The encapsulated fungus Cryptococcus neoformans is responsible for life- threatening disease, particularly in the context of compromised immunity, and current therapy is not adequate. Extracellular glycans define the interface between this opportunistic pathogen and its host;our long-term goal is to determine the biosynthetic pathways of these molecules and discover ways to interfere with them. The main virulence factor of C. neoformans is an extensive polysaccharide capsule composed of two polymers: glucuronoxylomannan (GXM) and glucuronoxylomanno-galactan (GXMGal;previously termed GalXM). We know the structures of these polysaccharides, but major questions remain concerning their synthesis and assembly into the capsule. In the preceding period of this project we purified, cloned, expressed, and mutated a novel xylosyltransferase (Cxt1p), and showed that it acts in synthesis of GXM, GXMGal, and glycolipids;we also discovered a related enzyme (Cxt2p). We further established how capsule is transported to the cell surface and demonstrated that cell wall alpha glucan is required for capsule display.
Aim I of the current application is to localize Cxt1p and Cxt2p within the cryptococcal cell and perform targeted experiments on glycan structure and virulence to address their role in cryptococcal biology.
Aim II proposes a biochemical approach to identify the machinery of capsule association with the cell surface, followed by phenotypic analysis of strains deficient in candidate proteins. These revised aims can be reasonably performed in two years. We plan creative new strategies to investigate novel features of C. neoformans glycobiology, with emphasis on the capsule. We will capitalize on our prior discoveries to gain greater understanding of cryptococcal biology and pathogenesis, identify targets for anti-fungal drug discovery, and elucidate glycan synthesis in eukaryotes.
PROJECT RELEVANCE / NARRATIVE This research is highly relevant to public health because the organism under study causes serious human illness for which current therapies are not adequate. The capsule of Cryptococcus neofor- mans is fundamental to its ability to cause disease, so understanding and inhibiting its synthesis and assembly may advance treatment of cryptococcal infection. Further, this work will add to basic sci- ence knowledge, which will contribute insights into the biology of eukaryotic organisms in general.
| Agustinho, Daniel P; Miller, Liza C; Li, Lucy X et al. (2018) Peeling the onion: the outer layers of Cryptococcus neoformans. Mem Inst Oswaldo Cruz 113:e180040 |
| Wang, Zhuo A; Li, Lucy X; Doering, Tamara L (2018) Unraveling synthesis of the cryptococcal cell wall and capsule. Glycobiology 28:719-730 |
| Li, Lucy X; Ashikov, Angel; Liu, Hong et al. (2017) Cryptococcus neoformans UGT1 encodes a UDP-Galactose/UDP-GalNAc transporter. Glycobiology 27:87-98 |
| Kumar, Pardeep; Heiss, Christian; Santiago-Tirado, Felipe H et al. (2014) Pbx proteins in Cryptococcus neoformans cell wall remodeling and capsule assembly. Eukaryot Cell 13:560-71 |
| Wang, Zhuo A; Griffith, Cara L; Skowyra, Michael L et al. (2014) Cryptococcus neoformans dual GDP-mannose transporters and their role in biology and virulence. Eukaryot Cell 13:832-42 |
| Wohlschlager, Therese; Buser, Reto; Skowyra, Michael L et al. (2013) Identification of the galactosyltransferase of Cryptococcus neoformans involved in the biosynthesis of basidiomycete-type glycosylinositolphosphoceramide. Glycobiology 23:1210-9 |
| Heiss, Christian; Skowyra, Michael L; Liu, Hong et al. (2013) Unusual galactofuranose modification of a capsule polysaccharide in the pathogenic yeast Cryptococcus neoformans. J Biol Chem 288:10994-1003 |
| Skowyra, Michael L; Doering, Tamara L (2012) RNA interference in Cryptococcus neoformans. Methods Mol Biol 845:165-86 |
| Reilly, Morgann C; Aoki, Kazuhiro; Wang, Zhuo A et al. (2011) A xylosylphosphotransferase of Cryptococcus neoformans acts in protein O-glycan synthesis. J Biol Chem 286:26888-99 |
| Kumar, Pardeep; Yang, Meng; Haynes, Brian C et al. (2011) Emerging themes in cryptococcal capsule synthesis. Curr Opin Struct Biol 21:597-602 |
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