Polysaccharide Capsule Export in the Fungal Pathogen Cryptococcus neoformans ABSTRACT Cryptococcus neoformans is an opportunistic pathogen, responsible for life-threatening disease in individuals who are immunocompromised due to AIDS or other conditions. The main virulence factor of Cryptococcus is an extensive polysaccharide capsule that surrounds its cell wall. This capsule is required for the fungus to cause disease. Little information is available about the intracellular formation of capsule components, and none about how they exit the cell and become associated with its surface. In this R21 application we propose to develop new approaches to exploration of these fundamental questions, using cell biological methods that have not yet been applied to C. neoformans. Specifically, we propose to directly investigate the nature of intracellular capsular material, taking advantage of a mutant strain we have engineered to accumulate secretory vesicles containing capsule- related polysaccharides. Aim I proposes to develop methods to purify and characterize the secretory vesicles that contain capsule precursors. Aim II proposes to isolate capsule-related polysaccharides from these vesicles, purify them, and apply analytical methods to define their glycan structures. Depending on the specific nature of the capsule precursors, the experiments may begin to address the presence of any non-carbohydrate components associated with the relevant glycans. The approaches that will be used to achieve these goals include subcellular fractionation, electron micros- copy, biochemical enzyme assays, immuno-detection, differential extraction and precipitation, a variety of chromatography methods, and glycan analysis. These exploratory studies will have a major impact on the field, as they will address a critical gap in our understanding of this organism's major virulence factor, break new ground in the methods available for study of C. neoformans, and open exciting areas for investigation of this important pathogen. / RELEVANCE This research is highly relevant to public health because the organism being investigated causes serious human illness, for which current therapies are not adequate. The process to be studied is fundamental to the ability of Cryptococcus neoformans to cause disease, so understanding and inhibiting it may advance treatment of cryptococcal infection. Further, this work will contribute to basic science knowledge, which will impact the area of pathogenic microbes as well as other areas of biology. [unreadable] [unreadable] [unreadable]