Blocking bacterial entry at the adhesion step reveals dynamic recruitment of membrane and cytosolic probes.

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 internalized. 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. Secondly, we demonstrate the feasibility of monitoring post-translationally modified proteins, e.g. 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 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. This article is protected by copyright. All rights reserved.