Abstract
Salmonella is a gram-negative facultative intracellular pathogen that is capable of infecting a variety of hosts. Inside host cells, most Salmonella bacteria reside and replicate within Salmonella-containing vacuoles. They use virulence proteins to manipulate the host cell machinery for their own benefit and hijack the host cytoskeleton to travel toward the perinuclear area. However, a fraction of bacteria escapes into the cytosol where they get decorated with a dense layer of polyubiquitin, which labels the bacteria for clearance by autophagy. More specifically, autophagy receptor proteins recognize the ubiquitinated bacteria and deliver them to autophagosomes, which subsequently fuse to lysosomes. Here, we describe methods used to infect HeLa cells with Salmonella bacteria and to detect their ubiquitination via immunofluorescence and laser scanning confocal microscopy.
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Acknowledgement
M.L. holds a predoctoral fellowship from the Fund for Scientific Research Flanders (FWO). Work in the authors’ lab is supported by grants from the FWO, “Belgian Foundation Against Cancer”, “Agency for Innovation by Science and Technology”, “Interuniversity Attraction Poles”, “Concerted Research Actions”, and “Group-ID Multidisciplinary research partnership” from Ghent University, VIB. The authors would like to acknowledge networking support by the Proteostasis COST Action (BM1307).
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Lork, M., Delvaeye, M., Gonçalves, A., Van Hamme, E., Beyaert, R. (2016). Monitoring Ubiquitin-Coated Bacteria via Confocal Microscopy. In: Matthiesen, R. (eds) Proteostasis. Methods in Molecular Biology, vol 1449. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3756-1_14
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DOI: https://doi.org/10.1007/978-1-4939-3756-1_14
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