Laccase Regulation and Virulence in Cryptococcus

隐球菌中的漆酶调节和毒力

• 批准号: 7022990
• 负责人: Peter Richard Williamson
• 金额: $ 26.49万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-07-01 至 2010-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7022990
• 关键词: Cryptococcus neoformans; fungal genetics; gel mobility shift assay; gene expression; genetic enhancer element; genetic regulation; laboratory mouse; miscellaneous oxidoreductase; molecular cloning; posttranslational modifications; temperature; transcription factor; virulence

DESCRIPTION (provided by applicant): Cryptococcus neoformans is a major pathogen in immunocompetent as well as immunocompromised patients including those with AIDS in both the developed as well as the developing world. Our long-term objective is to test the hypothesis that molecular regulators of the virulence factor laccase affect the virulence of Cryptococcus neoformans. The specific hypothesis behind the present proposal is that a virulence associated DEAD-box protein, Vad1, identified by insertional mutagenesis, is an important regulator of laccase and virulence in C. neoformans. This is based on the following observations. First, deletion of VAD1 results in loss of virulence and accelerated clearance of C. neoformans from lung in mouse models. Second, differential display has shown that deletion of VAD1 results in altered transcription of a number of genes in addition to laccase. Finally, deletion of one of the genes showing VAD1-dependent transcription, PCK1, exhibited attenuated virulence in a mouse model in spite of retained laccase activity. In Specific Aim 1, we will assess for functional conservation of VAD1 within the RCK/p54 protein subfamily of DEXD/H box proteins to which Vad1 exhibits significant homology. We plan to test this by overexpression of VAD1 in an RCK/p54 mutant of yeast, testing the role of VAD1 in haploid fruiting and mating, comparing the cellular localization of the Vad1 protein to members of the RCK/p54 subfamily and performing gel filtration of cellular extracts of cells expressing affinity-tagged Vad1 constructs to identify and characterize possible Vad1 multi-protein complexes. In Specific Aim 2, we will further define the role of VAD1 in laccase transcription. This will test our hypothesis that VAD1 is required for optimal expression of laccase. We will analyze VAD1 -mediated degradation of the transcriptional repressor, NOT1, and the role of NOT1 in suppression of laccase activity. In Specific Aim 3, we will analyze the host responses between wild-type and delta-vadl infections using an intratracheal model. Specifically, we will measure fungal cfu, leukocyte recruitment, cytokine production, antibody production and DTH response after infection with each strain. In Specific Aim 4, we will assess the role of 5 M4D7-dependent genes in cryptococcal virulence using an intratracheal and an intravenous mouse model. Completion of these specific aims will provide an integrated approach to understanding the role of VAD1 in the pathobiology of C. neoformans and may provide additional drug development targets for the treatment and prevention of cryptococcosis.

描述（由申请人提供）：新型隐球菌是免疫功能正常和免疫功能低下的患者（包括发达国家和发展中国家的艾滋病患者）的主要病原体。我们的长期目标是检验毒力因子漆酶的分子调节剂影响新型隐球菌毒力的假设。本提议背后的具体假设是，通过插入诱变鉴定出的毒力相关的 DEAD-box 蛋白 Vad1 是新型隐球菌漆酶和毒力的重要调节因子。这是基于以下观察。首先，在小鼠模型中，VAD1 的缺失导致毒力丧失并加速新型隐球菌从肺部的清除。其次，差异显示表明，除漆酶外，VAD1 的缺失还会导致许多基因的转录发生改变。最后，删除显示 VAD1 依赖性转录的基因之一 PCK1，尽管保留了漆酶活性，但在小鼠模型中表现出毒力减弱。在具体目标 1 中，我们将评估 DEXD/H 盒蛋白的 RCK/p54 蛋白亚家族中 VAD1 的功能保守性，Vad1 与该蛋白表现出显着的同源性。我们计划通过在酵母 RCK/p54 突变体中过表达 VAD1 来测试这一点，测试 VAD1 在单倍体子实体和交配中的作用，将 Vad1 蛋白的细胞定位与 RCK/p54 亚家族成员进行比较，并对表达亲和标记 Vad1 构建体的细胞提取物进行凝胶过滤，以鉴定和表征可能的 Vad1 多蛋白 复合物。在具体目标 2 中，我们将进一步定义 VAD1 在漆酶转录中的作用。这将检验我们的假设，即漆酶的最佳表达需要 VAD1。我们将分析 VAD1 介导的转录阻遏蛋白 NOT1 的降解，以及 NOT1 在抑制漆酶活性中的作用。在具体目标 3 中，我们将使用气管内模型分析野生型和 delta-vadl 感染之间的宿主反应。具体来说，我们将测量每种菌株感染后的真菌 cfu、白细胞募集、细胞因子产生、抗体产生和 DTH 反应。在具体目标 4 中，我们将使用气管内和静脉内小鼠模型评估 5 个 M4D7 依赖性基因在隐球菌毒力中的作用。这些具体目标的完成将为理解 VAD1 在新型隐球菌病理学中的作用提供一种综合方法，并可能为治疗和预防隐球菌病提供额外的药物开发目标。