Article Figures & Data
Figures
- FIG. 1.
Internalization of conidia by HAMs, as revealed by transmission electron microscopy (A to D), and MH-S cells, as revealed by scanning electron microscopy (E to G). Panels A to C show the early stages of engulfment after 5 min of contact between conidia and HAMs. Note the numerous lysosomes (LY) widely distributed throughout the cell. PV, pinocytic vesicles; M, mitochondria; N, nuclei. Also, note the double-layered cell wall (arrows) with the electron-dense pigmented outer layer of the conidium in panels A and B, the contact points (arrowheads) between conidia and AM pseudopodia in panels B and C, and the progressive engulfment of the conidium by AM pseudopodia. Panel D shows the late stage of engulfment after 90 min. Panels E to G show conidia bound to a macrophage analyzed 5 min after the beginning of ingestion. Note the extensions of pseudopodia towards the conidium and the contact of these pseudopodia with the conidial cell wall in panels F and G. Bars, 2 μm (A) and 1 μm (E to G).
- FIG. 2.
F-actin staining, with Texas Red-phalloidin, of MH-S cells (a, c, and e) and HAMs (b, d, and f) during the engulfment of conidia. Panels a and b show control cells. Note the phagocytic cup in panel c and the actin ring surrounding an ingested conidium in panel d (arrows). Panels e and f contain differential interference contrast (DIC) images of what is shown in panels c and d, respectively. Bar, 2 μm. (g) Kinetics of actin involvement in conidia phagocytosis. The percentage of actin-positive conidia was calculated as follows: (the number of conidia surrounded by an actin ring or cup stained with Texas Red-phalloidin/the total number of conidia associated to macrophages × 100. Data are averages from three independent experiments, and standard deviations are indicated with error bars. ♦, HAMs; ▪, MH-S cells. Infection times are in minutes.
- FIG. 3.
Effect of cytochalasin D and wortmannin treatment on MH-S cells. MH-S cells were treated for 30 min with 2 μM cytochalasin D (A to C) or 1 μM wortmannin (D to F), and ingestion of conidia was performed in the presence of the drug. Shown are scanning electron micrographs of cells at 5 (A) and 30 (D) min postingestion. Arrowheads in panel A indicate blebs (Bl) and balloons (Ba), which are probably nuclei on the top of treated cells. The arrows in panels A and D indicate uningested conidia. Also shown are cells at 60 min postingestion (B, C, E, and F). Cells were fixed, treated with anti-conidia antibody, stained with an FITC-conjugated antibody to label uningested conidia (C and F), permeabilized, and incubated with Texas Red-phalloidin to label actin (B and E). Note the brightly stained aggregates of actin and the lack of F-actin rich filopodia in panels B and E; nonphagocytosed conidia (arrows) can be seen on the surface of MH-S cells in panels C and F. Bars, 1 μm.
- FIG. 4.
Maturation of A. fumigatus-containing phagosomes in HAMs. HAMs were incubated with conidia (2:1 ratio of conidia to macrophages) and were fixed at 5, 10, 30, and 60 min after the beginning of phagocytosis. After permeabilization with saponin, cells were labeled at 10 min (a to c and e to g) or 60 min (d and h to p) postingestion with the specific monoclonal or polyclonal anti-TfR (1/1,000), anti-EEA1 (1/500), anti-Rab 7 (1/50), and anti-Lamp1 (1/100) antibodies and secondary antibodies conjugated to Texas Red. (a to c) Arrowheads indicate TfR-positive (a), EEA1-positive (b), and Rab 7-positive (c) staining surrounding the conidia. (d) Cells were labeled at 60 min postingestion, and the arrowhead indicates positive staining for Lamp1. (e to h) Corresponding DIC images. The arrowheads indicate conidia. Note that the conidia outside the cells (thin arrows in panels c, g, j, and n) were not labeled by the same antibodies. (i to k) Lack of labeling with the anti-TfR, anti-EEA1, and anti-Rab7 antibodies. (l) Cells were labeled with control IgG1 MAb. (m to p) Corresponding DIC images. Bar, 2 μm.
- FIG. 5.
Kinetics of maturation of A. fumigatus-containing phagosomes in AMs. Shown is the quantification of the immunolabeling of conidia ingested by HAMs (A) and MH-S cells (B) with antibodies directed against various endocytic markers as described in the legend to Fig. 4. The percentages shown are the number of phagosomes labeled with the antibody divided by the total number of phagosomes containing conidia. Data are means from three independent experiments, and standard deviations are indicated with error bars. Infection times are in minutes. •, percentage of ingestion; ✖, percentage of Lamp1-positive phagosomes; ♦, percentage of TrfR-positive phagosomes; ▴, percentage of Rab7-positive phagosomes; ▪, percentage of EEA1-positive phagosomes.
- FIG. 6.
Acidification of conidia-containing phagolysosomes. HAMs were incubated with the acidotropic probe LysoTracker Red DND-99 for 2 h, washed with RPMI complete medium, and incubated with conidia in RPMI complete medium containing LysoTracker. Control experiments were done in the presence of 250 nM bafilomycin A. (A) Negative control prior to conidial challenge. (B and C) At 4 h postinfection, the acidic pH in the phagolysosome was revealed by bright red fluorescence (arrows) surrounding the conidia (B) or impregnating the conidia (C). (D) Shown is a cell (arrow) with phagocytosed conidia incubated in the presence of bafilomycin A that is negative with LysoTracker. (E to H) Corresponding phase-contrast (E) and DIC (F to H) images. Bar, 2 μm.
- FIG. 7.
Estimation of the capacity of MH-S cells to kill A. fumigatus conidia. (A) Killing of conidia (estimated as the percentage of nongerminating conidia) (solid column) and phagosomal acidification (estimated as the percentage of A. fumigatus-containing phagosomes positive for LysoTracker labeling) (open columns) at 4 and 18 h following ingestion. Values represent means plus standard deviations from three experiments. (B) Effect of bafilomycin A on the capacity of MH-S cells to kill A. fumigatus conidia. Untreated control cells (open column) and cells treated with 250 nM bafilomycin A (solid column) were allowed to ingest conidia, and the percentages of killing following 18 h of ingestion are shown.
- FIG. 8.
Kinetics of cathepsin D acquisition by the conidia-containing phagosome. Phagocytosis of A. fumigatus by HAMs was initiated at 37°C after 30 min of incubation at 4°C with conidia at a 2:1 ratio of conidia to macrophages. (A) After different incubation times (5 to 60 min), cells were fixed, permeabilized, and stained with a pAb specific for cathepsin D and a secondary antibody conjugated with Texas Red (upper panels). Arrowheads indicate positive staining surrounding the conidia. Corresponding DIC images are also shown (lower panels). For the control, purified rabbit IgG was used instead of the anti-cathepsin D pAb. Bar, 2 μm. (B) Colocalization of Lamp1 and cathepsin D (arrowheads) at 30 and 60 min postinfection. Cells were labeled with the anti-Lamp1 MAb followed by the anti-cathepsin D pAb and successive labeling with FITC- and Texas Red-conjugated corresponding secondary antibodies. Arrowheads indicate staining surrounding conidia.
- FIG. 9.
Model of phagocytosis of A. fumigatus by AMs. V-ATPase, vacuolar ATPase.
