CRYPTOCOCCAL GLYCAN SYNTHESIS

摘要 新型隐球菌是一种毁灭性的机会致病菌， 每年的死亡人数，主要是在发展中国家。这种生物体的聚糖结构包括细胞壁复合物， 隐球菌荚膜是一种主要的维鲁， lence因素。这些分子是隐球菌生物学和发病机制的核心，但仍存在重大空白 我们对它们合成途径的理解。我们缺乏这方面的基本知识，导致错过行动- 定义基础生物学和探索有针对性的干预措施的必要性。我们研究的长期目标是 确定胶囊和其他隐球菌聚糖的生物化学途径， 我们的基本认识，并改善这种毁灭性感染的结果。在本提案中，我们将 重点关注在真菌毒力中起作用的糖活性蛋白。我们将深入研究他们的生物- 化学功能和细胞环境，包括结合伴侣和亚细胞定位。同时，我们 我建议实施一种系统的方法来鉴定在聚糖合成中起中心作用的新蛋白质 和毒力，通过利用基因组序列，新的突变体库，以及 创新的生化测定。我们提出的研究，以确定生物学功能和作用的发病机制 这些关键蛋白质中的一种从糖活性酶作为药物靶点的有效使用中获得动力，包括 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- jugates和隐球菌转运蛋白提供这些前体。在目标1中，我们将使用生物化学和 细胞生物学方法来确定底物，功能评估独特的结构域，定义结合部分， ners，并确定我们已经知道的影响隐球菌的糖活性蛋白的亚细胞定位， 毒性。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化。 转移酶基因然后，我们将使用聚糖分析来鉴定与毒力最相关的活性， 新的生化分析来表征蛋白质功能。 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。我们希望我们的生物化学和遗传学的结合 通过定义具有独特的致病微生物中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- 隐球菌聚糖合成的前景，并可能提出干预点，以促进抗真菌 疗法

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.