This application is for initial independent research on extracellular glycan synthesis and maturation in Cryptococcus neoformans. This opportunistic fungal pathogen is responsible for life threatening disease, particularly in the context of compromised immunity, and current therapy is inadequate. The long-term goal of the proposed research is to investigate novel features of C. neoformans glycobiology, which will lead to the identification of targets for anti-fungal drug discovery. The proposed studies will lead to greater understanding of cryptococcal biology, and will contribute to the elucidation of glycan synthesis in eukaryotes. Glucuronoxylomannan (GXM) is the dominant component of the cryptococcal polysaccharide capsule that is the major virulence factor of C. neoformans. Although the structure of GXM is known and some of its biological roles have been investigated, how this polymer is built is not understood. The unique structural features of GXM suggest attractive targets for drug discovery, but these require investigation of biosynthesis and development of biochemical assays. Efforts will be directed at studies of a C. neoformans mannosyl transferase (MT) recently discovered by the investigator which exhibits the unique specificity required for a role in GXM synthesis. This enzyme will be purified using chromatographic methods based on physical properties and affinity, and biochemically characterized to examine kinetics, substrate specificity, and processivity. A high throughput assay for MT activity will also be developed. Based on sequence derived from the purified protein the structural gene will be cloned, allowing examination of gene expression and regulation and disruption of the gene to assess function directly. A fusion protein will be expressed and used to generate antibody against the MT, which will be used to analyze protein expression, processing, posttranslational modification and localization. Other studies will focus on mutant strains of C. neoformans that have been found by the investigator to be defective in both MT activity and in capsule production. These strains provide an exciting link between these two processes, which may reflect a defect in the gene encoding the MT itself or in other genes involved in glycan synthesis or regulation. Experiments to define and study these defects will use gene cloning and expression, virulence studies and other methods mentioned above.
