Investigations on the Morphology, Kinetics and Mechanism of Cryptococcus neoformans Non-Lytic Exocytosis

Abstract

Cryptococcus neoformans is an environmental microbe that has evolved to become an intracellular pathogen in a variety of organisms. The infection cycle begins when dried yeast cells or spores are inhaled from the surrounding environment. After ingestion of a yeast particle by a macrophage, there are several different outcomes that can occur. The first outcome is that macrophages control the infection by killing the intracellular yeast. The second outcome is that the fungal burden is too great for the macrophage and the host cell lyses releasing cryptococcal cells into the extracellular environment. The third and most novel outcome is that yeast cells can be released from macrophages by two new mechanisms: non-lytic exocytosis or cell-to-cell transfer. This phenomenon was first described in 2006 and there is still basic information that has yet to be elucidated surrounding this newly observed exit strategy. Thus this thesis is built upon understanding morphological and cellular aspects of non-lytic exocytosis.;Using time-lapsed microscopy to observe infected macrophages, the first study characterized both the morphology and temporal kinetics surrounding this C. neoformans-macrophage interaction. Based on the amount of fungal cells expunged and the manner in which they were released, the process was categorized into three subcategories: Type I (complete non-lytic exocytosis), Type II (partial non-lytic exocytosis) and Type III (cell-cell transfer), and occurred during early stages of infection. The second study concentrated on annexin A2, a membrane-binding protein that aggregates cellular membranes to promote fusion. I showed that phagocytosis and non-lytic exocytosis were decreased in annexin A2 deficient primary murine macrophages. I also observed that the absence of this protein causes an enlargement of the cryptococcal capsule both in vitro and in vivo. In addition, there was a significant decrease in survival of knock out mice when challenged with C. neoformans.;Non-lytic exocytosis is a complex interaction and must involve a variety of cellular components to ensure the survival of both macrophage and fungal cell. This dissertation provides new information on the morphology of this phenomenon throughout a fungal infection and attempts to decipher specific factors that are part of the machinery that drives non-lytic exocytosis.