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Fig. 4. Intracellular trafficking of CIP50/LpdC and its role in mycobacterial survival. (A) J774 cells were infected with gentamycin killed (GK-), live (L-) BCG, M. smegmatis (Msmeg) or M. smegmatis expressing Mtb LpdC (rMsmeg). At 4 hours post-phagocytosis, cells were fixed, permeabilized and stained with N-7 mAb (anti-coronin-1) and Texas Red-conjugated goat anti-mouse IgG. Cells were also stained with anti-LpdC or normal rabbit serum (L-BCG/Irr) and Alexa Fluor 350-conjugated goat anti-rabbit IgG. The enlarged merge panels (L-BCG and rMsmeg), show colocalization (pink color) of coronin-1 (red) with LpdC (blue) in the phagosomal membrane and secreted LpdC (blue) surrounding the phagosome. In the right panel, the yellow signal indicates colocalization of L-BCG and coronin-1 in cells stained with normal rabbit IgG instead of anti-LpdC. Bar, 10 µm. (B) Quantification of LpdC-coronin-1 colocalization in phagosomes observed in 20-25 cells from two individual experiments. (C) Thin sections of BCG-infected cells were fixed with paraformaldehyde and glutaraldehyde and incubated sequentially with rabbit anti-LpdC IgG and gold-conjugated goat anti-rabbit IgG to visualize LpdC (15 nm gold). EM grids were examined with a Tecnai 12 electron microscope at magnification of 37000x (a), 59000x (b) and 97000x (c). The arrows indicate gold particles localized on the phagosomal membrane and the arrowheads indicate gold particles localized in the cytosol. (D) PKH26-stained cells (10⁷/culture plate) were allowed to ingest mycobacteria and phagosomes were prepared at 4 hours post-phagocytosis. Phagosome preparations were stained with normal rabbit IgG or specific antibodies to coronin-1 or LpdC. Preparations were washed and stained with Alexa Fluor 647-labeled secondary antibodies. After adequate compensations, blue fluorescence was analyzed on double (red and green fluorescent)-positive events, which are green fluorescent bacteria surrounded by a red phagosomal membrane (supplementary material Fig. S3); results are expressed as histograms. In each panel, the left histogram represents phagosomes stained with specific antibodies, and the histogram displaced to the right (red) represents phagosomes stained with normal IgG. Values represent the proportion of phagosomes expressing coronin-1 or LpdC.

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