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Quantitative Analysis Of Phagolysosome Fusion In Intact Cells: Inhibition By Mycobacterial Lipoarabinomannan And Rescue By An 1α,25-dihydroxyvitamin D3–phosphoinositide 3-kinase Pathway

Zakaria Hmama, Khalid Sendide, Amina Talal, Rosa Garcia, Karen Dobos, Neil E. Reiner

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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.