F-035. Intracellular Localization of Cryptococcus neoformans following Dendritic Cell Phagocytosis

K. L. Wozniak1, S. M. Levitz2;
1Univ. of Massachusetts, Worcester, MA, 2Univ. of Massachusetts Med. Sch., Worcester, MA.

Cryptococcus neoformans is an opportunistic fungal pathogen that primarily causes disease in immunocompromised individuals. Dendritic cells (DCs) can phagocytose C. neoformans in vitro and in vivo and present cryptococcal antigen ex vivo. However, early events following C. neoformans phagocytosis by DCs are unknown. We hypothesized that C. neoformans traffics to the endosome and then to the lysosome following phagocytosis by DCs. Confocal and electron microscopy were used to determine the intracellular location of C. neoformans upon phagocytosis by DCs. For this, murine bone marrow-derived DCs (BMDCs) or human monocyte-derived DCs (HDCs) were incubated with encapsulated C. neoformans and opsonizing antibody for 10, 20, 30, or 60 minutes. Following incubation, DCs were fixed, permeabilized, and intracellularly stained with antibodies against EEA1 (endosome) and LAMP-1 (late endosome/lysosome). In HDCs, studies were repeated using complement-sufficient autologous serum for opsonization of C. neoformans. Additionally, live imaging of cyptococcal entry into lysosomes was performed in DCs stained with lysotracker red. Finally, lysosomal extracts were purified from DCs and incubated with C. neoformans to determine their potential to kill C. neoformans. Results of confocal microscopy showed that C. neoformans trafficked to endosomal compartments of DCs within 10 minutes and to lysosomal compartments within 30 minutes post-incubation. In HDCs, complement opsonization produced similar results to antibody opsonization, with C. neoformans localized to endosomes within 20 minutes and to lysosomes within 60 minutes post-incubation. The results of confocal microscopy were confirmed by transmission electron microscopy. Live imaging of DCs stained with lysotracker showed that C. neoformans entered lysosomal compartments within 20 minutes following phagocytosis. Finally, lysosomal components isolated from DCs 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.