1. Blocking bacterial entry at the adhesion step reveals dynamic recruitment of membrane and cytosolic probes
- Author
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Frank Lafont, Elisabeth Werkmeister, Yann Ciczora, Sébastien Janel, Magali Soyer, Michka Popoff, Assemblage et réplication du virus de l'hépatite C (ARVHC), Centre National de la Recherche Scientifique (CNRS), Centre d’Infection et d’Immunité de Lille - INSERM U 1019 - UMR 9017 - UMR 8204 (CIIL), Centre National de la Recherche Scientifique (CNRS)-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille)-Université de Lille-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Pasteur de Lille, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP), Institut Cochin (IC UM3 (UMR 8104 / U1016)), Centre National de la Recherche Scientifique (CNRS)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut d’Électronique, de Microélectronique et de Nanotechnologie - UMR 8520 (IEMN), Centrale Lille-Institut supérieur de l'électronique et du numérique (ISEN)-Université de Valenciennes et du Hainaut-Cambrésis (UVHC)-Université de Lille-Centre National de la Recherche Scientifique (CNRS)-Université Polytechnique Hauts-de-France (UPHF), Institut Pasteur de Lille, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Lille-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille)-Centre National de la Recherche Scientifique (CNRS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Physique - IEMN (PHYSIQUE - IEMN), Centrale Lille-Institut supérieur de l'électronique et du numérique (ISEN)-Université de Valenciennes et du Hainaut-Cambrésis (UVHC)-Université de Lille-Centre National de la Recherche Scientifique (CNRS)-Université Polytechnique Hauts-de-France (UPHF)-Centrale Lille-Institut supérieur de l'électronique et du numérique (ISEN)-Université de Valenciennes et du Hainaut-Cambrésis (UVHC)-Université de Lille-Centre National de la Recherche Scientifique (CNRS)-Université Polytechnique Hauts-de-France (UPHF), We thank A. Bongiovanni from the BICeL Facility for access to systems and technical advice. We would like to thank J. Warein for expert technical assistance, P.-H. Puech for valuable discussions and T. Melin, P-E Milhiet and G Tran Van Nhieu for critical reading of the manuscript., FundingThis study was supported by funds from Univ. Lille1 to M.P. and ANR (09-MIEN-020-01, 10-EQPX-04-01, 16), FEDER (12,001,407) to F.L., and LAFONT, Frank
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Cell signaling ,[SDV.IB.IMA]Life Sciences [q-bio]/Bioengineering/Imaging ,Glycosylphosphatidylinositols ,Green Fluorescent Proteins ,Cell ,[SDV.BC.IC] Life Sciences [q-bio]/Cellular Biology/Cell Behavior [q-bio.CB] ,Adhesion force ,Polymerization ,03 medical and health sciences ,Cytosol ,0302 clinical medicine ,[SDV.BC.IC]Life Sciences [q-bio]/Cellular Biology/Cell Behavior [q-bio.CB] ,Cell Adhesion ,medicine ,Fluorescence microscope ,ubiquitin ,Humans ,Yersinia pseudotuberculosis ,Super-resolution microscopy ,Cytoskeleton ,ComputingMilieux_MISCELLANEOUS ,030304 developmental biology ,Calcium signaling ,TNF Receptor-Associated Factor 6 ,Host cell surface ,0303 health sciences ,Binding Sites ,biology ,Cell Membrane ,Ubiquitination ,Cell Biology ,General Medicine ,Adhesion ,biology.organism_classification ,[SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology ,Actins ,Cell biology ,Correlative microscopy ,[SDV.IB.IMA] Life Sciences [q-bio]/Bioengineering/Imaging ,medicine.anatomical_structure ,Adsorption ,[SDV.MP.BAC] Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology ,AFM ,Protein Processing, Post-Translational ,030217 neurology & neurosurgery ,HeLa Cells - Abstract
International audience; Background: Bacterial invasion covers two steps: adhesion and entry per se. The cell signalling response is triggered upon pathogen interaction at the cell surface. This response continues when the pathogen is internalised. It is likely that these two steps activate different molecular machineries. So far, it has not been possible to easily follow in physiological conditions these events separately. We thus developed an approach to uncouple adhesion from entry using atomic force microscopy (AFM)-driven force and fluorescence measurements.Results: We report nanometric-scale, high-resolution, functional dynamic measurements of bacterial interaction with the host cell surface using photonic and adhesion force analyses. We describe how to achieve a precise monitoring of iterative cell–bacterium interactions to analyse host cell signalling responses to infection. By applying this method to Yersinia pseudotuberculosis, we first unveil glycosylphosphatidylinositol-anchored protein domains recruitment to the bacterium cell surface binding site and concomitant cytoskeleton rearrangements using super-resolution fluorescence microscopy. Second, we demonstrate the feasibility of monitoring post-translationally modified proteins, for example, via ubiquitylation, during the first step of infection.Conclusion: We provide an approach to discriminate between cellular signalling response activated at the plasma membrane during host–pathogen interaction and that is triggered during the internalisation of the pathogen within the cell.Significance: This approach adds to the technological arsenal to better understand and fight against pathogens and beyond the scope of microbiology to address conceptual issues of cell surface signalling.
- Published
- 2019
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