Time-Resolved Fluorescence Microscopy Screens on Host Protein Subversion During Bacterial Cell Invasion.
BAR domain-containing proteins
Bacterial infection
High-content screening
Intracellular bacterial pathogens
Membrane trafficking
Rab GTPases
Shigella flexneri
Journal
Methods in molecular biology (Clifton, N.J.)
ISSN: 1940-6029
Titre abrégé: Methods Mol Biol
Pays: United States
ID NLM: 9214969
Informations de publication
Date de publication:
2022
2022
Historique:
entrez:
27
6
2022
pubmed:
28
6
2022
medline:
30
6
2022
Statut:
ppublish
Résumé
Intracellular bacterial pathogens have evolved a plethora of strategies to invade eukaryotic cells. By manipulating host signaling pathways, in particular vesicular trafficking, these microbes subvert host functions to promote their internalization and to establish an intracellular niche. During these events, host endomembrane compartments are dynamically reorganized. Shigella flexneri, the causative agent of bacillary dysentery, recruits components of the host recycling pathway and the exocyst of non-phagocytic enterocytes in the vicinity of its entry site to facilitate its access to the host cytosol. These factors are either dynamically tethered to in situ formed macropinosomes or to the bacteria-containing vacuole itself. The underlying interactions cannot readily be monitored as individual bacterial infection events take place without synchronicity using cellular infection models. Therefore, time-resolved screens by fluorescence microscopy represent a powerful tool for the study of host subversion. Such screens can be performed with libraries of fluorescently tagged host factors. Using the cytosolic pathogenic agent Shigella flexneri as a model, we provide detailed protocols for such medium-to-high throughput multidimensional imaging screening of the dynamic host-pathogen cross talk. Our workflow is designed to be easily adapted for the study of different host factor libraries and different pathogen models.
Identifiants
pubmed: 35759194
doi: 10.1007/978-1-0716-2449-4_8
doi:
Substances chimiques
Bacterial Proteins
0
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
113-131Informations de copyright
© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.
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