Unit: Biochemistry of Macromolecular Interactions

Director: Daniel LADANT

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. Basic knowledge on the mechanisms of toxin entry into eukaryotic target cells and its interaction with cellular effectors are exploited for various applications in vaccinology and biotechnology.

1- Structure-function studies of the CyaA toxin

The CyaA toxin is one of the major virulence factors of B. pertussis and 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 unravel 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. Recently we have characterized 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 (repeat in toxin), located in the C-terminal part of the toxin. These motifs are characteristics of a family of RTX cytolysins that are key virulence factors of various bacterial pathogens. Furthermore, the RTX repeat domain is directly involved in the binding of CyaA to its specific cellular receptor, the αMβ2 integrin. We produced various sub-fragments of the RTX domain that have been characterized functionally and structurally by biochemical and biophysical approaches (fluorescence circular dichroism, electrophysiology, limited proteolysis, etc…). Our results suggest a model for the structural changes induced by calcium binding.

2 - Recombinant CyaA toxin as a vaccine vehicle (in collaboration with the unit of C. Leclerc, Immune Regulation and Vaccinology, Institut Pasteur)

Earlier studies have shown that the CyaA toxin constitutes a potent non-replicating vector to deliver antigens into antigen presenting cells and induce specific cell-mediated immune responses. Antigens of interest can be genetically inserted into the catalytic domain of a detoxified recombinant CyaA that can be efficiently targeted in vivo to dendritic cells (DC) that express the toxin receptor, the αMβ2 integrin. In these professional antigen-presenting cells, the grafted antigens can be processed and presented to both MHC-class I and class II pathways to induce specific CD8+ and CD4+ T cell responses (see figure 1). Our recent studies have demonstrated, in two different preclinical models, the potential of this vector in immunotherapy of cancer. The first one, carried out within the European program THERAVAC, is focused on human melanoma antigens while the second one, carried out in collaboration with the Biotech company, BT-Pharma (Labège,France) , is focused on the immunotherapy of human papilloma virus associated tumors. We also showed that in addition to CD4 and CD8 T-cell reponses, the CyaA vector is able to trigger neutralizing humoral immune responses against the grafted antigen. A recombinant CyaA carrying the transactivating factor Tat (86 residues), from the human immunodeficiency virus (HIV), induced long-lasting neutralizing anti-Tat antibody responses in mice, as well as Th1-polarized CD4+ T-cells and HIV-Tat specific CD8+ T-cells. Hence, CyaA represents an attractive vaccine vehicle to elicit broad immunogenic responses against an antigen.

3 - Assembly of membrane proteins involved in cell division in Escherichia coli.

We previously described a sensitive genetic technique to detect, in E. coli, protein/protein interactions. This "bacterial two-hybrid system" (BACTH) is based on the functional complementation between two subdomains of the CyaA catalytic domain (figure 2). Furthermore, as it involves a cAMP-signaling cascade, this system appears to be particularly appropriate to study interactions between membrane proteins.

Recently we used the BACTH system to characterize interactions between the E. coli proteins involved in the cell division machinery. Formation of the bacterial division septum is catalyzed by a number of essential proteins (named Fts) that assemble in a hierarchical order into a ring-like structure at the future division site. Several of these Fts proteins are intrinsic transmembrane proteins, whose functions are largely unknown. To identify the molecular basis of septum assembly, the protein interaction network among selected membrane-associated E. coli cell division proteins was analyzed with the BACTH system. Our study indicated that most Fts proteins are able to interact with multiple partners . A deletion mapping analysis was carried out to establish the polypeptide regions that determined the specific associations of the Fts proteins with their partners. We also showed that the interactions between two Fts-hybrid proteins can be modulated by co-expression of a third Fts partner. Altogether, these data suggest that the cell division machinery assembly is driven by the cooperative association between the different Fts proteins to form a dynamic multi-protein structure at the septum site. The BACTH genetic assay was also used to identify novel interacting partners of Fts proteins. Characterization of the putative role of these components is underway. We are also using the BACTH system to analyze different membrane-associated, multi-protein complexes such as the maltose ABC transporters and/or the Type I secretion machinery od various Gram- bacteria.

Legends of Figure

Figure 1: Recombinant CyaA toxins can deliver antigens into antigen presenting cells.

Recombinant CyaA carrying an antigen (Ag) genetically inserted into the catalytic domain, binds to antigen-presenting cells (dendritic cells) as a result of its selective interaction with the aMb2 integrin receptor (j). After translocation across the plasma membrane of the APC, the catalytic domain and the grafted antigen are degraded by the proteasome into peptides that can reach the classical cytosolic MHC class I presentation pathway, to elicit specific cytotoxic T-cell responses. Alternatively, the recombinant CyaA can be endocytosed into. endosomes/lysosomes where the protein is proteolyzed. The released peptides, can enter the MHC class II presentation pathway, to elicit specific Th1 type CD4+ helper T-cell responses.

Figure 2: Bacterial two-hybrid system (BACTH) based on CyaA fragment complementation

In this genetic assay, polypeptides of interest (i.e. X and Y) are genetically fused to two complementary fragments (T25 and T18) of CyaA catalytic domain, 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 CyaA subdomains and restores enzymatic activity: the synthesized cAMP, can then activate gene transcription (via binding to the Catabolite Activator Protein, CAP) that confers a particular phenotype ("Cya+") to the host cell. This can be scored easily on appropriate indicator or selective media.

Keywords: toxin, adenylyl cyclase, vectorisation, Two-hybrid, cell division, vaccinology, biotechnology


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