Monitoring Ubiquitin-Coated Bacteria via Confocal Microscopy.
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.
Unit of Molecular Signal Transduction in Inflammation, Inflammation Research Center, VIB, Ghent, Belgium.|Department of Biomedical Molecular Biology, Ghent University, Technologiepark 927, Zwijnaarde, Ghent, 9052, Belgium.
Marie
M
Lork
Unit of Molecular Signal Transduction in Inflammation, Inflammation Research Center, VIB, Ghent, Belgium.|Department of Biomedical Molecular Biology, Ghent University, Technologiepark 927, Zwijnaarde, Ghent, 9052, Belgium.
Mieke
M
Delvaeye
Bio Imaging Core, Inflammation Research Center, VIB, Ghent, Belgium.
Amanda
A
Gonçalves
Bio Imaging Core, Inflammation Research Center, VIB, Ghent, Belgium.
Evelien
E
Van Hamme
Unit of Molecular Signal Transduction in Inflammation, Inflammation Research Center, VIB, Ghent, Belgium. rudi.beyaert@irc.vib-UGent.be.|Department of Biomedical Molecular Biology, Ghent University, Technologiepark 927, Zwijnaarde, Ghent, 9052, Belgium. rudi.beyaert@irc.vib-UGent.be.
Rudi
R
Beyaert
D000071182:Autophagosomes / Q000378:metabolism; D001343:Autophagy / Q000502:physiology; D001419:Bacteria / Q000378:metabolism / Q000472:pathogenicity; D003600:Cytosol / Q000378:metabolism / Q000382:microbiology; D005455:Fluorescent Antibody Technique; D006367:HeLa Cells; D054884:Host-Pathogen Interactions; D006801:Humans; D018613:Microscopy, Confocal; D012475:Salmonella / Q000378:metabolism / Q000472:pathogenicity; D025801:Ubiquitin / Q000378:metabolism*; D054875:Ubiquitination; D014617:Vacuoles / Q000378:metabolism / Q000382:microbiology
D025801:Ubiquitin
Autophagy; Bacteria; Bacterial clearance; Confocal microscopy; Host–pathogen interaction; Imaging; Immunofluorescence; Salmonella; Ubiquitination