Filling gaps in the cryptococcal wall with glycogen and a novel enzyme

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ABSTRACT Cryptococcus neoformans is a devastating opportunistic fungus that causes hundreds of thousands of deaths each year, mainly in developing countries. This pathogen is surrounded by a flexible wall that maintains cell integrity and anchors a protective polysaccharide capsule. The cell wall, which consists mainly of interlinked polysaccharides, is a compelling topic of study because it is required for viability, absent from the cells of mam- malian hosts, and a proven target for antifungal therapies. Almost a century ago, it was postulated that yeast walls contain glycogen, similar in structure to the intracellular storage molecule but linked to the cell wall itself. However, where this occurs in nature, as well as how this material is made and reaches the cell surface, have never been established, constituting major gaps in our knowledge of an essential structure. We recently discov- ered that a previously unstudied cryptococcal protein influences both glycogen synthesis and cell wall integrity, potentially providing a key to this area of research. This protein is also required for normal infection, suggesting a potential vulnerability in the wall that might be productively exploited. The long-term goal of our research is to define the biochemical pathways by which cryptococcal glycans are made, to advance our fundamental understanding and improve the outcome of this devastating infection. In this proposal we focus on the novel area of cell wall glycogen and an intriguing glycosyltransferase we have discov- ered, which we call GTX. In Aim 1 we will isolate and quantitate cell wall glycogen from C. neoformans grown in various environments, including host-like conditions, and determine how this material fits into the complex mesh of the cell wall. In Aim 2 we will use biochemical assays to define and characterize the activity of purified GTX, which we already have in hand. In Aim 3 we will assess the phenotypes and virulence characteristics of mutants lacking GTX. We will also localize this protein and identify its interacting partners. These studies will be enabled by our expertise in cryptococcal biology and advice from expert colleagues, which we will integrate into thoughtful and rigorous studies. Together, these experiments will define a new component of the cryptococcal cell wall and determine the activity of a novel protein implicated in pathogenesis. The major antifungal drugs that target cell walls are not effective against C. neoformans. It is therefore critical to find other vulnerabilities in the cryptococcal wall that can be productively exploited – our proposed studies are a focused step on this path. Completing them will advance our understanding of fungal cell wall construction, define a novel glycan synthetic process, and potentially suggest a point of intervention for antifungal therapy.