GLYCAN PRECURSOR TRANSPORT IN CRYPTOCOCCUS NEOFORMANS

描述(由申请人提供)：由新生隐球菌引起的脑膜炎每年导致超过625,000名免疫功能受损的人死亡。这种真菌的多糖壳和其他糖结合物对它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知之甚少。我们的长期目标是确定新生隐球菌糖合成的主要途径，这既是为了基本的生化理解，也是作为潜在的治疗靶点。多糖的各个组成部分来自核苷酸糖，这些核苷酸糖是作为生物合成反应底物的活性糖供体。这些带电化合物存在于细胞胞浆中，这意味着它们必须跨膜运输到分泌细胞器，大多数真核糖的合成都发生在那里。因此，完成这一任务的蛋白质，即核苷酸糖转运体(NSTs)，是促成隐球菌致病的关键生物合成途径的重要和限制性成分。这项建议的目的是确定一种新的核苷酸糖转运蛋白NSTX的功能，我们已经发现了它，并与真菌毒力密切相关。我们假设NSTX介导了一个或多个酸性单糖的活化供体的运输。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出的胶囊多糖、糖蛋白和糖脂的组成来确定缺失NSTX编码基因对隐球菌糖偶联物的影响。在第二个目标中，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新生葡萄球菌的生物化学、糖生物学和细胞生物学方面的独特优势。测定NSTX的活性将有助于确定新生葡萄球菌如何定位制造胶囊和其他与毒力有关的重要糖偶联物所需的前体，还可能解决这些细胞使用唾液酸的长期问题。这一应用具有创新性，提出了关于NSTX功能的新假说，并直接研究了具有独特和未被研究的糖偶联物的生物体中的核苷酸糖运输。这项研究意义重大，因为它有望促进我们对多糖合成过程中一个限制步骤的理解，而这一过程对人类的一种重要病原体至关重要。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.