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Biogenesis of Leishmania-harbouring parasitophorous vacuoles following phagocytosis of the metacyclic promastigote or amastigote stages of the parasites

¹ Unité d'Immunophysiologie et Parasitisme Intracellulaire, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15, France
² INSERM U411, UFR de Médecine Necker-Enfants Malades, Paris, France
³ Laboratoire de Cinémicrographie INSERM, Le Vésinet, France
⁴ Unité de Biologie des Interactions Cellulaires, Institut Pasteur, Paris, France

View larger version (91K): [in a new window]   Fig. 1. Mechanisms of entry of L. amazonensis metacyclic promastigotes into macrophages. (A,B) Microcinematography of phagocytic events. Promastigote cell bodies and flagella are indicated by white arrowheads and white arrows, respectively. (A) Parasite attachment occurs by the cell body. The promastigote is then progressively internalized through the formation of a long tubular pseudopod (black arrowheads). (B) The parasite binds to the macrophage via the tip of the flagellum. It then turns around, so that its cell body comes into contact with the cell surface of the macrophage. Ruffles are formed at this site (black arrowheads). The promastigote is finally ingested via the cell body. The numbers indicated in the right hand corners correspond to the time (in seconds) elapsed from the first contact of promastigotes with macrophages (time 0). QuickTime movie sequence versions of A and B are available online (http://jcs.biologists.org/supplemental ). (C,D) Scanning electron micrographs showing the internalization of promastigotes via the formation of a closely apposed pseudopod (C) or of ruffles (D). Macrophages were fixed after 10 minutes of contact with the parasites. Bars, 10 µm (A,B), 1 µm (C,D).

View larger version (57K): [in a new window]   Fig. 2. F-actin accumulation around parasites during or following phagocytosis. (A) Macrophages untreated () or treated with IFN- (, [UNK]) were infected with L. amazonensis metacyclic promastigotes (, ) or amastigotes ([UNK]) and then fixed and permeabilized at the different time points indicated. F-actin was stained with Alexa Fluor 488 phalloidin (green staining) and parasites with a mouse anti-Leishmania immune serum and a Texas Red conjugate (red staining) before examination of the cells by fluorescence microscopy. The percentages of promastigotes or amastigotes surrounded by F-actin were determined after counting about 100 parasites for each time point. Data are the means±s.d. of three experiments () or are from a single experiment (,[UNK]). (B,C) Fluorescence confocal microscopy of macrophages incubated for 10 minutes with L. amazonensis metacyclic promastigotes (B) or amastigotes (C). F-actin and parasites were stained as in A. A 3D reconstruction and an optical section (0.5 µm thickness) are shown in B and C, respectively. Bar, 2 µm.

View larger version (23K): [in a new window]   Fig. 3. Promastigote orientation shortly after internalization. Macrophages untreated (white bars) or treated with IFN- (hatched and black bars) were infected with L. amazonensis metacyclic promastigotes. They were fixed 30 minutes (white and hatched bars) or 60 minutes (black bars) after the addition of the parasites and permeabilized before staining with an anti-leishmanolysin or anti-Leishmania immune serum and adequate fluorochrome conjugates. Cell preparations were then examined by fluorescence microscopy. Three groups of intracellular parasites were distinguished according to their orientation as shown in the schemes below the histograms. Results are expressed as the means+s.d. of two (white bars) or three experiments (hatched bars) or are from a single experiment (black bars). Percentages were determined after counting 703, 2200 and 712 parasites, respectively.

View larger version (103K): [in a new window]   Fig. 4. Light microscopy analysis of PV formation. Macrophages treated with IFN- were infected with L. amazonensis metacyclic promastigotes (A-D) or amastigotes (E-H). At the time points indicated, macrophages were fixed and then examined by phase-contrast microscopy. In each panel, some parasites are indicated by white arrowheads. Bar, 10 µm.

View larger version (21K): [in a new window]   Fig. 5. Kinetics of endocytic `marker' association with phagosomes containing initially L. amazonensis metacyclic promastigotes. Macrophages pre-exposed (A,B) or not exposed (C) to IFN- were infected at a multiplicity of four parasites/host cell. Association of the following molecules with parasite-containing phagosomes was examined as a function of time: transferrin receptor, EEA1, rab7p, macrosialin, lamp-1, cathepsin B, cathepsin D and MHC class II molecules. At the time points indicated, cell preparations were fixed, permeabilized and incubated with immunological reagents before analysis by fluorescence microscopy. For each experiment and at each time point, the percentages of parasite-containing compartments displaying the molecules listed above were determined after counting about 100 to 200 organelles. Each value represents the mean+s.d. of two to eight experiments (A,B) or is from a single experiment representative of two separate experiments (C).

