Unit: Biochemistry of Macromolecular Interactions

Director: LADANT Daniel

We are studying the structure/function relationships of a bacterial toxin, the adenylate cyclase (CyaA) toxin produced by Bordetella pertussis, the causative agent of whooping cough. The CyaA toxin, one of the major virulence factors of this organism, is able to enter into eukaryotic cells where, upon activation by endogenous calmodulin, it catalyzes high-level synthesis of cAMP that in turn alters cellular physiology. This toxin exhibits several remarkable characteristics and constitutes an attractive model system to analyze the molecular mechanisms underlying protein-protein and protein-membrane interactions. Basic knowledge on the mechanisms of toxin entry into eukaryotic target cells and its interaction with cellular effectors is exploited for various applications in vaccinology and biotechnology.

Interaction of the CyaA toxin with its cellular receptor (Cécile Bauche, in collaboration with C. Leclerc, Unité de Biologie des Régulations Immunitaires, Institut Pasteur, Paris, and P.Sebo, Microbiology Institute, Praha, Czech Republic)

The CyaA toxin is a 1706 residues-long bifunctional protein that belongs to the RTX (repeat in toxin) protein family. The calmodulin-activated, catalytic domain is located in the 400 amino-proximal residues, whereas the carboxy-terminal 1306 residues are responsible for the binding of the toxin to the target cells and the translocation of the catalytic domain across the cytoplasmic membrane of these cells. We have previously shown that, in vivo, CyaA uses the αMβ2 integrin (CD11b/CD18) as a specific cellular receptor. During the last year, we demonstrated that functional interaction of CyaA with cells that express CD11b/CD18+ is dependent upon the specific post-translational acylation of the toxin. Furthemore, we showed that a main integrin binding site of CyaA is located in its RTX repeat domain. Current work is now aimed at further defining critical residues and/or structures involved in toxin/receptor interactions.

Interaction of the CyaA toxin with model membranes (Cécile Bauche, in collaboration with J. Chopineau, Université de Technologie de Compiègne, Compiègne, France).

One of the most striking properties of the CyaA toxin is its ability to translocate its N-terminal catalytic domain directly across the plasma membrane of the target cells. Our main objective is to delineate the molecular mechanisms of the translocation of the catalytic domain across the plasma membrane of the eukaryotic cells. To study the interaction of CyaA with model membrane we used a novel type of biomimetic phospholipid bilayers that are covalently tethered to the sensing surface of an optical biosensor instrument. Binding properties of CyaA to these model membranes were characterized by surface plasmon resonance spectroscopy, and shown to be similar to those observed with intact cells. Binding properties of various CyaA variants and of different fragments of CyaA, as well as the effects of membrane bilayer composition are currently evaluated. The goal now is to elaborate a functional assay to follow the translocation of the catalytic domain across the supported bilayer. This will open the way for detailed biochemical and biophysical characterization of this process.

Recombinant CyaA toxin as a vaccine vehicle (Cécile Bauche, in collaboration with C. Leclerc, Institut Pasteur, and B. Van den Eynde, Ludwig Institute for Cancer Research, Brussels, Belgium)

As a result of its interaction with the CD11b/CD18 integrin, CyaA is efficiently targeted to dendritic cells (DC) and we showed earlier that this protein constitutes a potent non-replicating vector to deliver antigens into DC to induce specific cell-mediated immune responses. Recombinant CyaA toxins carrying human melanoma epitopes genetically inserted into the catalytic domain were produced and shown to induce strong anti-melanoma CTL responses in transgenic mice. Besides, human DC treated with these recombinant CyaAs were shown to efficiently process and present the melanoma epitopes to human CTL clones. These observations suggest that these CyaA molecules might be used to induce antitumoral CTL in humans.

Adenylate cyclase as a screening tool to study protein-protein interactions. (Gouzel Karimova, Nathalie Dautin)

CyaA is an interesting model of a bacterial enzyme activated by an eukaryotic protein, calmodulin. Its catalytic domain (AC) presents an original, modular structure, made of two complementary fragments (T25 and T18) that are both required for enzymatic activity. We previously designed a sensitive genetic technique ("bacterial two-hybrid", BTH), based on the functional complementation between these two fragments, to detect protein/protein interactions in vivo in Escherichia coli. In 2003, we exploited this methodology to analyze the assembly of membrane proteins. The BTH system was used in particular to study the associations between various E. coli membrane proteins that are involved in the cell division process (Fts proteins). Our results established that these different essential proteins are linked to each other by a network of interactions that involve specific sub-domain(s) of each partner. The BTH system was also successfully applied to study other membrane protein complexes such as ABC transporters. These data indicate that this methodology is particularly appropriate to analyze membrane protein complexes.

AC-based genetic assays for HIV protease activity (Nathalie Dautin, Gouzel Karimova, Marilyne Davi)

The modular structure of AC was also used to design a genetic screen for site-specific proteolytic activities, in particular, to characterize the protease of the human immunodeficiency virus (HIV). This approach has been used to analyze the structure-function relationships of HIV protease and to screen HIV protease variants resistant to the anti-protease inhibitors used in AIDS-treatments. In 2003, this genetic test has been further implemented and evaluated as a potential diagnostic test to detect, in patients undergoing highly active anti-retroviral therapy (HAART), the emergence of HIV variants harboring antiprotease-resistant proteases.

In vitro selection for enzymatic activity : a model study using adenylate cyclase. (D. Ladant, in collaboration with H. Strobel and J.L. Jestin, Unité de Chimie Organique, Institut Pasteur)

An in vitro enzyme selection approach has been established, using AC as a model enzyme. By using a "tailor-made" biotinylated ATP analog and an antibody fragment that specifically recognizes cAMP, filamentous phages displaying on their surface an enzymatically active AC form could be selected. This in vitro selection procedure should be applicable, in principle, to many types of catalysts acting on small molecules.

Catabolite repression in E. coli. (Agnes Ullmann, Gouzel Karimova)

As a continuation of A. Ullmann's earlier projects, a novel cAMP receptor mutant that confers cAMP-independent expression and total relief of catabolite repression of the catabolic operons in E. coli has been isolated and characterized.

Keywords: toxin, adenylate cyclase, vectorization, two-hybrid, protease, Vaccinology, biotechnology

Activity Reports 2003 - Institut Pasteur

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