Quantification of Cytosolic vs. Vacuolar Salmonella in Primary Macrophages by Differential Permeabilization.

Détails

ID Serval
serval:BIB_837C61C4E7B0
Type
Article: article d'un périodique ou d'un magazine.
Collection
Publications
Titre
Quantification of Cytosolic vs. Vacuolar Salmonella in Primary Macrophages by Differential Permeabilization.
Périodique
Journal of Visualized Experiments
Auteur(s)
Meunier E., Broz P.
ISSN
1940-087X (Electronic)
ISSN-L
1940-087X
Statut éditorial
Publié
Date de publication
2015
Peer-reviewed
Oui
Numéro
101
Pages
e52960
Langue
anglais
Résumé
Intracellular bacterial pathogens can replicate in the cytosol or in specialized pathogen-containing vacuoles (PCVs). To reach the cytosol, bacteria like Shigella flexneri and Francisella novicida need to induce the rupture of the phagosome. In contrast, Salmonella typhimurium replicates in a vacuolar compartment, known as Salmonella-containing vacuole (SCV). However certain mutants of Salmonella fail to maintain SCV integrity and are thus released into the cytosol. The percentage of cytosolic vs. vacuolar bacteria on the level of single bacteria can be measured by differential permeabilization, also known as phagosome-protection assay. The approach makes use of the property of detergent digitonin to selectively bind cholesterol. Since the plasma membrane contains more cholesterol than other cellular membranes, digitonin can be used to selectively permeabilize the plasma membrane while leaving intracellular membranes intact. In brief, following infection with the pathogen expressing a fluorescent marker protein (e.g. mCherry among others), the plasma membrane of host cells is permeabilized with a short incubation in digitonin containing buffer. Cells are then washed and incubated with a primary antibody (coupled to a fluorophore of choice) directed against the bacterium of choice (e.g. anti-Salmonella-FITC) and washed again. If unmarked bacteria are used, an additional step can be done, in which all membranes are permeabilized and all bacteria stained with a corresponding antibody. Following the staining, the percentage of vacuolar and cytosolic bacteria can be quantified by FACS or microscopy by counting single or double-positive events. Here we provide experimental details for use of this technique with the bacterium Salmonella typhimurium. The advantage of this assay is that, in contrast to other assay, it provides a quantification on the level of single bacteria, and if analyzed by microscopy provides the exact number of cytosolic and vacuolar bacteria in a given cell.

Mots-clé
Animals, Cell Membrane/microbiology, Cell Membrane Permeability/drug effects, Cytosol/microbiology, Digitonin/pharmacology, Flow Cytometry/methods, Fluorescein-5-isothiocyanate/chemistry, Intracellular Membranes/microbiology, Luminescent Proteins/chemistry, Macrophages/microbiology, Mice, Phagosomes/microbiology, Salmonella typhimurium/isolation & purification, Vacuoles/microbiology
Pubmed
Web of science
Création de la notice
25/10/2017 11:05
Dernière modification de la notice
20/08/2019 15:43
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