Isolation of F. novicida-Containing Phagosome from Infected Human Monocyte Derived Macrophages

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.

27-Hydroxycholesterol Promotes the Transfer of Astrocyte-Derived Cholesterol to Neurons in Co-cultured SH-SY5Y Cells and C6 Cells

“…Since CYP46A1 mobilization from the ER to the PM could increase 24S-OHC levels, we speculated that CYP46A1 mobilization was involved in 27-OHC-induced 24S-OHC generation in SH-SY5Y cells. By cell fraction separation combined with Western blot, N cadherin and KDEL were selected as marker proteins of PM and ER, respectively (Marecic et al, 2017;Ode et al, 2017), we found that with the increase of 27-OHC treatment concentration, the protein expression of CYP46A1 gradually decreased in the ER and increased in the PM (Figures 5F,G). Consistent with this, immunofluorescence staining suggested that 27-OHC down-regulated CYP46A1 expression in the ER but up-regulated the expression in the PM (Figures 5H,I).…”

Nutrition and Bipartite Metabolism of Intracellular Pathogens

“…In order to replicate in the host cytosol, F. tularensis induced ATG5-independent macroautophy mediated by the type VI secretion system (Fig. 3) [4,66,67]. This process is required for intracellular replication of F. tularensis but failure to replicate due to inhibition of macroautophagy could be rescued with the addition of excess nonessential amino acids or pyruvate [4].…”