Abstract
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.
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Acknowledgments
The authors of this manuscript wish to thank the entire DIHP unit and particularly the following lab members Laurent AUDRY, Laura BARRIO, and Magdalena GIL and Sonja KUHN for their fruitful discussion, constructive criticism, technical support, and mentorship during this project. We are particularly grateful to Emmanuel Boucrot for sharing the BAR-domain library for our work (Chan Wah Hak, 2018). The DIHP unit is supported by an ERC grant (ERC-CoG-Endosubvert), and the ANR (StopBugEntry, HBPSensing), and it is part of the two LabExes IBEID and Milieu Interieur.
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Sanchez, L., Chang, YY., Mellouk, N., Enninga, J. (2022). Time-Resolved Fluorescence Microscopy Screens on Host Protein Subversion During Bacterial Cell Invasion. In: Kufer, T.A., Kaparakis-Liaskos, M. (eds) Effector-Triggered Immunity. Methods in Molecular Biology, vol 2523. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2449-4_8
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DOI: https://doi.org/10.1007/978-1-0716-2449-4_8
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