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  Director : LADANT Daniel (ladant@pasteur.fr)


  abstract

 

We are studying the molecular mechanisms that underlie protein-protein and protein-membrane interactions using as a model system a bacterial toxin, the adenylate cyclase (AC or CyaA) produced by Bordetella pertussis, the causative agent of whooping cough. 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.



  report

cale

1 - Mechanisms of entry of the CyaA toxin into eukaryotic cells (Cécile Bauche)

The CyaA toxin, one of the major virulence factors of this organism, presents several striking characteristics: it is secreted by the virulent bacteria and it 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. The CyaA toxin is a 1706 residues-long bifunctional protein. 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. Our main objective is to analyze the molecular mechanisms of the invasion of target cells by the CyaA toxin. Indeed, this protein is endowed with the unique capability of delivering its N-terminal catalytic domain directly across the plasma membrane of eukaryotic cells. During the last year, our work has been focused on the characterization of the role of calcium, a key effector of toxin entry into target cells. CyaA is able to bind about 40 calcium ions on repeated motifs called RTX, located in the C-terminal part of the toxin. Various sub-fragments of the RTX domain have been produced and characterized functionally and structurally by spectroscopic approaches (fluorescence and circular dichroism, in collaboration with A. Chaffotte, Unité de Repliement et Modélisation des Protéines, Institut Pasteur), which allowed us to define the structural changes induced by calcium binding.

2 —Recombinant CyaA toxin as a general vaccine vehicle (Cécile Bauche, in collaboration with C. Leclerc, Unité de Biologie des Régulations Immunitaires, Institut Pasteur)

We showed earlier in collaboration with Claude Leclerc and her team (Unité de Biologie des Régulations Immunitaires, Institut Pasteur), that the CyaA toxin constitutes a potent non-replicating vector to deliver antigens into antigen presenting cells and induce specific cell-mediated immune responses: recombinant CyaA toxins carrying defined CD8+ T-cell epitopes genetically inserted into the catalytic domain, are able to stimulate strong cytotoxic T- cell (CTL) responses in mice. Recently we have demonstrated that, in vivo, CyaA is efficiently targeted to dendritic cells (DC) as a result of its interaction with the CD11b/CD18 integrin, previously identified as a specific cellular receptor for CyaA. Hence, antigens genetically or chemically coupled to CyaA, can be delivered to these professional antigen-presenting cells. Selective targeting of the CyaA toxin to DC could explain the remarkable efficacy of this vector able to trigger cytotoxic T-cell responses independently of CD4+ T-cell help. Current work is focused on the biochemical characterization of the interaction of the CyaA with its CD11b/CD18 receptor as well as the engineering of recombinant CyaA proteins carrying various viral and tumoral antigens.

3 — Adenylate cyclase as a signal transducer in Escherichia coli : development of screening technologies for protein-protein interactions. (Gouzel Karimova, Nathalie Dautin)

B. pertussis adenylate cyclase (AC) represents an original model of a bacterial enzyme activated by an eukaryotic protein, calmodulin. Previous work indicated that the catalytic domain of AC is made of two complementary fragments, named T25 and T18, that are both required for enzymatic activity. We took advantage of the modular structure of the AC catalytic domain to design a sensitive genetic technique to detect either protein/protein interactions. In this "bacterial two-hybrid system" (BTH), polypeptides of interest are genetically fused to the two AC subdomains, T25 and T18, and the hybrid proteins are co-expressed in an E. coli cya strain. Association of the two chimeric polypeptides allows functional complementation between the two AC subdomains and restores AC enzymatic activity: the synthesized cAMP, can then activate gene transcription thus conferring a particular phenotype ("Cya+") to the host cell. This can be scored easily on appropriate indicator or selective media.

In 2002, we showed that this methodology is particularly suited 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). These proteins have been intensely studied by genetic and cell-biology techniques but the molecular basis of their selective assembly in the septum are largely ignored. Results of two-hybrid analysis revealed a network of interactions between these different essential proteins. Current work is now focused on the delineation of the specific domain(s) of interactions between each partners. These results should yield novel insights into the fundamental processes of bacterial cell division

5 - AC-based genetic assays for protease activity in E. coli: detection of HIV protease resistance to protease inhibitors. (Nathalie Dautin, Sébastien Franconi)

The modular stucture of AC was also used to design a genetic screen for site-specific proteolytic activities. This assay is based on the inactivation, upon specific proteolysis, of a chimeric AC modified by the insertion between the two AC subdomains ,T25 and T18, of a polypeptide, corresponding to the cleavage site of the protease of interest. When expressed in an E. coli cya strain with the cognate protease, the modified AC is cleaved and inactivated. The resulting change in the phenotype of the host cells ("Cya+" -> "Cya-") can be easily detected on indicator or selective media. This genetic system was designed, in particular, to assay the protease of the human immunodeficiency virus (HIV), and we showed earlier that this test is sensitive enough to detect HIV protease variants that are resistant to the anti-protease inhibitors used in AIDS-treatments. In 2002, in collaboration with F. Clavel (Hôpital Bichat, Paris) we initiated a study to evaluate the potentialities of this genetic assay as a diagnostic test for the detection, in patients undergoing highly active anti-retroviral therapy (HAART), of the emergence of HIV variants harboring antiprotease-resistant proteases.

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

This work is a continuation of A. Ullmann's projects started in the seventies. We isolated and characterized a novel cAMP receptor mutant which confers cAMP-independent expression and total relief of catabolite repression of the catabolic operons in E. coli. The two specific mutations responsible for these effects were identified.

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



  publications

puce Publications of the unit on Pasteur's references database


  personnel

  Office staff Researchers Scientific trainees Other personnel
    KARIMOVA Gouzel, Institut Pasteur, Researcher,karimova@pasteur.fr

LADANT Daniel, CNRS, DR2,ladant@pasteur.fr
BAUCHE Cécile, Postdoc

DAUTIN Nathalie, PhD student

ULLMANN Agnès, Emeritus CNRS

FRANCONI Sébastien, Technician (from 01/02/02 to 04/08/02)
 

Activity Reports 2002 - Institut Pasteur
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