F-032. The Cryptococcus neoformans Vacuole Has Characteristics of an Autophagosome
Cryptococcus neoformans is an encapsulated yeast that is a relatively frequent cause of meningitis. Macrophages are the most important effector cells in the immune response to this infection, as is the case with other facultative intracellular pathogens. However, C. neoformans does not inhibit phagolysosomal maturation nor evades its vacuole (CnV), implying that there must be some other mechanism used by macrophages to control it. We have investigated whether autophagy, a conserved tool used to recycle cellular material, is involved in control of intracellular C. neoformans. Time-lapse confocal imaging of the interaction between macrophage-like J774 cells and C. neoformans revealed that several hours after phagocytosis, the CnV became positive for the autophagosome marker LC3 coupled to enhanced green fluorescent protein (EGFP). However, no cup-shaped sequestration membranes (hallmarks of autophagosome formation) could be observed; instead, this giant autophagosome appeared to form by sequential fusion of small autophagic vacuoles (Av) with the CnV. These fusion events were also apparent by transmission electron microscopy, which revealed fusion of the outer membrane of double-membraned vesicles with the CnV membrane. To evaluate the maturation of both Cnv and Avs, we included in the live-cell imaging experiments the lysosomal marker 10 kDa Dextran - Texas Red. We have observed that lysosomal fusion with the CnV occurred prior to its fusion with the Avs. We could also determine that these Avs had not undergone maturation and fusion with lysosomes prior to fusion with the CnV. To rule out artifacts due to EGFP aggregation, we fixed C. neoformans infected J774 cells and stained them with anti-LC3 antibody, which marked the CnV in a fraction of the cells. Similar results were obtained with murine peritoneal macrophages. In conclusion, the CnV acquires the autophagy marker LC3 after maturation and that this autophagosome is not formed by the typical sequestration membranes. We hypothesize that autophagy is involved on the outcome of macrophage infection by C. neoformans.