Figures & data
Figure 1. Effect of C. neoformans on the antimicrobial activity of the G. mellonella hemolymph. (A) Groups of 10 G. mellonella were infected with C. neoformans H99 strain (106 cells/G. mellonella) or with PBS (C), and then they were incubated at 37°C. The lytic activity of the hemolymph was evaluated as described in Material and Methods after different timepoints. Asterisks denote statistical difference between the sample and the corresponding control G. mellonella larvae treated with PBS. (B) G. mellonella larvae were infected with C. neoformans H99 or C. albicans SC5314 strain (105 cells per G. mellonella) and incubated at 37°C overnight. Then, the lytic of the hemolymph was evaluated. Asterisks denote statistical difference between the sample and the control G. mellonella treated with PBS.
Figure 2. Role of the capsule on the accumulation of antimicrobial peptides in the hemolymph. (A) Galleria mellonella larvae were infected with approximately 105 cells from B3501 or cap59 strains as described in Material and Methods, and placed at 37°C for 24 h. Lytic activity of the hemolymph was assessed as described in M&M. Asterisks denote statistical difference. (B) Groups of 10 G. mellonella were infected with 106 H99 cells prepared in water or with different amounts of purified GXM. In parallel, G. mellonella were injected with sterile water. After 24 h of incubation at 37°C, the antimicrobial activity of the hemolymph was determined. Asterisks indicate statistical difference between the sample and the control treated with water. (C) Cells from H99 strain with different capsule size were obtained after growth in Sabouraud (Control, small capsule) or 10% Sabouraud, pH 7.3 (Induced, large capsule). Then, G. mellonella were infected with 105 cells from these cultures or with PBS, and lytic activity in the hemolymph was determined after 24 h of incubation at 37°C. Asterisks denote statistical difference between the sample and the larvae treated with PBS.
Figure 3. Behavior of C. neoformans inside hemocytes. Galleria mellonella were infected with 106 cryptococcal cells (H99 strain) and after 3 h of incubation at 37°C, the hemolymph was collected and hemocytes placed in a 96-well plate under the microscope. (A) Intracellular replication event; (B) Non lytic exocytosis of the cryptococcal cells from the hemocytes. In each case, the time difference between the pictures of the panels is 9 min.
Figure 4. Early melanization of G. mellonella after infection with C. neoformans and C. albicans. Groups of 10 G. mellonella larvae were injected with PBS (A), C. albicans SC5314 strain (106 cells/G. mellonella) or C. neoformans H99 (106 cells/G. mellonella). Quantification of hemolymph melanization as described in M&M. Asterisks denote statistical difference between the sample and the control G. mellonella treated with PBS.
Figure 5. Structural features of the C. neoformans capsule in vitro and after passage through G. mellonella. Cells from H99 strain were obtained in vitro with different capsule sizes or from G. mellonella. (A) Cells with small capsule size after growth in vitro, (B) cells with enlarged capsule after incubation in 10% Sabouraud pH 7.3, (C) cells isolated from G. mellonella. These cells were incubated with different mAbs to the capsule (18B7, 2H1 and 2D10). mAb 18B7 was directly conjugated to Alexa488, and for the other 2, specific GAM-IgG (for 2H1) or GAM-IgM (for 2D10) were used. In each case, a representative cell is shown, including both light microscopy (left panel) and fluorescence (right panel). Scale bar in upper left panel applies to the rest of the pictures. (D) Quantification of the fluorescence of the cells shown in A, B and C by flow cytometry. Black histograms correspond to cells to which no primary anticryptococcal mAb has been added (negative controls), and green histograms indicate the fluorescence intensity of the cells to which both anti-cryptococcal and secondary Abs have been added. In all cases, straight line corresponds to cells grown in Sabouraud (Sab, small capsule in vitro), dotted line to cells incubated in 10% Sabouraud pH 7.3 (MOPS, large capsule in vitro), and filled histogram to yeast cells isolated from G. mellonella (Gal).
