Figure 1.
Morphological features of giant cells from infected mice.
A) Cells grown in vitro in Sabouraud medium. B) Cell obtained from the lungs of a mouse infected with C. neoformans (105 cells/mouse) 5 weeks earlier. C) Photomicrographs of a giant cell illustrating the presence of multiple vesicles (highlighted with arrows) within giant cells. Scale bar, 10 µm. D, E and F, vacuole staining with MDY-64 dye. Yeast cells isolated from the lung of mice four weeks after infection with 105 yeast cells were stained with the specific vacuole marker MDY-64 as described in Materials and Methods. Localization of the signal was performed by confocal microscopy. D) Cell of regular size. E and F) Giant cells. In all the cases, regular light microscopy, fluorescence and the merge of both images are shown. Scale bars in the left panels applies to the corresponding middle and right panels. G, H and I, transmission electron microscopy of the capsule and cell wall of regular and giant cells. Cells grown in vitro or giant cells obtained from the lungs of mice three weeks after infection were fixed and processed for TEM. G, cell grown in vitro; H, giant cell; I, magnification of the cell wall region of the giant cell shown in B. CP, capsule; CW, cell wall; CY, cytoplasm. The rectangle indicates the width of the cell wall. J,K, staining of giant cells with anti-melanin mAb. J, light microscopy; K, fluorescence. Scale bar, 10 µm.
Figure 2.
Scanning electron microscopy of cells grown in vitro and of giant cells.
A) Cell in vitro grown in Sabouraud. B-F) Giant cells isolated from lung. Scale bar in panel B (10 µm) also applies to panels C-F.
Figure 3.
Capsule release by γ-radiation.
Cells of in vitro-enlarged capsule (A) and giant cells (B) were exposed to γ-irradiation, suspended in India ink, and observed under the microscope. Images of representative cells are shown before (control, left panel) and after irradiation (γ-irradiation, right panel).
Figure 4.
Differences in capsular structure between regular and giant cells.
Yeast cells from lung extracts were incubated in mouse serum and labelled with antibodies to C3 and GXM. A) cell of a regular size; B) giant cell. Light microscopy and complement localization (green fluorescence) and capsule edge (red fluorescence) are shown. C–H) capsular features shown by fluorescence. C–F) Indirect immunofluorescence with GXM-binding mAbs. MAb 18B7 labelling of cells of regular size (C) or giant cells (D–F) isolated from lung of infected mice. Light microscopy and fluorescence pictures are shown for each cell. G–H) Binding of wheat germ agglutinin to C. neoformans. The presence of chitin-like structures in the capsule was studied by the binding of WGA to the cells as described in Materials and Methods. G) Cells grown in Sabouraud and then transferred to 10% Saboraud buffered at pH 7.3 with 50 mM MOPS buffer to induce capsule enlargement. H) A representative giant cell isolated from the lungs of infected mice. Arrows indicate the major regions where WGA binds to the cells. Scale bars in C–D in light microscopy panels denote 5 microns and apply to the fluorescence images.
Figure 5.
Time lapse images of replicating giant cells in vitro.
Giant cells were obtained from lungs of four weeks-infected mice and placed on Sabouraud plates. After 18 h, the growing colonies were monitored under the microscope. The digital videos were processed with Windows Movie Maker software. This software allowed the conversion of the videos into single pictures, each one represented as a different frame of the video. The number in each picture corresponds to the time in seconds. A, B and C correspond to three different budding events. Corresponding videos are included as supporting information files.
Figure 6.
Giant cell formation in vitro.
Cells from H99 strain were grown in Sabouraud, washed and labelled with mouse serum. C3 deposition was then detected by immunofluorescence (A). After incubation in mouse serum, cryptococcal cells were transferred to minimal medium (MM) for four days, and C3 was detected (B). Scale bar in A and B, 10 µm, and apply to the corresponding fluorescence panels. C) Cells were grown and labelled as in A (Day 0 sample), transferred to Sabouraud or MM for 4 days at 37°C, and the cell size was measured in the C3 labelled population (“MM, C3 labelled” and “Sab, C3 labelled”) and in the whole population, including both C3 labelled and unlabelled cells. The average and the standard deviation (error bars) are plotted and p-values for the highlighted comparisons are shown. At least, 20–50 cells were counted, except in the “Sab, C3 labelled” samples, where C3 positive cells were rarely identified due to the overgrowth of the culture. Kruskall-Wallis test was used to assess statistical differences. D) Scatter representation showing all the cells plotted in panel B. The line in each sample denotes the average of the distribution.
