A Dual Microscopy-Based Assay To Assess Listeria monocytogenes Cellular Entry and Vacuolar Escape.
Listeria monocytogenes is a Gram-positive bacterium and a facultative intracellular pathogen that invades mammalian cells, disrupts its internalization vacuole, and proliferates in the host cell cytoplasm. Here, we describe a novel image-based microscopy assay that allows discrimination between cellular entry and vacuolar escape, enabling high-content screening to identify factors specifically involved in these two steps. We first generated L. monocytogenes and Listeria innocua strains expressing a β-lactamase covalently attached to the bacterial cell wall. These strains were then incubated with HeLa cells containing the Förster resonance energy transfer (FRET) probe CCF4 in their cytoplasm. The CCF4 probe was cleaved by the bacterial surface β-lactamase only in cells inoculated with L. monocytogenes but not those inoculated with L. innocua, thereby demonstrating bacterial access to the host cytoplasm. Subsequently, we performed differential immunofluorescence staining to distinguish extracellular versus total bacterial populations in samples that were also analyzed by the FRET-based assay. With this two-step analysis, bacterial entry can be distinguished from vacuolar rupture in a single experiment. Our novel approach represents a powerful tool for identifying factors that determine the intracellular niche of L. monocytogenes.
Institut Pasteur, Unité des Interactions Bactéries-Cellules, Paris, France INSERM, U604, Paris, France INRA, USC2020, Paris, France.
Juan J
JJ
Quereda
Institut Pasteur, Unité des Interactions Bactéries-Cellules, Paris, France INSERM, U604, Paris, France INRA, USC2020, Paris, France pizarroj@pasteur.fr.
Javier
J
Pizarro-Cerdá
Institut Pasteur, Unité des Interactions Bactéries-Cellules, Paris, France INSERM, U604, Paris, France INRA, USC2020, Paris, France.
Damien
D
Balestrino
Institut Pasteur, Unité Dynamique des Interactions Hôte-Pathogène, Paris, France.
Alexandre
A
Bobard
Institut Pasteur, Imagopole, Paris, France.
Anne
A
Danckaert
Institut Pasteur, Imagopole, Paris, France.
Nathalie
N
Aulner
Institut Pasteur, Imagopole, Paris, France.
Spencer
S
Shorte
Institut Pasteur, Unité Dynamique des Interactions Hôte-Pathogène, Paris, France.
Jost
J
Enninga
Institut Pasteur, Unité des Interactions Bactéries-Cellules, Paris, France INSERM, U604, Paris, France INRA, USC2020, Paris, France.
Pascale
P
Cossart
D001426:Bacterial Proteins / Q000378:metabolism; D003593:Cytoplasm / Q000382:microbiology*; D031541:Fluorescence Resonance Energy Transfer; D006367:HeLa Cells; D006576:Heterocyclic Compounds, 4 or More Rings / Q000378:metabolism; D054884:Host-Pathogen Interactions; D006801:Humans; D008087:Listeria / Q000201:enzymology / Q000378:metabolism; D008089:Listeria monocytogenes / Q000201:enzymology / Q000502:physiology*; D008856:Microscopy, Fluorescence; D014617:Vacuoles / Q000382:microbiology*; D001618:beta-Lactamases / Q000378:metabolism
D001426:Bacterial Proteins; C524948:CCF4-AM; D006576:Heterocyclic Compounds, 4 or More Rings; D001618:beta-Lactamases