Cryptococcus neoformans is an opportunistic fungal pathogen with a high mortality rate. Its major virulence factors are primarily composed of well-characterized glycans; however, the enzymatic steps involved in synthesizing these glycans are largely unknown. The long-term objective of the proposed research is to define unknown steps in glycan synthesis pathways of this organism, mediated by glycosyltransferase enzymes (GTs), that are important for virulence. I propose two complementary approaches to achieve this objective. First, the virulence of GT gene deletion mutants will be compared to that of wild-type C. neoformans to prioritize candidates for study. Mutants lacking GTs of greatest interest will be assessed phenotypically, to suggest biological roles of the GTs, and their glycans analyzed for defects, to suggest specific biochemical roles of the GTs. In parallel, GTs of interest will be recombinantly expressed and their enzymatic activity determined using novel biochemical assays. Together, these approaches will define the roles of GTs that are important in virulence, contributing to our understanding of the basic biology of this pathogen and potentially revealing novel drug targets. Through the successful completion of the proposed research, together with additional formal and informal training, the PI will become technically adept in molecular microbiology, glycobiology, and protein biochemistry. In addition, the PI will cultivate professional skills tailored to a future career in academia by presenting original work at seminars, conferences, and in written form for publication. By facilitating her participation in these predoctoral training opportunities, the Ruth L. Kirschstein Predoctoral Individual National Research Service Award will enable the PI to develop as an independent scientist, in keeping with its mission.
C. neoformans is a deadly fungal pathogen that primarily afflicts immunocompromised people. Enzymes inside the cryptococcal cell generate many polysaccharides that enable it to cause disease. The proposed research will define the roles of three of these enzymes in synthesizing polysaccharides, revealing fundamental biological pathways in this organism that could suggest new strategies for drug targeting.
