title: Quantitative analysis of phagolysosome fusion in intact cells: inhibition by mycobacterial lipoarabinomannan and rescue by an 1alpha,25-dihydroxyvitamin D3-phosphoinositide 3-kinase pathway.
abstract: Macrophage cell membranes were labeled with PKH26 and subsequently incubated with latex beads to generate phagosomes surrounded by a red-fluorescent membrane suitable for flow cytometry. Following cell disruption and partial purification of phagosomes, these vesicles were readily distinguished from both cell debris and free beads released from disrupted vacuoles. Flow cytometry analysis of phagosomes stained with specific mAbs and FITC-labeled secondary antibodies showed progressive acquisition of both Rab7 and LAMP-1 consistent with movement along the endocytic pathway. Alternatively, macrophages were preloaded with the lysosomal tracer FITC-dextran before membrane labeling with PKH and incubation with latex beads. Phagosome-lysosome fusion was then quantified on the basis of the colocalization of red and green signals. Using these flow cytometry-based systems, we showed that co-internalization of beads with lysates of Mycobacterium tuberculosis, but not lysates from the nonpathogenic organism Mycobacterium smegmatis, markedly decreased phagosome acquisition of Rab7 and LAMP-1 and vesicle fusion with FITC-dextran-loaded lysosomes. Inhibition of phagolysosome fusion could be attributed, at least in part, to the mycobacterial cell wall glycolipid lipoarabinomannan, and further analysis showed complete rescue of phagosome maturation when cells were pretreated with vitamin D3 before exposure to lipoarabinomannan. Moreover, the ability of vitamin D3 to reverse the phenotype of phagosomes in the presence of the glycolipid was completely abrogated by LY-294002, suggesting that vitamin D3 promotes phagolysosome fusion via a phosphoinositide 3-kinase signaling pathway. These findings establish a robust platform technology based on labeling of phagocyte cell membranes and flow cytometry capable of supporting broad-based screens to identify microbial and other bioactive compounds that influence phagosome biology.
authors.affiliation: Division of Infectious Diseases, Department of Medicine, The University of British Columbia and Vancouver Hospital Health Sciences Center, D452-HP, 2733 Heather Street, Vancouver, BC, V5Z 3J5 Canada. hmama@interchange.ubc.ca
authors.forename: Zakaria
authors.initials: Z
authors.lastname: Hmama
authors.forename: Khalid
authors.initials: K
authors.lastname: Sendide
authors.forename: Amina
authors.initials: A
authors.lastname: Talal
authors.forename: Rosa
authors.initials: R
authors.lastname: Garcia
authors.forename: Karen
authors.initials: K
authors.lastname: Dobos
authors.forename: Neil E
authors.initials: NE
authors.lastname: Reiner
mesh_terms: D015703:Antigens, CD / Q000378:metabolism; D015153:Blotting, Western; D002117:Calcitriol / Q000378:metabolism*; D002454:Cell Differentiation; D002462:Cell Membrane / Q000378:metabolism; D002473:Cell Wall / Q000378:metabolism; D005434:Flow Cytometry; D006017:Glycolipids / Q000737:chemistry; D006801:Humans; D008070:Lipopolysaccharides / Q000737:chemistry*; D051907:Lysosomal Membrane Proteins; D008247:Lysosomes / Q000737:chemistry* / Q000378:metabolism; D008264:Macrophages / Q000378:metabolism; D008856:Microscopy, Fluorescence; D009161:Mycobacterium / Q000378:metabolism*; D020102:Mycobacterium smegmatis / Q000378:metabolism; D009169:Mycobacterium tuberculosis / Q000378:metabolism; D010587:Phagocytosis; D010588:Phagosomes / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D020691:rab GTP-Binding Proteins / Q000378:metabolism; D000090802:rab7 GTP-Binding Proteins
chemical_list: D015703:Antigens, CD; D002117:Calcitriol; D006017:Glycolipids; D008070:Lipopolysaccharides; D051907:Lysosomal Membrane Proteins; D019869:Phosphatidylinositol 3-Kinases; D020691:rab GTP-Binding Proteins; D000090802:rab7 GTP-Binding Proteins; C050016:lipoarabinomannan; C000716463:rab7 GTP-binding proteins, human