Virulence Factor Trafficking in Cryptococcus

隐球菌毒力因子贩运

• 批准号: 6925206
• 负责人: Peter Richard Williamson
• 金额: $ 4.51万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-04-01 至 2006-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6925206
• 关键词: Cryptococcus neoformans; cell capsule; cell wall; chimeric proteins; clathrin; confocal scanning microscopy; fungal genetics; gene mutation; green fluorescent proteins; immunoelectron microscopy; intracellular transport; laboratory mouse; miscellaneous oxidoreductase; protein localization; protein sequence; protein transport; urease; virulence

DESCRIPTION (provided by applicant): Cryptococcus neoformans is a major pathogen in AIDS patients causing life-threatening meningoencephalitis in a large number of HIV+ individuals. Furthermore, groups classically at risk for cryptococcosis, the elderly, transplant recipients and those immunosuppressed with steroids or chemotherapy, are continually rising. Previous studies by us and others have shown a strong association between laccase-dependent melanin production by C. neoformans in vitro and virulence of the organism in experimental animals. We have shown that laccase is a cell wall-associated virulence factor by multiple lines of evidence: 1) localization by immuno-electron microscopy using an anti-laccase monoclonal antibody, 2) localization to the periphery of a green-fluorescent protein-tagged recombinant laccase by confocal microscopy, 3) localization of enzyme activity and laccase immunoreactive protein to cell wall preparations of C. neoformans. We have also developed a method of insertional mutagenesis and have isolated numerous laccase-deficient mutants. The overall objective of this proposal is to test the hypothesis that accurate cell wall targeting of laccase is required for the virulence of Cryptococcus neoformans. Detailed understanding of virulence-related trafficking may allow the identification of other novel virulence factors which share these same pathways and may allow the identification of unique trafficking inhibitors that disrupt the function of multiple virulence factors with a single agent. In Specific Aim 1, we will identify targeting-specific amino acid sequences in laccase. We will use confocal microscopy to compare the cellular localization of expressed green fluorescent protein-tagged laccase in C. neoformans with an identical fusion protein containing deletions of the putative N-terminal vacuolar targeting sequence E25SPT or C-terminal clathrin-coated pit sequence Y426GFNNI. Additional deletions will be performed within the laccase N-terminus and C-terminus of the fusion protein to determine if other targeting sequences may be present. In Specific Aim 2, we will identify genes and inhibitors involved in laccase trafficking by studying laccase trafficking after mutation of sentinel genes involved in actin-dependent, clathrin-dependent and vacuolar-dependent clathrin-independent trafficking pathways. In Specific Aim 3 we will identify genes involved in laccase trafficking by studying laccase-deficient insertional mutants which show secretory defects in laccase secretion. Each of the mutants identified in Specific Aims 2 and 3 will then be assessed for production of other virulence-related factors such as capsule, urease, phospholipase and virulence in a mouse model.

描述（由申请方提供）：新型隐球菌是一种主要的 艾滋病患者中的病原体导致危及生命的脑膜脑炎， 大量的HIV+患者。此外，传统上有风险的群体 隐球菌病，老年人，移植受者和免疫抑制者 使用类固醇或化疗的患者人数不断上升。我们以往研究所 其他研究表明，依赖漆酶的黑色素 生产C。体外新形虫和体内微生物毒力 实验动物我们已经证明，漆酶是一种细胞壁相关的 毒力因子的多线证据：1）定位， 使用抗漆酶单克隆抗体的免疫电子显微术，2） 定位于绿色荧光蛋白标记的重组体的外周 用共聚焦显微镜观察漆酶; 3）酶活性和漆酶定位 免疫反应蛋白对C.新人类我们还 开发了一种插入诱变的方法，并分离出许多 漆酶缺陷突变体。本提案的总体目标是测试 假设漆酶的准确细胞壁靶向是必需的， 新型隐球菌的毒力。详细了解 与毒品有关的贩运可能有助于查明其他新的 毒力因子共享这些相同的途径，并可能允许 鉴定破坏生物体功能的独特贩运抑制剂， 多个毒力因子与一个单一的代理。具体目标1： 鉴定漆酶中靶向特异性氨基酸序列。我们将使用 共聚焦显微镜比较表达的绿色的细胞定位 荧光蛋白标记的C.漆酶具有相同融合的新型人 含有假定的N-末端空泡靶向缺失的蛋白质 序列E25 SPT或C-末端网格蛋白包被的小坑序列Y 426 GFNNI。 将在漆酶N-末端内进行另外的缺失， 融合蛋白的C-末端，以确定其它靶向序列是否可以 要在场在具体目标2中，我们将确定相关的基因和抑制剂 通过研究突变后的漆酶运输， 参与肌动蛋白依赖性，网格蛋白依赖性和 空泡依赖的网格蛋白独立的运输途径。具体目标3 我们将通过研究， 显示漆酶分泌缺陷的漆酶缺陷插入突变体 分泌物然后，将在特定目的2和3中鉴定的每种突变体， 评估其他毒性相关因素的产生，如胶囊， 尿素酶、磷脂酶和小鼠模型中的毒力。