CRYPTOCOCCAL GLYCAN SYNTHESIS

隐球菌聚糖合成

基本信息

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
项目类别：
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

项目摘要

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.

抽象的新型隐球菌是一种毁灭性的机会致病菌，可导致数十万每年都有死亡病例，主要发生在发展中国家。该生物体的聚糖结构包括细胞壁成分隐球菌荚膜的成分、糖蛋白、糖脂和多糖，隐球菌荚膜是一种主要病毒伦斯因子。这些分子对于隐球菌生物学和发病机制至关重要，但仍存在重大差距我们对它们的合成途径的理解。我们缺乏这方面的基础知识，导致错失良机定义基础生物学和探索有针对性的干预的机会。我们研究的长期目标是确定胶囊和其他隐球菌聚糖的生化途径，以推进我们对这一毁灭性感染的基本了解并改善其结果。在本提案中，我们将重点关注在真菌毒力中发挥作用的糖活性蛋白。我们将对其生物进行深入研究化学功能和细胞环境，包括结合伙伴和亚细胞定位。与此同时，我们提议实施一种系统方法来识别聚糖合成中的核心蛋白质和毒力，通过利用基因组序列、新的突变体文库和in- 创新的生化测定。我们提出的研究旨在定义发病机制中的生物学功能和作用这些关键蛋白质中的一些从糖活性酶作为药物靶标的有效使用中获得了推动力，包括在真菌感染的背景。我们建议研究糖基转移酶，它利用活化前体中的糖来构建糖基提供这些前体的jugates和隐球菌转运蛋白。在目标 1 中，我们将使用生化和细胞生物学方法确定底物，功能评估独特的结构域，定义结合部分 ners，并确定我们已知影响隐球菌的糖活性蛋白的亚细胞定位卡尔毒力。在目标 2 中，我们将在小鼠感染模型中测试缺失菌株，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识别与毒力和毒力最相关的活动表征蛋白质功能的新生化测定。在整个项目中，我们将设计和进行良好控制的实验，并严格解释它们，我们在隐球菌生物学方面的专业知识背景。我们期望生化和遗传的结合通过定义具有独特特征的病原微生物的基本糖生物学来推进该领域的方法聚糖。完成这些研究将填补我们对真菌生物学理解的主要空白，将关键定义为：隐球菌聚糖合成的影响，并可能提出促进抗真菌治疗的干预点治疗。