Virulence Factor Trafficking in Cryptococcus

描述(申请人提供)：新生隐球菌是一种主要 艾滋病患者致命性脑膜脑炎的病原体调查 大量HIV+的个人。此外，处于典型风险的群体 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人数在持续上升。我们之前的研究 还有一些研究表明，依赖漆酶的黑色素之间存在强烈的联系 新生隐孢子菌的体外生产及其毒力测定 实验动物。我们已经证明了漆酶是一种与细胞壁相关的 毒力因子通过多条证据：1)通过以下方式定位 使用抗漆酶单抗的免疫电子显微镜，2) 绿色荧光蛋白标记重组体的外周定位 漆酶的共聚焦显微镜研究，3)酶活性和漆酶的定位 新生隐球菌细胞壁免疫反应蛋白制剂。我们还有 开发了一种插入突变的方法，并分离出了许多 漆酶缺失突变体。这项提案的总体目标是测试 假设漆酶需要准确的细胞壁靶向 新生隐球菌的毒力。详细了解 与毒力相关的贩运可能会允许识别其他小说 毒力因子共享这些相同的途径，并可能使 确定独特的人口贩运抑制物，破坏其功能 单一毒剂的多种毒力因子。在具体目标1中，我们将 鉴定漆酶中靶向特异的氨基酸序列。我们将使用 共聚焦显微镜比较绿色表达的细胞定位 融合完全相同的新生隐形杆菌荧光蛋白标记漆酶 含有可能的N端空泡靶向缺失的蛋白质 序列E25SPT或C端包被网状蛋白的PIT序列Y426GFNNI。 额外的缺失将在漆酶N-末端和 以确定其他靶向序列是否可以 一定要在场。在具体目标2中，我们将确定涉及的基因和抑制物 通过研究漆酶突变后的漆酶转运 Sentinel基因参与肌动蛋白依赖、网状蛋白依赖和 依赖液泡的胞膜蛋白非依赖性转运途径。在特定目标中3 我们将通过研究确定与漆酶运输有关的基因 漆酶分泌缺陷的漆酶缺失插入突变体 分泌物。然后，在特定目标2和3中确定的每个突变体将是 评估其他与毒力相关的因子的产生，如胶囊， 小鼠模型中的尿素酶、磷脂酶和毒性。

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.