Our research unit studies bacterial protein toxins. These virulence factors produced by highly pathogenic bacteria are largely responsible for the pathophysiological outcomes of the acute phase of infection. It is essential to define the mode of action of toxins and their function in order to predict the pathogenic potential of bacterial isolates and to adapt therapeutic strategies. The study of these remarkably powerful proteins is also an invaluable source of information on basic biological processes and allows us to consider their use as therapeutic agents. This is the case for Clostridium neurotoxins, one group of toxins studied in the laboratory, that are widely used to block cholinergic nerve transmission and thereby block involuntary muscle contractions.
Our research focuses on the study of the mode of action of bacterial toxins in relationship to their impact on the barrier function of the epithelium and endothelium. We determine the intimate molecular mechanisms that allow toxins to breach or cross these barriers to promote their spreading or the spreading of bacteria in the tissues. Our study models include toxins that target the actin cytoskeleton and its upstream regulators, that we study through multidisciplinary approaches encompassing Cell Biology, Biochemistry and Physics. Our studies enable us to develop new diagnostic tools in particular for neurotoxins, as well as new therapeutic strategies in vaccinology. The study of cellular targets of toxins also provides valuable information on molecular processes that are impaired in a large number of human diseases among which inflammatory diseases and cancer.
Project toxins and epithelial barrier:
A large number of pathogenic bacteria, notably toxigenic strains of bacteria, reside or pass through the digestive compartment. Some toxins act systemically. Their passage through the epithelial barrier, the first essential step in the process of systemic dissemination, remains to be elucidated. This process of toxin translocation is an essential step especially in food botulism and in intestinal colonization. Our recent studies highlight the implication of a specific mechanism of transcytosis of botulinum toxins type A and B and the involvement of different enteric cell types that we seek to better characterize. In parallel, we develop diagnostic strategies for rapid determination of isoforms of botulinum toxins in order to better manage patients.
Project toxins and endothelial barrier:
We have discovered a new mode of permeabilization of the endothelium barrier by induction of transcellular tunnels in response to a group of toxins capable of disrupting the organization of the actin cytoskeleton. This involves the transient opening within cells of tunnels of several microns in diameter that are associated with the appearance of edema and hemorrhage symptoms, as well as the dissemination of bacteria in tissues. Transient opening of tunnels is also observed during the diapedesis of immune cells through the endothelium. Our multidisciplinary studies with the physicists of the Institut Curie and the Institut Pasteur of Lille have allowed us to define that this phenomenon is similar to the so-called process of dewetting of a viscous film. In reaction to enlargement of these tunnels, the cells form a rigid actin cable, which encircles the periphery to limit their opening. Cells also produce membrane ruffles, which extend over the tunnels to close them. The dysfunction of this system of guard correlates with an induction of hemorrhage.
Project toxins targeting the actin cytoskeleton regulators:
A large number of bacterial toxins catalyze post-translational modifications of small GTPases of the Ras superfamily. Some Gram-negative bacteria such as Escherichia coli, Yersinia pseudotuberculosis, and Bordetella spp produce deamidase toxins capable of catalyzing the post-translational modification of a critical glutamine residue of Rho GTPases into a glutamic acid. This type of targeted modification of Rho GTPases is analogous to a somatic mutation leading to the permanent activation of Rho GTPases. We have discovered a cellular regulation by ubiquitination and proteasomal degradation that ensures a proper control of the level of activation of these GTPases. We currently decipher the elements of this signaling system considering its importance in the control of cell division and migration, cohesion of epithelia and inflammatory reactions as well as its involvement in the mechanism of action of toxins. A growing number of studies point to the implication of a dysfunction of this system in infectious, inflammatory and tumorigenesis processes. We study in parallel the mode of action of the glucosylating toxins of Clostridium, which induce the inactivation of these GTPases.
Project neurotoxins and countermeasures:
EuroBioTox is a large European project focusing on biological toxins of potential bioterrorism risk involving 60 expert institutions from 23 countries. Recent incidents in France (Paris, May 2018) and in Germany (Cologne, June 2018) on thwarted biorrorist attacks highlighted that extremists take an interest in the production and dissemination of biological toxins. Therefore, there is an urgent need to strengthen European capabilities to cope with a potential intentional release of biological toxins.
It focuses on four main objectives:
1) Evaluate currently available analytical tools, methods, reagents, reference samples and matrices using the expertise of the 13 core partners
2) Improve validation and testing capabilities for biological toxin analysis across the EU.
3) Enable laboratories across the EU to further improve their analytical skills in detecting biological toxins: To this end, eleven interlaboratory exercises targeting the different toxins are planned involving Pasteur Institute for Botulinum toxins testing. Linked with this task is the establishment of a European repository for validation of toxin detection methods which makes available proprietary tools and reagents available among the 13 core partners. Pasteur Institute has recently produced a full range of reference samples available among the 60 partner laboratories.
4) Replacement of animal experiments for the detection of BoNT, led by Pasteur Institute (Michel Popoff): This goal comprises a cross-validation study to compare different existing in vitro or ex vivo replacement methods available in the consortium side-by-side to identify robust, sensitive and ethical alternative approaches that can be spread within the whole EuroBioTox network.
Overall, the project is implemented by the 13 core partners in collaboration with 47 outer network partners with the goal to consolidate and expand a network of laboratories focusing on biological toxins transferring know-how from experts to practitioners.