Propriétés mécaniques et fonctionnelles des cellules épithéliales respiratoires exposées à une toxine bactérienne : l’adénylate cyclase

Abstract : The increase in respiratory infections involving virulent factors of bacterial origin has become a major public health issue. A better knowledge of the cell respiratory response in the course of the initial cell invasion by bacterial toxins is important from the pathophysiological and therapeutical point of views.The purpose of this work is to decipher the cellular and molecular mechanisms involved in the exposition of respiratory epithelial cells to the adenylate cyclase toxin (CyaA) produced by Bordetella pertussis which is the whooping cough agent. We have chosen this toxin for its multiple capacities of penetrating a wide range of eukaryotic cells. Indeed, this toxin enables direct translocation of its catalytic domain across the plasma membrane of target cells using the endogen calmoduline to increase the cAMP rate at supraphysiological levels. However, the effects of these changes on mechano-chemical signaling (mechanotransduction) pathways remain largely unknown while it affects cellular functions and cell integrity. So, we perform an evaluation of cellular functions as well as mechanical and adhesion properties of respiratory epithelial cells exposed to CyaA toxin in order to detect some critical modifications in the mechanotransduction processes.In a preliminary study aiming at defining physiopathological concentrations of CyaA toxin used in our experiments, we determined the cell viability degree for 3 concentrations of CyaA toxin (0.5; 5 and 10 nM). We found that the smallest concentration (0.5 nM) did not affect cell viability whereas inducing supraphysiological cAMP levels in less than one hour.Then, we assessed the effects of CyaA toxin on cell migration and repair phenomenon, on ciliary beating and on cell permeability of epithelial cells representative of the different levels of the respiratory tract. The toxin induces a decrease in cell migration and repair, an increase in cell permeability suggesting a weakening of lateral cell-cell junctions.Immunostaining was performed on intracellular and interfacial structures of alveolar epithelial cells exposed to the 3 concentrations of CyaA toxin. Results show that CyaA toxin is able to induce cytoskeleton remodeling and a decrease in the number of focal adhesions. Finally, a refined analysis of mechanical properties and adhesion parameters was performed on the same cells by 2 techniques of micro/nanomanipulation modified to permit at the same time, an evaluation of cell adhesion and cell rigidity (Atomic Force Microscopy with indentation and force spectroscopy to characterize the number of bond during adhesion reinforcement and multiscale Magnetic Twisting Cytometry). To evaluate the role of cAMP on cellular and molecular changes, we tested the enzymatically inactive form of CyaA toxin called CyaAE5 which could not permit to increase the intracellular cAMP rate.The AFM experiments have revealed that the main effect of CyaA toxin is to decrease the number of associated integrin-ligand bounds (meaning an alteration of clustering) while, at the smallest concentration of CyaA toxin, we observe an increase in cell rigidity with an individual bound reinforcement, a result consistent with MTC results. Nevertheless, CyaE5 does not exhibit such cellular effects. On the whole, these results suggest that CyaA toxin affects the mechanotransduction pathways of cells exposed to the toxin, a result which is in agreement with the expected effects of cAMP increase (notably cytoskeleton remodeling, lateral junction alteration and inhibition of Rac1 expression) what brings a new vision of the cytotoxicity induced by the adenylate cyclase toxin.
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Christelle Angely. Propriétés mécaniques et fonctionnelles des cellules épithéliales respiratoires exposées à une toxine bactérienne : l’adénylate cyclase. Biochimie, Biologie Moléculaire. Université Paris-Est, 2018. Français. ⟨NNT : 2018PESC0058⟩. ⟨tel-02272458⟩

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