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First published online April 16, 2004
doi: 10.1242/10.1242/jcs.01072

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Quantitative analysis of phagolysosome fusion in intact cells: inhibition by mycobacterial lipoarabinomannan and rescue by an 1,25-dihydroxyvitamin D3–phosphoinositide 3-kinase pathway

¹ 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
² Laboratoire d'Immunologie, Faculté des Sciences Dhar Mahraz, Université Mohamed Ben Abdallah. BP 1796, Atlas, Fès, 30000, Morocco
³ Department of Microbiology, Colorado State University, Fort Collins, CO 80523, USA

View larger version (36K): [in a new window]   Fig. 1. Flow cytometry analysis of latex bead phagosome. (A) PKH-labeled THP-1 cells adherent to coverslips and ingesting latex beads (4 µm) were fixed and mounted on microscopy slides. Samples were examined at the optimal wavelengths for PKH26 (551 excitation, 567 nm emission) using a PL-Apochromat 63x/1.4 oil immersion objective on a Zeiss Axioplan II microscope. Latex beads are surrounded by a red fluorescent membrane. Bar, 5 µm. (B) In (a), equal volumes of 1, 2 and 4 µm latex beads were mixed and analyzed by flow cytometry. Beads of different sizes were localized by plotting forward (FS) versus side (SS) scatter channels on a linear scale. (b) Postnuclear supernatant from THP-1 homogenate mixed with 4 µm beads and analyzed by flow cytometry using similar SS and FS parameter settings as in (a). Large latex beads (R1 window) were readily discriminated from cell debris. (d) Red fluorescence (FL2) of partially purified phagosomes from PKH labeled cells that have ingested 4 µm latex beads. Events were recorded from the R1 window, which is the predicted position of phagosomes based on the localization of free beads in (b). (c) Fluorescence of latex beads alone (background autofluorescence), allowing for positioning of window R2 in (d) to include signals from true phagosomes only. A comparison of (c) and (d) shows that a significant fraction of latex beads were released from phagosomes during homogenization and centrifugation. (C) Phagosomes were sorted using a Becton Dickinson FACStar plus flow cytometer on the basis of positive red fluorescence (gate R2 shown in Bd); 5 million sorted early (30 minutes) and late (2 hours) phagosomes were pelleted and phagosome membrane proteins were solubilized in 1xLaemmli buffer. Samples were separated by SDS-PAGE along with 100 µg of total cell lysate (WCL). Proteins were transferred to nitrocellulose membranes and probed with antibodies against pleckstrin, Rab5 and Rab7.

View larger version (55K): [in a new window]   Fig. 2. Flow cytometry analysis allows the quantitative evaluation of phagosome maturation. PKH-stained cells were allowed to ingest latex particles, and phagosomes were prepared after 30 minutes, 1 hour and 2 hours. Phagosome preparations were fixed/permeabilized and stained with irrelevant antibodies or specific antibodies to LAMP-1 (A) or Rab7 (B). Preparations were washed and stained with FITC-labeled secondary antibodies. After adequate green/red compensations, green fluorescence was analyzed on positive red fluorescent events, which correspond to particles surrounded by a red phagosomal membrane (gate R3), and the results were expressed as green fluorescence histograms. In each panel, the histogram on the left represents phagosomes stained with irrelevant antibodies, and the histogram displaced to the right represents phagosomes stained with specific antibodies. The proportion of phagosomes expressing LAMP-1 (A) and Rab7 (B) increases as a function of maturation. Values represent the averages of two independent experiments.

View larger version (31K): [in a new window]   Fig. 3. Quantitative analysis of phagosome-lysosome fusion by flow cytometry. THP-1 cells were differentiated with PMA in the presence of 1 mg/ml F-DXT then washed and chased for 4-5 hours. F-DXT-loaded cells were stained with PKH and allowed to ingest latex beads for the indicated times. (A) Adherent cells on coverslips were then examined by fluorescence microscopy at 2 hours post phagocytosis, as described in Fig. 1A. Color images of merged green (F-DXT) and red (PKH) fluorescence signals show colocalization (yellow), which indicates substantial fusion between phagosomes containing latex beads and F-DXT-loaded lysosomes. The inset shows isolated phagolysosomes in post-nuclear cell homogenates, which were examined by flow cytometry in B. Bar, 5 µm. (B) Phagosomes were prepared 30 minutes, 1 hour and 2 hours after phagocytosis and analyzed by flow cytometry. Green fluorescence was analyzed on positive red fluorescent events, which corresponded to particles surrounded by a phagosomal membrane (gate R3), and the results were expressed as green fluorescence histograms. In each panel, histograms on the left represent PKH-labeled phagosomes isolated from cells without F-DXT loading, and histograms on the right represent phagosomes from cells loaded with F-DXT. The frequencies of phagosomes colocalizing with F-DXT and MFIs increased as a function of time. Values represent the averages of two independent

View larger version (37K): [in a new window]   Fig. 4. Mycobacterial lysate inhibits phagosome maturation and fusion with lysosomes. (A,B) PKH-labeled cells were exposed to latex beads in the presence of 100 µg/ml of BSA (control) or a soluble fraction derived from whole-cell lysates of either M. tuberculosis (Mtb) or M. smegmatis (M.smeg). Phagosomes were isolated at 1 hour (A) and 2 hours (B) post phagocytosis, permeabilized and incubated with antibodies to Rab7 and LAMP-1, respectively, and samples were analyzed by flow cytometry as described in Fig. 2. (C) F-DXT-loaded THP-1 cells were stained with PKH and allowed to ingest latex beads in the presence of BSA, or a soluble fraction derived from whole-cell lysates of either M. tuberculosis or M. smegmatis. Phagosomes were prepared at 2 hours post phagocytosis and analyzed by FACS as described in Fig. 3. Values represent the averages of two independent experiments.

View larger version (19K): [in a new window]   Fig. 5. Mannose capped mycobacterial glycolipid inhibits lysosomal delivery to phagosomes. F-DXT-loaded THP-1 cells were stained with PKH and allowed to ingest latex beads in the presence of BSA (control) or various concentrations of soluble cell wall fraction (CW), LAM or PIM. Phagosomes were prepared at 2 hours post phagocytosis and analyzed by flow cytometry as described in Fig. 3. The data shown are means±s.d. of three separate experiments. The value on the top of each bar represent the proportion of phagosomes fusing with lysosomes.

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