View larger version (95K): [in a new window]   Fig. 6. Immunofluorescence labeling of late endosome/lysosome `markers' associated with parasite-containing organelles at different time points after infection of IFN--treated macrophages with L. amazonensis metacyclic promastigotes. Macrophages were processed for fluorescence microscopy 10 minutes (A), 30 minutes (B-D), 2 hours (E) or 18 hours (F) after the addition of the parasites. Cell preparations were incubated with immune sera or Abs directed against rab7p (A), macrosialin (B), lamp-1 (C,E), cathepsin B (D,F) and then with adequate fluorescein conjugates (green staining). Except in B, macrophage nuclei and parasite nuclei and kinetoplasts were stained with propidium iodide (red staining). In B, the parasites are indicated by arrows. Sections (0.3-0.5 µm thickness) obtained by confocal microscopy are shown. The micrographs are representative of three to eight experiments. Bars, 2 µm.

View larger version (22K): [in a new window]   Fig. 7. PV biogenesis in IFN--treated macrophages infected with either L. amazonensis stationary phase or metacyclic promastigotes. Macrophages were processed as described in the legend to Fig. 5 to determine the association of the transferrin receptor, rab7p, lamp-1 and cathepsin B with phagosomes as a function of time. Results obtained after phagocytosis of stationary phase and metacyclic promastigotes are represented by broken and solid lines, respectively.

View larger version (22K): [in a new window]   Fig. 8. PV biogenesis in IFN--treated macrophages infected with L. major metacyclic promastigotes. Macrophages were processed as described in Fig. 5 to determine the association of the different endosome/lysosome `markers' with phagosomes as a function of time. Results are the means±range of two experiments.

View larger version (48K): [in a new window]   Fig. 9. (A) Kinetics of delivery of late endosome/lysosome-associated FDex into parasite-containing organelles formed after phagocytosis of metacyclic promastigotes. IFN--treated macrophages were incubated with FDex for 2 hours. After washings, they were chased either for 160 minutes or overnight before infection with L. amazonensis (LV79) or L. major (NIH173) metacyclic promastigotes. Macrophages were fixed and permeabilized at various times post-infection and the parasites they contained were counted after staining with either a mouse anti-Leishmania immune serum and a Texas Red conjugate or propidium iodide. For each experiment and at each time point, the percentage of FDex-positive, parasite-containing compartments was determined after counting about 100 organelles. Results are from a single experiment (LV79) or are the means + or - range of two experiments (NIH173). (B-E) Confocal microscopy of macrophages loaded with FDex, chased overnight in FDex-free medium and then infected with L. amazonensis metacyclics. Analysis was done 30 minutes (B,C) or 18 hours (D,E) after adding parasites. (B,D) and (C,E) are the differential interference contrast (DIC) and the fluorescence images of the same cells, respectively. Optical sections (0.3-0.5 µm thickness) are shown. The position of the parasites is indicated by arrows. Bar, 2 µm.

View larger version (153K): [in a new window]   Fig. 10. Fusion of HRP-loaded late endosomes/lysosomes with parasite-containing compartments as observed by electron microscopy. IFN--treated macrophages were incubated with HRP for 30 minutes. After extensive washings, they were chased for 160 minutes before infection with L. amazonensis metacyclic promastigotes. Thirty minutes after infection, macrophages were fixed and processed for peroxidase cytochemistry. (A) HRP is located in numerous late endosomes/lysosomes (arrows) as well as in the lumen of the tight promastigote-harbouring compartment (arrowheads). (B,C) Details of A showing the fusion of HRP-containing late endosomes/lysosomes with the phagolysosomal compartment (arrows). The presence of HRP in the flagellar pocket of the parasite is also observed (arrowheads). Bars, 1 µm (A), 0.2 µm (B,C).

View larger version (22K): [in a new window]   Fig. 11. Kinetics of endocytic `marker' association with phagosomes formed after internalization of L. amazonensis amastigotes. IFN--treated macrophages were infected at a multiplicity of four parasites/host cell. At various times post-infection, they were processed as described in Fig. 5 and the association of the following molecules with parasite-containing compartments was examined: transferrin receptor, EEA1, rab7p, macrosialin, lamp-1, cathepsin B and MHC class II molecules. For each experiment and at each time point, the percentages of parasite-containing organelles displaying the molecules listed above were determined after counting about 100 organelles. Data represent the means + or - range of two separate experiments.

View larger version (40K): [in a new window]   Fig. 12. Confocal microscopy analysis of the association of late endosome/lysosome `markers' with early phagosomes formed after internalization of L. amazonensis amastigotes. IFN--pre-treated macrophages were infected (four parasites/host cell) and 30 minutes later processed for immunofluorescence microscopy. Cell preparations were incubated with immune sera or Abs directed against rab7p (A), lamp-1 (B) or cathepsin B (C) and then with adequate fluorescein conjugates (green staining). In B and C, cells were also stained with propidium iodide to visualize macrophage nuclei and parasite nuclei and kinetoplasts (red staining). Optical sections (0.3-0.5 µm thickness) are shown. The micrographs are representative of two separate experiments. Bars, 2 µm.