Role of glycan precursor transporters in Cryptococcus neoformans virulence

 描述（由申请人提供）：新型隐球菌是一种包囊化的机会性真菌病原体，每年在全球感染超过100万人，并导致超过60万人死亡。目前的治疗是不充分的高发病率，死亡率和复发率，尽管昂贵和有毒的抗真菌干预。隐球菌多糖是真菌存活和发病的关键决定因素，使其成为有吸引力的治疗靶点。用于合成大多数糖缀合物（包括多糖胶囊）的活化供体分子是核苷酸糖。这些高度带电的分子通常在胞质溶胶中产生，然后由核苷酸糖转运蛋白（NST）转运到分泌途径中，在此发生大多数聚糖生物合成。尽管它们在聚糖合成中起关键作用，但整套隐球菌NST的身份和调控仍然未知。我们知识中的这一重大空白严重限制了我们操纵这种重要病原体的关键生物合成过程的能力。本提案的目的是确定三种新的核苷酸糖转运蛋白的功能，命名为NSTX，NSTH和NSTG，这是通过与已知的NST同源性鉴定的，并与真菌毒力有关。这三个NST的缺失显着减少胶囊的合成，并在相当大的温度和应力敏感性的结果。此外，nstX和nstH突变体在小鼠中建立感染的能力的严重缺陷使得这些转运蛋白成为药物发现的有吸引力的靶标。在目标1中，我将使用两种互补的方法来确定这些NST中每一种的底物。我将用半完整的隐球菌细胞直接测定放射性标记的核苷酸糖进入分泌途径的转运。我还将比较野生型和突变型细胞的糖缀合物组成，以确定潜在的底物。在目标2中，我将研究缺失菌株毒力降低的机制。我将检查巨噬细胞的吞噬作用和清除在体外，并确定在体内感染的动力学为每个突变体。这项工作将促进我们对聚糖生物合成及其毒力要求的理解。因此，它将为基础糖生物学，隐球菌生物学和发病机制的进一步研究，以及潜在的抗真菌药物的发现奠定基础。

 DESCRIPTION (provided by applicant): Cryptococcus neoformans is an encapsulated opportunistic fungal pathogen that infects over one million people annually and kills over 600,000 individuals per year worldwide. Current treatments are inadequate with high rates of morbidity, mortality, and relapse despite expensive and toxic antifungal interventions. Cryptococcal glycans are crucial determinants of fungal survival and pathogenesis, making them attractive therapeutic targets. The activated donor molecules for synthesis of most glycoconjugates, including the polysaccharide capsule, are nucleotide sugars. These highly charged molecules are typically made in the cytosol and then transported by nucleotide sugar transporters (NSTs) into the secretory pathway, where most glycan biosynthesis occurs. Despite their key role in glycan synthesis, the identity and regulation of the complete set of cryptococcal NSTs remains unknown. This major gap in our knowledge severely limits our ability to manipulate critical biosynthetic processes in this important pathogen. The objective of this proposal is to determine the function of three novel nucleotide sugar transporters, designated NSTX, NSTH, and NSTG, which were identified by homology to known NSTs and have been implicated in fungal virulence. Deletion of these three NSTs markedly reduces capsule synthesis and results in considerable temperature and stress sensitivity. The profound defects in the ability of nstX and nstH mutants to establish infection in mice, furthermore, make these transporters an attractive target for drug discovery. In Aim 1, I will determine the substrate(s) of each of these NSTs using two complementary approaches. I will directly assay transport of radiolabeled nucleotide sugars into the secretory pathway with semi-intact cryptococcal cells. I will also compare glycoconjugate composition of wild-type and mutant cells to determine potential substrates. In Aim 2, I will investigate the mechanism underlying the decreased virulence of the deletion strains. I will examine macrophage phagocytosis and clearance in vitro, and define the dynamics of infection in vivo for each mutant. This work will advance our understanding of glycan biosynthesis and its requirement for virulence. It will thereby set the stage for further studies of fundamental glycobiology, cryptococcal biology and pathogenesis, and potential antifungal agent discovery.