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Infect. Immun. doi:10.1128/IAI.00660-08
Copyright (c) 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

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

Department of Medicine, Division of Infectious Diseases, University of Massachusetts Medical School, Worcester, MA USA

^* To whom correspondence should be addressed. Email: Stuart.Levitz{at}umassmed.edu.

	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 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 minutes and to lysosomal compartments within 30 minutes post-incubation. In HDCs, studies were repeated using complement-sufficient autologous plasma for opsonization of C. neoformans. These data showed similar results to antibody opsonization, with C. neoformans localized to endosomes within 20 minutes and to lysosomes within 60 minutes post-incubation. Additionally, live real-time imaging studies demonstrated that C. neoformans entered lysosomal compartments within 20 minutes following initiation of phagocytosis. 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.