2.50
Hdl Handle:
http://hdl.handle.net/10033/621042
Title:
Isolation of F. novicida-Containing Phagosome from Infected Human Monocyte Derived Macrophages.
Authors:
Marecic, Valentina; Shevchuk, Olga; Ozanic, Mateja; Mihelcic, Mirna; Steinert, Michael; Jurak Begonja, Antonija; Abu Kwaik, Yousef; Santic, Marina
Abstract:
Francisella is a gram-negative bacterial pathogen, which causes tularemia in humans and animals. A crucial step of Francisella infection is its invasion of macrophage cells. Biogenesis of the Francisella-containing phagosome (FCP) is arrested for ~15 min at the endosomal stage, followed by gradual bacterial escape into the cytosol, where the microbe proliferates. The crucial step in pathogenesis of tularemia is short and transient presence of the bacterium within phagosome. Isolation of FCPs for further studies has been challenging due to the short period of time of bacterial residence in it and the characteristics of the FCP. Here, we will for the first time present the method for isolation of the FCPs from infected human monocytes-derived macrophages (hMDMs). For elimination of lysosomal compartment these organelles were pre-loaded with dextran coated colloidal iron particles prior infection and eliminated by magnetic separation of the post-nuclear supernatant (PNS). We encountered the challenge that mitochondria has similar density to the FCP. To separate the FCP in the PNS from mitochondria, we utilized iodophenylnitrophenyltetrazolium, which is converted by the mitochondrial succinate dehydrogenase into formazan, leading to increased density of the mitochondria and allowing separation by the discontinuous sucrose density gradient ultracentrifugation. The purity of the FCP preparation and its acquisition of early endosomal markers was confirmed by Western blots, confocal and transmission electron microscopy. Our strategy to isolate highly pure FCPs from macrophages should facilitate studies on the FCP and its biogenesis.
Affiliation:
Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany.
Citation:
Isolation of F. novicida-Containing Phagosome from Infected Human Monocyte Derived Macrophages. 2017, 7:303 Front Cell Infect Microbiol
Journal:
Frontiers in cellular and infection microbiology
Issue Date:
2017
URI:
http://hdl.handle.net/10033/621042
DOI:
10.3389/fcimb.2017.00303
PubMed ID:
28725638
Type:
Article
Language:
en
ISSN:
2235-2988
Appears in Collections:
publications of the research group modell systems for infections and immunity (MSYS)

Full metadata record

DC FieldValue Language
dc.contributor.authorMarecic, Valentinaen
dc.contributor.authorShevchuk, Olgaen
dc.contributor.authorOzanic, Matejaen
dc.contributor.authorMihelcic, Mirnaen
dc.contributor.authorSteinert, Michaelen
dc.contributor.authorJurak Begonja, Antonijaen
dc.contributor.authorAbu Kwaik, Yousefen
dc.contributor.authorSantic, Marinaen
dc.date.accessioned2017-08-04T08:32:52Z-
dc.date.available2017-08-04T08:32:52Z-
dc.date.issued2017-
dc.identifier.citationIsolation of F. novicida-Containing Phagosome from Infected Human Monocyte Derived Macrophages. 2017, 7:303 Front Cell Infect Microbiolen
dc.identifier.issn2235-2988-
dc.identifier.pmid28725638-
dc.identifier.doi10.3389/fcimb.2017.00303-
dc.identifier.urihttp://hdl.handle.net/10033/621042-
dc.description.abstractFrancisella is a gram-negative bacterial pathogen, which causes tularemia in humans and animals. A crucial step of Francisella infection is its invasion of macrophage cells. Biogenesis of the Francisella-containing phagosome (FCP) is arrested for ~15 min at the endosomal stage, followed by gradual bacterial escape into the cytosol, where the microbe proliferates. The crucial step in pathogenesis of tularemia is short and transient presence of the bacterium within phagosome. Isolation of FCPs for further studies has been challenging due to the short period of time of bacterial residence in it and the characteristics of the FCP. Here, we will for the first time present the method for isolation of the FCPs from infected human monocytes-derived macrophages (hMDMs). For elimination of lysosomal compartment these organelles were pre-loaded with dextran coated colloidal iron particles prior infection and eliminated by magnetic separation of the post-nuclear supernatant (PNS). We encountered the challenge that mitochondria has similar density to the FCP. To separate the FCP in the PNS from mitochondria, we utilized iodophenylnitrophenyltetrazolium, which is converted by the mitochondrial succinate dehydrogenase into formazan, leading to increased density of the mitochondria and allowing separation by the discontinuous sucrose density gradient ultracentrifugation. The purity of the FCP preparation and its acquisition of early endosomal markers was confirmed by Western blots, confocal and transmission electron microscopy. Our strategy to isolate highly pure FCPs from macrophages should facilitate studies on the FCP and its biogenesis.en
dc.language.isoenen
dc.rights.urihttp://creativecommons.org/licenses/by-nc-sa/4.0/*
dc.titleIsolation of F. novicida-Containing Phagosome from Infected Human Monocyte Derived Macrophages.en
dc.typeArticleen
dc.contributor.departmentHelmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany.en
dc.identifier.journalFrontiers in cellular and infection microbiologyen
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