GLYCAN PRECURSOR TRANSPORT IN CRYPTOCOCCUS NEOFORMANS

Washington University, Saint Louis MO

Investigators

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Abstract

DESCRIPTION (provided by applicant): Meningitis caused by Cryptococcus neoformans kills over 625,000 immunocompromised individuals each year. The polysaccharide capsule and other glycoconjugates of this fungus are critical to its survival in the environment and its succes as a serious pathogen of humans. Despite the importance of these structures, we have only limited knowledge of how they are made. Our long-term goal is to define the major pathways of glycan synthesis in C. neoformans, both for fundamental biochemical understanding and as potential targets for therapy. The individual components of glycans come from nucleotide sugars, which are activated sugar donors that serve as substrates in biosynthetic reactions. These charged compounds are present in the cell cytosol, which means that they must be transported across membranes into the secretory organelles where most eukaryotic glycan synthesis occurs. The proteins that accomplish this, nucleotide sugar transporters (NSTs), are thus essential and limiting components of key biosynthetic pathways that contribute to cryptococcal pathogenesis. The objective of this proposal is to determine the function of a novel nucleotide sugar transporter, NSTX, which we have discovered and strongly implicated in fungal virulence. We hypothesize that NSTX mediates transport of the activated donor of one or more acidic monosaccharides. We will test this hypothesis through two aims. In the first aim, we will determine the effects of deleting the gene encoding NSTX on cryptococcal glycoconjugates by comparing the compositions of capsule polysaccharides, glycoproteins, and glycolipids isolated from wild type and mutant cells. In the second aim, we will directly determine the in vitro transport activity of NSTX. These complementary aims play to the unique strengths of the research team in biochemistry, glycobiology, and cell biology of C. neoformans. Determining the activity of NSTX will help establish how C. neoformans localizes the precursors required to make capsule and other important glycoconjugates implicated in virulence, and may also settle a long-standing question about sialic acid use by these cells. The application is innovative in terms of proposing a novel hypothesis about NSTX function and direct studies of nucleotide sugar transport in an organism with unique and understudied glycoconjugates. It is significant because it is expected to advance our understanding of a limiting step in glycan synthetic processes that are vital to an important pathogen of humans. This exploratory work will further be of broad impact because it will set the stage for future studies of fundamental glycobiology, cryptococcal biology, and pathogenesis.