Role of glycan precursor transporters in Cryptococcus neoformans virulence

聚糖前体转运蛋白在新型隐球菌毒力中的作用

• 批准号: 9251645
• 负责人: Lucy Li
• 金额: $ 4.9万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-03-01 至 2019-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9251645
• 关键词: Acetylgalactosamine; Amaze; Anabolism; Antifungal Agents; Biochemical; Biological Assay; Biology; Cell Wall; Cells; Characteristics; Charge; Clinical; Complement; Cryptococcus neoformans; Cytidine Monophosphate N-Acetylneuraminic Acid; Cytosol; Data; Defect; Drug Targeting; Encapsulated; Exhibits; GDP-mannose transporter; Genes; Glycobiology; Glycoconjugates; Glycolipids; Glycoproteins; Goals; Golgi Apparatus; Growth; Guanosine Diphosphate Mannose; Homeostasis; Homologous Protein; Human; Immune response; In Vitro; Individual; Industrial fungicide; Infection; Intervention; Knowledge; Lipids; Mediating; Modification; Monosaccharides; Morbidity - disease rate; Mus; Organ; Pathogenesis; Pathway interactions; Phagocytes; Phagocytosis; Pharmaceutical Preparations; Phenotype; Polysaccharides; Process; Proteins; Radiolabeled; Regulation; Relapse; Role; Saccharomyces cerevisiae; Stress; Techniques; Temperature; Testing; Uridine Diphosphate Glucuronic Acid; Uridine Diphosphate Xylose; Virulence; Work; base; capsule; carbohydrate structure; design; drug discovery; experimental study; glycosylation; in vivo; insight; killings; macrophage; mortality; mutant; novel; pathogen; public health relevance; sugar; sugar nucleotide; therapeutic target

 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.

 描述(由申请人提供)：新生隐球菌是一种封装的机会性真菌病原体，每年感染100多万人，每年导致全球60多万人死亡。尽管进行了昂贵和有毒的抗真菌干预，但目前的治疗方法还不够充分，发病率、死亡率和复发率都很高。隐球菌多糖是真菌存活和致病的关键决定因素，使它们成为有吸引力的治疗靶点。包括多糖胶囊在内的大多数糖偶联物的活性供体分子都是核苷酸糖。这些高电荷分子通常是在胞浆中产生的，然后由核苷酸糖转运体(NST)运输到分泌途径，在那里大多数糖的生物合成发生。尽管它们在多糖合成中起着关键作用，但隐球菌NSTs的全套识别和调控仍然未知。我们知识中的这一重大差距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。这项建议的目的是确定三个新的核苷酸糖转运蛋白的功能，命名为NSTX，NSTH和NSTG，它们被鉴定为与已知的NSTs同源性，并与真菌毒力有关。这三个NSTs的缺失显著减少了包膜的合成，并导致了相当大的温度和应力敏感性。NSTX和nstH突变体在建立小鼠感染的能力方面存在严重缺陷，而且使这些转运蛋白成为有吸引力的药物发现的目标。在目标1中，我将使用两种互补的方法确定每一种NST的底物(S)。我将用半完整的隐球菌细胞直接检测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共轭组成，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。我将在体外检测巨噬细胞的吞噬和清除能力，并确定每个突变体在体内的感染动力学。这项工作将促进我们对多糖的生物合成及其毒力要求的理解。因此，它将为进一步研究基础糖生物学、隐球菌生物学和发病机制以及潜在的抗真菌药物发现奠定基础。