Filling gaps in the cryptococcal wall with glycogen and a novel enzyme

用糖原和一种新型酶填充隐球菌壁的间隙

• 批准号: 10648839
• 负责人: Tamara L Doering
• 金额: $ 23.4万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-01 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10648839
• 关键词: Address; Affect; Antifungal Agents; Antifungal Therapy; Area; Basic Science; Binding; Biochemical; Biochemical Pathway; Biological; Biological Assay; Biology; Candida; Carbohydrates; Cell Fraction; Cell Survival; Cell Wall; Cell membrane; Cell surface; Cells; Cessation of life; Characteristics; Chitin; Chitosan; Communities; Complex; Cryptococcus; Cryptococcus neoformans; Dangerousness; Developing Countries; Drug Targeting; Engineering; Environment; Enzymes; Future; Genes; Glucans; Glycogen; Goals; Growth; HIV; Hand; Human; Individual; Infection; Intervention; Investigation; Knowledge; Link; Location; Mammalian Cell; Mammals; Mannose; Measures; Microbe; Morbidity - disease rate; Nature; Organism; Pathogenesis; Phagocytes; Pharmaceutical Preparations; Phenotype; Physiological; Polymers; Polysaccharides; Process; Productivity; Proteins; Public Health; Reaction; Reporting; Research; Resistance; Role; Saccharomyces; Severity of illness; Stress; Structure; System; Testing; Traction; Virulence; Visit; Work; Yeast Model System; Yeasts; capsule; experimental study; extracellular; fascinate; flexibility; fungus; gene product; glycosyltransferase; improved outcome; insight; mutant; neglect; novel; pathogen; pathogenic fungus; patient population; protein purification; resilience; screening; synthetic enzyme

ABSTRACT Cryptococcus neoformans is a devastating opportunistic fungus that causes hundreds of thousands of deaths each year, mainly in developing countries. This pathogen is surrounded by a flexible wall that maintains cell integrity and anchors a protective polysaccharide capsule. The cell wall, which consists mainly of interlinked polysaccharides, is a compelling topic of study because it is required for viability, absent from the cells of mam- malian hosts, and a proven target for antifungal therapies. Almost a century ago, it was postulated that yeast walls contain glycogen, similar in structure to the intracellular storage molecule but linked to the cell wall itself. However, where this occurs in nature, as well as how this material is made and reaches the cell surface, have never been established, constituting major gaps in our knowledge of an essential structure. We recently discov- ered that a previously unstudied cryptococcal protein influences both glycogen synthesis and cell wall integrity, potentially providing a key to this area of research. This protein is also required for normal infection, suggesting a potential vulnerability in the wall that might be productively exploited. The long-term goal of our research is to define the biochemical pathways by which cryptococcal glycans are made, to advance our fundamental understanding and improve the outcome of this devastating infection. In this proposal we focus on the novel area of cell wall glycogen and an intriguing glycosyltransferase we have discov- ered, which we call GTX. In Aim 1 we will isolate and quantitate cell wall glycogen from C. neoformans grown in various environments, including host-like conditions, and determine how this material fits into the complex mesh of the cell wall. In Aim 2 we will use biochemical assays to define and characterize the activity of purified GTX, which we already have in hand. In Aim 3 we will assess the phenotypes and virulence characteristics of mutants lacking GTX. We will also localize this protein and identify its interacting partners. These studies will be enabled by our expertise in cryptococcal biology and advice from expert colleagues, which we will integrate into thoughtful and rigorous studies. Together, these experiments will define a new component of the cryptococcal cell wall and determine the activity of a novel protein implicated in pathogenesis. The major antifungal drugs that target cell walls are not effective against C. neoformans. It is therefore critical to find other vulnerabilities in the cryptococcal wall that can be productively exploited – our proposed studies are a focused step on this path. Completing them will advance our understanding of fungal cell wall construction, define a novel glycan synthetic process, and potentially suggest a point of intervention for antifungal therapy.

抽象的 新型隐球菌是一种毁灭性的机会性真菌，可导致数十万人死亡 每年，主要在发展中国家，这种病原体被维持细胞的柔性壁包围。 完整性并锚定保护性多糖胶囊，其主要由相互连接的细胞壁组成。 多糖是一个引人注目的研究课题，因为它是生存所必需的，而哺乳动物细胞中却没有它。 大约一个世纪前，人们就假设酵母是马里的宿主，并且是抗真菌疗法的经过验证的目标。 细胞壁含有糖原，其结构与细胞内储存分子相似，但与细胞壁本身相连。 然而，这种现象在自然界中发生的地点，以及这种材料是如何制造并到达细胞表面的，目前还不清楚。 从未建立过，这构成了我们对基本结构的认识的重大空白。 发现以前未经研究的隐球菌蛋白会影响糖原合成和细胞壁完整性， 可能为该领域的研究提供关键，这表明这种蛋白质也是正常感染所必需的。 隔离墙中可能被有效利用的潜在漏洞。 我们研究的长期目标是确定隐球菌聚糖的生化途径 旨在增进我们对这种毁灭性感染的基本认识并改善其结果。 建议我们重点研究细胞壁糖原的新领域和我们发现的有趣的糖基转移酶- ered，我们称之为 GTX。在目标 1 中，我们将从生长于其中的新型隐球菌中分离和定量细胞壁糖原。 各种环境，包括类似宿主的条件，并确定该材料如何适应复杂的网格 在目标 2 中，我们将使用生化物质来定义和表征分析纯化的 GTX 的活性， 在目标 3 中，我们将评估突变体的表型和毒力特征。 我们还将定位这种蛋白质的相互作用并确定其合作伙伴。 通过我们在隐球菌生物学方面的专业知识和专家同事的建议，我们将把它们融入深思熟虑的 和严格的研究一起，这些实验将定义隐球菌细胞壁的新组成部分和 确定与发病机制有关的新型蛋白质的活性。 主要针对细胞壁的抗真菌药物对新型隐球菌无效。 找到可以有效利用的隐球菌墙中的其他漏洞——我们提出的研究是 完成它们将加深我们对真菌细胞壁结构的理解， 定义了一种新的聚糖合成过程，并可能提出抗真菌治疗的干预点。