Discrepancy in assessment of phagosome-lysosome fusion with two lysosomal markers in murine macrophages infected with Candida albicans

Abstract

Phagosome-lysosome fusion (P-LF) was studied in cultured mouse resident peritoneal macrophages after phagocytosis of Candida albicans. The macrophages were labeled with acridine orange (AO), the electronopaque colloidal Thorotrast, or both markers. After phagocytosis of heat-killed C. albicans, both markers were delivered to more than 95% of phagosomes. After ingestion of viable C. albicans by labeled cells, delivery of AO to phagosomes was highly suppressed (90%), and yet Thorotrast delivery was almost universal. After phagocytosis and 60 min of incubation, about 10 to 20% of the yeasts were killed, and a similar fraction of phagosomes was stained by the fluorescent marker. The evidence from Thorotrast transfer and assessment of yeast viability indicates that C. albicans largely resists intracellular killing by resident macrophages in the face of entirely uninhibited P-LF. We infer that AO must transfer to nearly all of the phagosomes but that it is evidently recognizable only in those in which the yeasts have been killed or possibly severely injured. This conclusion constitutes yet another limitation in the usefulness of AO for studying P-LF.