blue) is inserted into the plasma membrane and, then, the N-terminal catalytic domain (green) is directly translocated across the membrane into the cytosol of the cells.

In 2006, our studies were focused primarily on the characterization of the C-terminal moiety of CyaA. This domain, made of about 40 nonapeptide motifs known as RTX (for Repeat in ToXin), is involved in the interaction with the integrin receptor and in binding of calcium, an essential co-factor for toxin entry into target cells. We have shown, by a combination of spectroscopic and biochemical approaches, that upon calcium binding, the RTX domain undergoes a large conformational change from an extended, highly solvated conformation into a compact and stable structure. These data have implications on the biogenesis and function of the toxin.

Besides, CyaA, as shown previously in collaboration with C. Leclerc and her team, can be used as an efficient vaccine vehicle. Antigens can be genetically grafted into CyaA and delivered in vivo to dendritic cells - that express the αMβ2 integrin receptor - where they are processed and presented to both MHC-class I and MHC-class II molecules, to elicit specific CD8+ and CD4+ T cell responses. Recent data indicated that CyaA can also induce potent neutralizing humoral immune responses against the grafted antigen(s).

II In vivo analysis of protein-protein interactions

Our second research theme is the study of protein-protein interaction in vivo with a CyaA-based two-hybrid screening technology (BACTH), previously designed in our group. This genetic assay is based on the functional complementation between two CyaA fragments to restore a cAMP-signaling cascade in an Escherichia coli cya strain. Currently, we are mainly interested to study the assembly of the cell division machinery of E. coli, by combining in vivo interaction assays (using BACTH system) with genetic approaches and in situ protein localization (using GFP fusions). In 2006, we characterized a putative novel component of the cell division apparatus, YmgF, a 72 residue-long polypeptide with 2 trans-membrane segments. YmgF had been isolated by two-hybrid screening and was found to interact with many other cell division proteins. It might serve as an adaptor protein to modulate septum assembly.



Publications 2006 of the unit on Pasteur's references database

Activity Reports 2006 - Institut Pasteur
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