Dr. Tamara Doering's laboratory at the Washington University Medical School studies the pathogenic yeast Cryptococcus neoformans, which causes opportunistic infections in immunocompromised individuals. Her lab investigates unique biochemical aspects of cryptococcal biology that are potential targets for antifungal chemotherapy.
Dr. Doering first encountered the OUHSC Laboratory for Genomics and Bioinformatics when she came to the campus as a seminar guest. Among scheduled meetings with colleagues in microbiology and glycobiology she spoke to Dr. Allison Gillaspy and discussed a new bioinformatics collaboration her lab had begun with Dr. Michael Brent at Washington University's Department of Computer Science. These studies addressed a difficulty of annotating the cryptococcal genome sequence, due to the numerous small introns in each gene. The collaboration combined computational gene prediction by the Brent lab (using their TWINSCAN software) with experimental validation by RT-PCR performed in the Doering lab. This required sequencing the RT-PCR products, which was performed in the OUHSC lab; the collaboration was quite successful.
Most research in the Doering lab focuses on the C. neoformans polysaccharide capsule required for virulence. The chemical structure of the capsule is known, but almost nothing is known about synthesis/assembly of this unique protective structure. The capsule is composed of several polysaccharides ranging in size up to 2 million Daltons molecular weight, with a variety of glycosidic linkages. Biosynthesis is complex, requiring cellular machinery to generate the appropriate sugars and their activated donors, to correctly link the sugars together, to assemble the polymers and to associate them with the external cell wall. To tackle this complex problem, Doering uses biochemistry, cell and molecular biology approaches. Many experiments amplify sequences from cryptococcal RNA, followed by DNA sequencing at OUHSC for confirmation. Another powerful technique, RNA interference, specifically down-regulates gene expression, and is being applied for forward genetics in cryptococcus by an interference library approach. This again involves extensive DNA sequencing, performed at OUHSC. Together, these approaches are elucidating the biosynthesis of a fascinating glycoconjugate as well as aspects of fungal biology that may reveal new drug targets.
Contributed by Tamara Doering, Ph.D.
