Temporal Kinetics and Quantitative Analysis of Cryptococcus

Cryptococcus neoformans is a facultative intracellular pathogen and the causative agent of cryptococcosis, a disease that is often fatal to those with compromised immune systems. C. neoformans has the capacity to escape phagocytic cells through a process known as nonlytic exocytosis whereby the cryptococcal cell is released from the macrophage into the extracellular environment, leaving both the host and pathogen alive. Little is known about the mechanism behind nonlytic exocytosis, but there is evidence that both the fungal and host cells contribute to the process. In this study, we used time-lapse movies of C. neoformans-infected macrophages to delineate the kinetics and quantitative aspects of nonlytic exocytosis. We analyzed approximately 800 macro-phages containing intracellular C. neoformans and identified 163 nonlytic exocytosis events that were further characterized into three subcategories: type I (complete emptying of macrophage), type II (partial emptying of macrophage), and type III (cell-to-cell transfer). The majority of type I and II events occurred after several hours of intracellular residence, whereas type III events occurred significantly (P< 0.001) earlier in the course of macrophage infection. Our results show that nonlytic exocytosis is a morphologically and temporally diverse process that occurs relatively rapidly in the course of macrophage infection. Cryptococcus neoformans is a facultative intracellular pathogenthat causes life-threatening disease in immunocompromised individuals (1, 2). Individuals can become infected after inhala-tion of desiccated yeast cells or spores (3) from the environment. Most human infections are asymptomatic, but immunocompro