Research Abstract:
We are interested in the fundamental biology and host interactions of the pathogenic fungus Cryptococcus neoformans, which causes serious opportunistic infections in immunocompromised individuals. Our goal is to elucidate unique aspects of cryptococcal biology that are of biological interest and may suggest targets for badly needed antifungal chemotherapy.
The main virulence factor of C. neoformans is its extensive polysaccharide capsule, a unique protective structure that is required for virulence. The chemical structure of the capsule is known, but almost nothing is known about its synthesis and assembly. We are taking several approaches to understanding capsule biosynthesis, and to investigating interactions of this pathogen with host cells.
One approach is biochemical. We develop assays for enzymes involved in capsule construction in order to purify and study them or to identify them genetically. We are also investigating the synthesis and intracellular transport of nucleotide sugar precursors that are required for the sugar transferase reactions that link capsule components. Another approach is genetic: generating and studying mutants with defects in capsule synthesis. In these studies we can take advantage of the well-sequenced cryptococcal genome for reverse genetic studies, using RNA interference to specifically down-regulate gene expression, or generating mutant strains. Taking a forward genetic strategy, we can also screen for mutants of interest after mutagenesis or exposure of cells to an RNA interference library.
Other experiments employ methods of cell biology to address the question of how new capsular material is incorporated into the existing structure, how capsule matures, and how capsular material associates with the cell. We hope in the future to address the molecular mechanisms of these fascinating processes. We have also recently begun applying high throughput screening to the question of host:pathogen interactions. Finally, we are investigating gene regulation in C. neoformans, taking advantage of new technologies and approaches in computational biology.
Selected Publications:
Doering TL. How sweet it is! Capsule formation and cell wall biogenesis in Cryptococcus neoformans. Annual Reviews of Microbiology 2009 Volume 63 (In Press).
Yoneda A and Doering TL. An unusual organelle in Cryptococcus neoformans links luminal pH and capsule biosynthesis. Fungal Genet Biol. 2009 (In Press).
Heiss C, Klutts JC, Wang Z, Doering TL, and Azadi P. The structure of Cryptococcus neoformans galactoxylomannan contains beta-D-glucuronic acid. Carbohydrate Research 2009 344: 915-920.
Klutts JC and Doering TL. Cryptococcal xylosyltransferase 1 (Cxt1p) from Cryptocococus neoformans plays a direct role in the synthesis of capsule polysaccharides. J. Biol. Chem. 2008 283: 14327-14334.
Reese AJ, Yoneda A, Breger JA, Beauvais A, Liu H, Griffith CL, Bose I, Kim M-J, Skau C, Yang S, Sefko JA, Osumi M, Latge J-P, Mylonakis E, and Doering TL. Loss of cell wall alpha(1-3) glucan affects Cryptococcus neoformans from ultrastructure to virulence. Molec Microbio 2007 63:1385-1398. [Cover image.]
Last Updated: 07/17/2009 |