CRYPTOCOCCAL GLYCAN SYNTHESIS

隐球菌聚糖合成

• 批准号: 10153676
• 负责人: Tamara L Doering
• 金额: $ 49.09万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-06-01 至 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10153676
• 关键词: Antifungal Agents; Antifungal Therapy; Antigens; Area; Basic Science; Binding; Biochemical; Biochemical Genetics; Biochemical Pathway; Biological; Biological Assay; Biological Process; Biology; Carbohydrates; Carrier Proteins; Cell Wall; Cells; Cessation of life; Characteristics; Clinical; Complex; Cryptococcus; Cryptococcus neoformans; Data; Developing Countries; Development; Disease; Drug Targeting; Engineering; Ensure; Enzymes; Exhibits; Future; Genome; Genomics; Glycobiology; Glycoconjugates; Glycolipids; Glycoproteins; Glycosyltransferase Gene; Goals; Growth; Human; Image; Immune response; Infection; Intervention; Knowledge; Libraries; Maintenance; Masks; Metabolic; Methods; Modeling; Molecular; Mus; Mycoses; Organism; Pathogenesis; Pathway interactions; Play; Polysaccharides; Process; Productivity; Proteins; Proteomics; Public Health; Reaction; Research; Role; Signal Transduction; Site; Structure; System; Testing; Therapeutic; Transferase; Virulence; Virulence Factors; Work; Yeast Model System; base; capsule; cryptococcal polysaccharide; design; enzyme activity; experimental study; fungus; genetic approach; glycosylation; glycosyltransferase; improved outcome; insight; macromolecule; mutant; novel; novel strategies; opportunistic pathogen; pathogen; pathogenic microbe; protein function; sugar; sugar nucleotide; synthetic enzyme

ABSTRACT Cryptococcus neoformans is a devastating opportunistic pathogen that causes hundreds of thousands of deaths each year, mainly in developing countries. The glycan structures of this organism include cell wall com- ponents, glycoproteins, glycolipids, and polysaccharides of the cryptococcal capsule, which is a major viru- lence factor. These molecules are central to cryptococcal biology and pathogenesis, yet there are major gaps in our understanding of their synthetic pathways. We lack basic knowledge in this area, leading to missed op- portunities to define basic biology and explore targeted intervention. The long-term goal of our research is to determine the biochemical pathways by which capsule and other cryptococcal glycans are made, to advance our fundamental understanding and improve the outcome of this devastating infection. In this proposal we will focus on glycoactive proteins that play roles in fungal virulence. We will perform in-depth studies of their bio- chemical functions and cellular context, including binding partners and subcellular localization. In parallel we propose to implement a systematic approach for identifying novel proteins that are central in glycan synthesis and virulence, shifting current practice by taking advantage of genome sequence, new mutant libraries, and in- novative biochemical assays. Our proposed studies to define the biological functions and roles in pathogenesis of these key proteins gains impetus from the validated use of glycoactive enzymes as drug targets, including in the context of fungal infections. We propose to study glycosyltransferase enzymes that use sugars from activated precursors to build glycocon- jugates and cryptococccal transporters that provide those precursors. In Aim 1 we will use biochemical and cell biological approaches to determine substrates, functionally assess unique domains, define binding part- ners, and determine subcellular localization of glycoactive proteins that we already know influence cryptococ- cal virulence. In Aim 2 we will test deletion strains in mouse infection models to prioritize potential glycosyl- transferase genes. We will then use glycan analysis to identify the activities most implicated in virulence and new biochemical assays to characterize protein function. Throughout the project we will design and conduct well-controlled experiments and interpret them rigorously, in the context of our expertise in cryptococcal biology. We expect our combination of biochemical and genetic approaches to advance the field by defining fundamental glycobiology in a pathogenic microbe with unique glycans. Completing these studies will fill major gaps in our understanding of fungal biology, define critical as- pects of cryptococcal glycan synthesis, and potentially suggest points of intervention to advance antifungal therapy.

抽象的 加密赛车是一种破坏性的机会性病原体，可导致数十万个 每年死亡，主要是发展中国家。该生物的聚糖结构包括细胞壁com- 加密摄氏胶囊的碳，糖蛋白，糖胶质和多糖，这是主要的毒素 透镜因子。这些分子对于加密局球生物学和发病机理至关重要，但是存在主要的差距 在我们对它们的合成途径的理解中。我们在这一领域缺乏基本知识，导致错过 定义基本生物学并探索有针对性的干预措施的港口。我们研究的长期目标是 确定制造胶囊和其他隐球菌的生化途径，以促进 我们的基本理解并改善了这种毁灭性感染的结果。在这个建议中，我们将 专注于在真菌毒力中扮演角色的糖活性蛋白。我们将对他们的生物进行深入研究 化学功能和细胞环境，包括结合伴侣和亚细胞定位。并行 建议实施一种系统的方法来识别在聚糖合成中核心的新型蛋白质 和毒力，通过利用基因组序列，新的突变图库和内部来改变当前的实践 新的生化测定。我们提出的研究定义生物学功能和发病机理的作用 在这些关键蛋白中，从糖活性酶作为药物靶标的验证的使用中获得了动力 真菌感染的背景。 我们建议研究使用活化前体的糖来构建糖基 - 糖基转移酶 提供这些前体的搭配和加密cccal转运蛋白。在AIM 1中，我们将使用生化和 细胞生物学方法来确定底物，在功能上评估独特域，定义结合部分 - 并确定我们已经知道会影响加密蛋白的糖活性蛋白的亚细胞定位 毒力。在AIM 2中，我们将测试小鼠感染模型中的缺失菌株，以优先考虑潜在的糖基 - 转移酶基因。然后，我们将使用聚糖分析来确定与毒力和毒力相关的活动 新的生化分析以表征蛋白质功能。 在整个项目中，我们将设计和进行控制良好的实验，并严格解释它们 我们在隐球菌生物学方面的专业知识的背景。我们期望我们的生化和遗传结合 通过在具有独特的致病微生物中定义基本糖生物学来推进现场的方法 聚糖。完成这些研究将填补我们对真菌生物学的理解的主要空白，定义关键的AS- 隐球菌聚糖合成的果皮，并有可能提出干预点以促进抗真菌 治疗。