GLYCAN PRECURSOR TRANSPORT IN CRYPTOCOCCUS NEOFORMANS

描述（由申请人提供）：由新型隐球菌引起的脑膜炎每年导致超过625,000名免疫功能低下者死亡。这种真菌的多糖胶囊和其他糖缀合物对其在环境中的生存和作为人类严重病原体的成功至关重要。尽管这些结构很重要，但我们对它们是如何制造的了解有限。我们的长期目标是确定C. neoformans中糖合成的主要途径，既用于基本的生化理解，也作为潜在的治疗靶点。聚糖的单个组分来自核苷酸糖，核苷酸糖是生物合成反应中作为底物的活化糖供体。这些带电荷的化合物存在于细胞质中，这意味着它们必须穿过细胞膜进入分泌细胞器，在那里大多数真核聚糖合成发生。完成这一过程的蛋白质，核苷酸糖转运蛋白（NSTs），因此是促进隐球菌发病的关键生物合成途径的必要和限制性组分。本提案的目的是确定一种新的核苷酸糖转运蛋白NSTX的功能，我们已经发现并与真菌毒力密切相关。我们假设NSTX介导一种或多种酸性单糖的激活供体的运输。我们将通过两个目标来检验这一假设。在第一个目标中，我们将通过比较从野生型和突变型细胞中分离的胶囊多糖、糖蛋白和糖脂的组成来确定删除编码NSTX的基因对隐球菌糖缀合物的影响。在第二个目的中，我们将直接测定NSTX的体外转运活性。这些互补的目标发挥了研究团队在生物化学，糖生物学和新生C.细胞生物学方面的独特优势。确定NSTX的活性将有助于确定新形态C.如何定位制造胶囊和其他与毒力有关的重要糖缀合物所需的前体，并可能解决关于这些细胞使用唾液酸的长期问题。该应用具有创新性，因为它提出了关于NSTX功能的新假设，并直接研究了具有独特且未充分研究的糖缀合物的生物体中核苷酸糖的转运。这项研究意义重大，因为它有望促进我们对对人类重要病原体至关重要的聚糖合成过程的限制步骤的理解。这项探索性工作将进一步产生广泛的影响，因为它将为未来基础糖生物学、隐球菌生物学和发病机制的研究奠定基础。

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.