Cryptococcus neoformans enters the endolysosomal pathway of dendritic cells and is killed by lysosomal components

Abstract

Cryptococcus neoformans is an opportunistic fungal pathogen that primarily causes disease in immunocompromised individuals. Dendritic cells (DCs) can phagocytose C. neoformans, present cryptococcal antigen, and kill C. neoformans. However, early events following C. neoformans phagocytosis by DCs are not well defined. We hypothesized that C. neoformans traffics to the endosome and the lysosome following phagocytosis by DCs and is eventually killed in the lysosome. Murine bone marrow-derived DCs (BMDCs) or human monocyte-derived DCs (HDCs) were incubated with live, encapsulated C. neoformans yeast cells and opsonizing antibody. Following incubation, DCs were intracellularly stained with antibodies against EEA1 (endosome) and LAMP-1 (late endosome/lysosome). As assessed by confocal microscopy, C. neoformans trafficked to endosomal compartments of DCs within 10 min and to lysosomal compartments within 30 min postincubation. For HDCs, the studies were repeated using complement-sufficient autologous plasma for the opsonization of C. neoformans. These data showed results similar to those for antibody opsonization, with C. neoformans localized to endosomes within 20 min and to lysosomes within 60 min postincubation. Additionally, the results of live real-time imaging studies demonstrated that C. neoformans entered lysosomal compartments within 20 min following the initiation of phagocytosis. The results of scanning and transmission electron microscopy demonstrated conventional zipper phagocytosis of C. neoformans by DCs. Finally, lysosomal extracts were purified from BMDCs and incubated with C. neoformans to determine their potential to kill C. neoformans. The extracts killed C. neoformans in a dose-dependent manner. This study shows that C. neoformans enters into endosomal and lysosomal pathways following DC phagocytosis and can be killed by lysosomal components.

FIG. 1.

Localization of C. neoformans to early endosomes and lysosomes following phagocytosis by murine DCs. Murine DCs were purified and incubated with encapsulated C. neoformans cells and Oregon green 488-labeled opsonizing antibody. DCs were then fixed, permeabilized, and stained with anti-EEA1 antibody (Alexa 568) or anti-LAMP-1 antibody (Alexa 568). Cells were imaged by confocal microscopy. (A) Line graph of average percentages of C. neoformans organisms inside EEA1- and LAMP-1-positive compartments of DCs following phagocytosis at 10, 20, 30, and 60 min postincubation. Data shown are representative of the results of six to eight independent experiments performed with BMDCs and three independent experiments performed with HDCs. Three to four images (including z-stack images) were obtained for each time point. (B) Representative confocal images of C. neoformans organisms in EEA1-positive compartments at 20 min postincubation. The arrows (in the red and green merged image) point to the two C. neoformans organisms in EEA1-positive compartments. (C) Representative confocal images of C. neoformans organisms shown in LAMP-1-positive compartments at 60 min postincubation. Scale bar = 11.9 μm. In panels B and C, “Merge” panels at far right show merged images of bright-field, red, and green panels.

FIG. 2.

Live imaging of DC phagocytosis of C. neoformans organisms and phagolysosomal fusion. Murine DCs were incubated with encapsulated C. neoformans cells, Oregon green 488-labeled opsonizing antibody, and LysoTracker red. Live cells were examined by confocal microscopy at 37°C, with the zero-minute time point indicative of when phagocytosis commenced. Images shown are representative of 10 observations of BMDCs phagocytosing C. neoformans cells. In 8 out of 10 of these observations, including the one shown in the figure, C. neoformans yeast was found in LysoTracker red-positive (lysosomal) compartments within 20 min following phagocytosis. Scale bar = 11.9 μm.

FIG. 3.

Localization of C. neoformans to early endosomes and lysosomes following serum opsonization and phagocytosis by HDCs. HDCs were incubated with encapsulated C. neoformans in the presence of complement-sufficient autologous plasma. Following incubation, HDCs were fixed, permeabilized, and stained with anti-EEA1 antibody (Alexa 568) or anti-LAMP-1 antibody (Alexa 568). (A) Line graph of average percentages of C. neoformans organisms inside EEA1- and LAMP-1-positive compartments of HDCs following phagocytosis at 10, 20, 30, and 60 min postincubation. These data are from three independent experiments. Three to four images (including z-stack images) were obtained at each time point. (B) Representative confocal images of C. neoformans organisms in EEA1-positive compartments at 20 min postincubation. (C) Representative confocal images of C. neoformans organisms in LAMP-1-positive compartments at 30 min postincubation. Scale bar = 11.9 μm. In panels B and C, “Merge” panels at far right show merged images of bright-field, red, and green panels.

FIG. 4.

Results of electron microscopy of C. neoformans phagocytosis by DCs. Following culture, BMDCs were purified and incubated for specified times with encapsulated C. neoformans cells and opsonizing antibody. DCs were then fixed and examined by SEM or TEM. (A) SEM of two C. neoformans yeast cells shown in the process of being phagocytosed by a DC at 10 min postincubation. Arrow points to a pseudopod from the DC attached to the yeast cell. Original magnification, ×19,000. Scale bar = 4 μm. (B) SEM of two C. neoformans yeast cells being phagocytosed by a DC at 10 min postincubation. Original magnification, ×16,000. Scale bar = 5 μm. (C) Close-up of the boxed area from panel B demonstrating a C. neoformans yeast cell partially covered by the “flap” of a DC pseudopod (arrow). Original magnification, ×50,000. Scale bar = 1 μm. (D) One C. neoformans yeast cell is seen inside a membrane-bound compartment of a DC, while another is just beginning to be phagocytosed (arrow) at 50 min postincubation. Original magnification, ×7,100. Scale bar = 2 μm. (E) A C. neoformans yeast cell is seen inside a membrane-bound compartment of a DC at 20 min postincubation. Original magnification, ×19,500. Scale bar = 0.5 μm. (F) Close-up of the boxed area from panel E demonstrating a C. neoformans cell surrounded by a contiguous endosomal membrane (arrows). Original magnification, ×40,000. Scale bar = 200 nm.

FIG. 5.

Killing of C. neoformans by lysosomal extracts from BMDCs. C. neoformans yeast cells were incubated in lysosomal buffer with the indicated concentrations of lysosomal extracts for 24 h at 37°C, following which the numbers of CFU in the wells were determined as described in Materials and Methods. The numbers of CFU in the inoculum are also shown. Data shown are means ± standard errors of the means of the results of four independent experiments, with each condition performed in duplicate. An asterisk indicates a significant difference compared to the results for 0% extract (P < 0.0001).