ABSTRACT Cryptococcus neoformans is a devastating opportunistic pathogen that causes hundreds of thousands of deaths each year, mainly in developing countries. The glycan structures of this organism include cell wall com- ponents, glycoproteins, glycolipids, and polysaccharides of the cryptococcal capsule, which is a major viru- lence factor. These molecules are central to cryptococcal biology and pathogenesis, yet there are major gaps in our understanding of their synthetic pathways. We lack basic knowledge in this area, leading to missed op- portunities to define basic biology and explore targeted intervention. The long-term goal of our research is to determine the biochemical pathways by which capsule and other cryptococcal glycans are made, to advance our fundamental understanding and improve the outcome of this devastating infection. In this proposal we will focus on glycoactive proteins that play roles in fungal virulence. We will perform in-depth studies of their bio- chemical functions and cellular context, including binding partners and subcellular localization. In parallel we propose to implement a systematic approach for identifying novel proteins that are central in glycan synthesis and virulence, shifting current practice by taking advantage of genome sequence, new mutant libraries, and in- novative biochemical assays. Our proposed studies to define the biological functions and roles in pathogenesis of these key proteins gains impetus from the validated use of glycoactive enzymes as drug targets, including in the context of fungal infections. We propose to study glycosyltransferase enzymes that use sugars from activated precursors to build glycocon- jugates and cryptococccal transporters that provide those precursors. In Aim 1 we will use biochemical and cell biological approaches to determine substrates, functionally assess unique domains, define binding part- ners, and determine subcellular localization of glycoactive proteins that we already know influence cryptococ- cal virulence. In Aim 2 we will test deletion strains in mouse infection models to prioritize potential glycosyl- transferase genes. We will then use glycan analysis to identify the activities most implicated in virulence and new biochemical assays to characterize protein function. Throughout the project we will design and conduct well-controlled experiments and interpret them rigorously, in the context of our expertise in cryptococcal biology. We expect our combination of biochemical and genetic approaches to advance the field by defining fundamental glycobiology in a pathogenic microbe with unique glycans. Completing these studies will fill major gaps in our understanding of fungal biology, define critical as- pects of cryptococcal glycan synthesis, and potentially suggest points of intervention to advance antifungal therapy.