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.

摘要 新生隐球菌是一种毁灭性的机会性真菌，导致数十万人死亡。 每年，主要是在发展中国家。这种病原体被一层柔韧的壁所包围，以维持细胞 完整，并锚定一个保护性的多糖胶囊。细胞壁，主要由相互连接的 多糖，是一个引人注目的研究主题，因为它是生存所必需的，在主母细胞中不存在。 马里是东道主，也是公认的抗真菌疗法的目标。大约一个世纪前，人们认为酵母 细胞壁含有糖原，其结构类似于细胞内的存储分子，但与细胞壁本身相连。 然而，在自然界中发生这种情况的地方，以及这种材料是如何制成并到达细胞表面的，都有 从来没有建立过，这构成了我们对基本结构的认识的重大空白。我们最近发现了- 一种以前从未研究过的隐球菌蛋白既影响糖原合成，也影响细胞壁完整性， 潜在地为这一研究领域提供了一把钥匙。这种蛋白质也是正常感染所必需的，提示 墙中的一个潜在漏洞，可能会被有效地利用。 我们研究的长期目标是确定隐球菌多糖通过哪些生化途径 以促进我们对这一毁灭性感染的基本认识和改善结果。在这 建议我们专注于细胞壁糖原的新领域和我们发现的一种有趣的糖基转移酶-- ERD，我们称之为GTX。在目标1中，我们将分离和定量新生隐球菌胞壁糖原。 各种环境，包括类似宿主的条件，并确定该材质如何适应复杂的网格 在细胞壁上。在目标2中，我们将使用生化分析来定义和表征纯化的GTX的活性， 我们手中已经有了。在目标3中，我们将评估突变体的表型和毒力特征。 缺乏GTX。我们还将对该蛋白质进行定位，并确定其相互作用伙伴。这些研究将被启用 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，我们将把这些建议整合到深思熟虑的 和严谨的研究。这些实验将共同定义一种新的隐球菌细胞壁成分，并 确定一种与发病机制有关的新蛋白质的活性。 针对细胞壁的主要抗真菌药物对新生葡萄球菌无效。因此，至关重要的是 在隐球菌墙中找到可以有效利用的其他漏洞-我们建议的研究是 在这条道路上有重点的一步。完成它们将促进我们对真菌细胞壁结构的理解， 定义了一种新的多糖合成过程，并潜在地提出了抗真菌治疗的干预点。