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© © Institut Pasteur/Matteo Bonazzi et Edith Gouin
Visualization of actin comet tails stained with two different antibodies against actin (Green and Red) in PtK2 cell infected with Listeria monocytogenes (Blue) (unité Intéractions Bactéries-Cellules en collaboration avec la Plate-Forme )d'Imagerie Dynamique)
Scientific Fields
Diseases
Organisms
Applications
Technique
Starting Date
10
Apr 2016
Status
Ongoing
Members
7
Structures
1
Publications
1

About

We developed a novel image-based microscopy assay which allows to discriminate Listeria monocytogenes cellular entry from vacuolar escape, enabling high-content screening to identify factors specifically involved in these two steps. We generated a L. monocytogenes strain expressing a β-lactamase covalently attached to the bacterial cell wall, which can cleave a Förster resonance energy transfer (FRET) probe CCF4 loaded in the cytoplasm of target cells: the probe will be only cleaved by bacteria which escape their phagosomes. To discriminate between non-invading bacteria and bacteria trapped in phagosomes we perform subsequently a differential immunofluorescence staining to distinguish extracellular versus total bacterial populations in samples that were also analyzed by the FRET-based assay.

 

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