Figure 7.
Determination of DNA content by cytometry and DAPI staining.
Fungal cell samples were obtained from the lungs of one mouse 3 weeks after challenge with 105 C. neoformans cells. As a control, cells grown for 24 hours in Sabouraud were also used. The cells were fixed after incubation at 60°C for 45 minutes. Propidium iodide was immediately added at 10 µg/mL DNA content and the labelling was determined by the propidium iodide fluorescence intensity. Matched samples were subjected to cytometry without propidium iodide. A) FSC/SSC plot of cells grown in Sabouraud. B) FSC/SSC of cryptococcal cells from lungs. Region 1 delimits all the cells whose size and complexity are not found in cultures grown in vitro and presumably cover all the cells with increased cell size. C) Propidium iodide fluorescence intensity of the four samples analyzed: in vivo isolated cells (plus and minus propidium iodide) and in vitro cells (plus and minus propidium iodide). D) Propidium iodide fluorescence intensity of the cells from the lung present in R1. E–F) DAPI staining. Yeast cells obtained from the lungs of mice infected with 105 yeast cells were stained with DAPI. E) cell of regular size; F) giant cell. Corresponding light microscopy and fluorescence images are shown. Scale bar in the light microscopy panels apply to the fluorescence images.
Figure 8.
Giant cell formation depends on cAMP, but not Ras1.
C57BL/6J mice were infected with H99, ras1 (Ras1 mutant) and cac1 (adenylate cyclase mutant) strains (106 per mouse). After three weeks, the mice were sacrificed and fungal cells were isolated. Representative pictures of fungal cells are shown. A) India ink microscopy, scale bar in left panel applies to all the pictures. B) Cell size distribution of wild type, cac1 mutant and its reconstituted strain (cac1/CAC1) after 5 days of growth in minimal medium at 37°C. The cells were suspended in India Ink to delimit the capsule, and the diameter of the cells (capsule included) was measured microscopically. C) Forward Scatter/Side Scatter plot of cac1 mutants after incubation in minimal medium. The cells described in B were analysed by flow cytometry to obtain the corresponding forward scatter (FSC, correlated with cell size) and side scatter (SSC, correlated with cell complexity).
Figure 9.
Parameters of disease in mice infected with C. neoformans and giant cell proportion in the lungs.
CD1 mice were infected with different inocula of C. neoformans, and different parameters were monitored during the course of the infection. A) Body weight of mice infected with a low dose (∼104 cells, black line, closed symbols, n = 3) or a high dose (106, grey line, grey symbols, n = 3) of C. neoformans. A control mouse, injected with PBS, is also shown (grey line, open symbols). B) Proportion of the lung weight in respect to body weight, as an indicator of lung inflammation. C) CFUs from the lungs of mice described in A and B. D) Cell size of C. neoformans in vitro and in vivo. Cell size (capsule included) was measured in fungal samples obtained from mice infected with high or low C. neoformans doses (see Figure 9A-C). As a control, cell size was measured in cells grown in vitro in Sabouraud or in 10% Sabouraud buffered at pH 7 with 50 mM MOPS to induce capsule enlargement. The number expresses the proportion of giant cells in each sample, defined as cell with a diameter above 30 µm. The line in each distribution represents the average of the population. E) Results of the CART analysis. Left panel, prediction of the correlation between inflammation and total fungal cell size. A cell size lower than 35.92 µm was strongly associated with increased inflammation. Right panel, corresponding ROC curve, showing a region under the curve of 0.8.
Figure 10.
Histological sections of mice infected with different C. neoformans inocula.
Tissue sections from mice described in Figure 9 were stained with hematoxylin and eosin. Tissue section of control mouse (upper left panel) and three different mice infected with a low C. neoformans inocula. Mouse 1, upper right panel; mouse 2, middle and lower left panels; mouse 3, middle and lower right panels (for mice 2 and 3, two different regions are shown). For each panel, a magnification of a region is shown, which is delimited by an inset. Scale bar in upper left magnification, 25 µm, and applies to the rest of amplified panels.
Table 1.
Fungal cell size and proportion of giant cells in 16-weeks old mice infected with low and high doses of H99 strain.