CRYPTOCOCCAL GLYCAN SYNTHESIS

摘要 新生隐球菌是一种毁灭性的机会性病原体，导致数十万人 每年死亡人数最多，主要发生在发展中国家。这种生物体的糖链结构包括细胞壁复合体-- 隐球菌胶囊的主要成分、糖蛋白、糖脂和多糖是一种主要的病毒。 伦斯因数。这些分子是隐球菌生物学和发病机制的核心，但仍存在重大差距。 在我们对它们合成途径的理解上。我们缺乏这方面的基本知识，导致错过了行动- 定义基础生物学和探索有针对性的干预措施的机会。我们研究的长期目标是 确定制造胶囊和其他隐球菌多糖的生化途径，以促进 我们对这一毁灭性感染的基本认识和改善结果。在本提案中，我们将 重点研究在真菌毒力中起作用的糖活性蛋白。我们将深入研究它们的生物-- 化学功能和细胞环境，包括结合伙伴和亚细胞定位。同时，我们 建议实施一种系统的方法来识别在多糖合成中起核心作用的新蛋白质 和毒力，改变目前的做法，利用基因组序列，新的突变文库和在- 创新的生化分析。我们建议的研究以确定在发病机制中的生物学功能和作用 这些关键蛋白的动力来自糖活性酶作为药物靶点的有效使用，包括在 真菌感染的背景。 我们建议研究糖基转移酶，这种酶利用活化的前体中的糖来构建糖蛋白- 提供这些前体的酸枣仁和隐球菌转运蛋白。在目标1中，我们将使用生化和 细胞生物学方法来确定底物，从功能上评估独特的结构域，定义结合部分- Ners，并确定我们已知的影响隐球菌糖活性蛋白的亚细胞定位。 加州的毒力。在目标2中，我们将在小鼠感染模型中测试缺失菌株，以确定潜在的糖基- 转移酶基因。然后我们将使用葡聚糖分析来确定与毒力和 用于表征蛋白质功能的新生化分析方法。 在整个项目中，我们将设计和进行受控良好的实验，并严格解释它们，在 我们在隐球菌生物学方面的专业知识。我们希望我们的生化和遗传结合 通过定义一种独特致病微生物的基本糖生物学来推进这一领域的方法 多聚糖。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- 隐球菌多糖合成的前景，并潜在地提出了促进抗真菌的干预点 心理治疗。

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.