Naslov
ISOLATION OF F. NOVICIDA-CONTAINING PHAGOSOME FROM INFECTED HUMAN MONOCYTE DERIVED MACROPHAGES

Autori
Marecic, Valentina ; Shevchuk, Olga ; Ozanic, Mateja ; Mihelcic, Mirna ; Steinert, Michael ; Jurak Begonja, Antonija ; Abu Kwaik, Yousef ; Santic, Marina

Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, prošireni sažetak, znanstveni

Izvornik
Acta Microbiologica et Immunologica Hungarica / Szabo, D - Budimpešta : Academic Kiado, 2017, 146-147

Skup
CEFORM

Mjesto i datum
Keszthely, Mađarska, 19.10.2017. - 21.10.2017

Vrsta sudjelovanja
Predavanje

Vrsta recenzije
Međunarodna recenzija

Ključne riječi
phagocytosis, organelle purification, pathogen-containing phagosomes, Francisella, human macrophages

Sažetak
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 minutes 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.

Izvorni jezik
Engleski

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