Biochemistry of Macromolecular Interactions - CNRS URA2185  


  HEADDr Daniel LADANT / ladant@pasteur.fr
  MEMBERSBENEDIC Marie-Bénédicte, Institut Pasteur
CHENAL, Alexandre, Institut Pasteur, Chargé de Recherche, KARIMOVA Gouzel, Institut Pasteur, Chargée de Recherche, LADANT Daniel, CNRS, DR2
ROBICHON Carine, Postdoctoral fellow, robichon@pasteur.fr SPIACZKA-KARST Johanna, Postdoctoral fellow, SOTOMAYOR Ana-Cristina, Ph. D. student, ULLMANN Agnès, DREM CNRS
DAVI Marilyne, Institut Pasteur, technician


  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 CyaA toxin: structure-function studies and application in vaccinology

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 unravel the molecular mechanisms of CyaA intoxication of target cells. Indeed, this protein is endowed with the unique capability of delivering its N-terminal catalytic domain directly across the plasma membrane of eukaryotic cells (Figure 1). Besides, CyaA is efficiently targeted in vivo to dendritic cells (DC) as a result of its interaction with the aMb2(CD11b/CD18) integrin, 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. In 2008, we have characterized the structural and hydrodynamic properties of the CyaA RTX Domain (RD) that mediates the binding of the toxin to its CD11b/CD18 receptor. This 701 amino acid-long domain contains about 40 « Repeat in ToXin » (RTX) motifs that are tandemly repeated calcium-binding nonapeptide sequences present in many cytolysins from Gram negative bacteria. We showed that, in the absence of calcium, RD was natively disordered, weakly stable and highly hydrated. Calcium-binding induced compaction and dehydration of RD, along with the formation of stable secondary and tertiary structures. The calcium-induced conformational switch between unfolded conformations of apo-RD and stable structures of holo–RD is likely to be a key property for the biological function of the CyaA toxin: in the low calcium environment of the bacterial cytosol, the intrinsically disordered character of the protein may facilitate its secretion through a dedicated type Isecretion machinery. In the extracellular medium, calcium binding can then trigger the folding of the polypeptide into its functional state.The intrinsic disorder of RTX motifs in the absence of calcium may be a common property of many cytolysins that are secreted by the type I secretion machineries of Gram negative bacteria.

II in vivo analysis of protein-protein interaction in the bacterial cell division machinery.

Our second research theme is the study of the assembly of multi-molecular membrane-associated protein complexes in vivo, in particular by exploiting a CyaA-based two-hybrid screening technology (BACTH), previously designed in our group. We are currently exploiting this genetic assay in combination with genetic approaches and in situ protein localization (using fusions to the Green Fluorescent Protein) to characterize the assembly of the cell division machinery of E. coli . In 2008, we identifiedYmgF, a 72 residue-long integral membrane protein of unknown function as a potential novel component of the divisome. YmgFwas found by two-hybrid screening, to associate with many E. coli cell division proteins, and to localise to the E. coli division septum. Although not essential for cell viability, YmgF was able to restore the viability in non-permissive conditions of ftsQ(ts) thermosensitive mutants. Other studies were focused on the characterization of the association of FtsL with FtsB and FtsQ, into a ternary complex that is central in the assembly of the cell division machinery.

Figure 1 : Schematic model for CyaA toxin entry into target cells

A 3 stage model for CyaA invasion of target cells : (i) binding of CyaA to CD11b/CD18 receptor at the target cell surface ; (ii) insertion of hydrophobic segments of CyaA into the plasma membrane of target cells ; (iii) translocation of CyaA catalytic domain across the plasma membrane of target cells. In the cytosol CyaA interacts with calmodulin (CaM) that stimulates its catalytic activity to produce supra-physiological amount of cAMP.

Keywords: toxin, adenylate cyclase, vectorisation, two-hybrid, cell division, vaccinology, biotechnology

Bima.jpg

Figure 1 : Schematic model for CyaA toxin entry into target cells
A 3 stage model for CyaA invasion of target cells : (i) binding of CyaA to CD11b/CD18 receptor at the target cell surface ; (ii) insertion of hydrophobic segments of CyaA into the plasma membrane of target cells ; (iii) translocation of CyaA catalytic domain across the plasma membrane of target cells. In the cytosol CyaA interacts with calmodulin (CaM) that stimulates its catalytic activity to produce supra-physiological amount of cAMP.



  Publications

G. Karimova, N. Dautin, and D. Ladant (2005)Interaction network among Escherichia coli membrane proteins involved in cell division as revealed by bacterial two-hybrid analysis. J. Bact.. 187:2233-2243(PMID: 15774864)

C. Bauche, A. Chenal, O. Knapp, C. Bodenreider, R. Benz, A. Chaffotte, and D. Ladant (2006) Structural and functional characterization of an essential RTX sub-domain of Bordetella pertussisadenylate cyclase toxin. J Biol Chem. 281 :16914-16926 (PMID: 16627468)

F. Dal Molin, F. Tonello, D. Ladant, I. Zornetta, I. Zamparo, G. Di Benedetto, M. Zaccolo, and C. Montecucco(2006) Cell entry and cAMP imaging of anthrax edema toxin. EMBO. J. 25:5405-5413 (PMID: 17082768)

P. Berraondo, C. Nouzé, X. Préville, D. Ladant, and C. Leclerc(2007) Eradication of Large Tumors in Mice by a Tritherapy Targeting the Innate, Adaptive, and Regulatory Components of the Immune System. Cancer Research67:8847-8855 (PMID: 17875726)

S.R. Paccani, F.D. Molin, M. Benagiano, D. Ladant, M.M. D'Elios, C.Montecucco, and C.T.Baldari (2008) Suppression of T lymphocyte activation and chemotaxis by the adenylate cyclase toxin of Bordetella pertussis.Infect. Immun.76:2822-2832 (PMID: 18426886)





Activity Reports 2009 - Institut Pasteur
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