Doering Lab Research
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 fungal virulence. The capsule can be seen surrounding the trio of cells below and extending as a meshwork from the cell wall in a higher resolution image (below at right). This. We are taking several approaches to understanding how capsule components are made and assembled to form this unusual surface coat, 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 experiments 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 studies 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 exits from and associates with the cell. (Some insight in this area has come from the secretion mutant shown below left.) We hope in the future to address the molecular mechanisms of these fascinating processes. We have also recently become interested in adapting high throughput screening to the question of host:pathogen interactions, depicted below right (host nuclei are blue and fungi are green or yellow). Finally, we are investigating gene regulation in C. neoformans, taking advantage of new technologies and approaches in computational biology.