Laccase Regulation and Virulence in Cryptococcus

描述（由申请人提供）：新型隐球菌是发达国家和发展中国家免疫功能正常和免疫功能低下患者（包括艾滋病患者）的主要病原体。我们的长期目标是测试毒力因子漆酶的分子调节因子影响新生隐球菌毒力的假设。本文提出的具体假设是，通过插入突变鉴定出的毒力相关DEAD-box蛋白Vad1是新生C.中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的缺失会导致毒力的丧失和新生梭状菌的加速清除。其次，差异显示显示，VAD1的缺失导致除漆酶外许多基因的转录发生改变。最后，在小鼠模型中，删除一个显示vad1依赖性转录的基因PCK1，尽管保留漆酶活性，但显示出减弱的毒力。在Specific Aim 1中，我们将评估VAD1在DEXD/H box蛋白的RCK/p54蛋白亚家族中的功能保护，其中VAD1具有显著的同源性。我们计划通过在酵母RCK/p54突变体中过表达VAD1来验证这一点，测试VAD1在单倍体结实和交配中的作用，比较VAD1蛋白与RCK/p54亚家族成员的细胞定位，并对表达亲和标记VAD1构建物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Specific Aim 2中，我们将进一步定义VAD1在漆酶转录中的作用。这将验证我们的假设，即VAD1是漆酶最佳表达所必需的。我们将分析VAD1介导的转录抑制因子NOT1的降解，以及NOT1在抑制漆酶活性中的作用。在Specific Aim 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，我们将测量每个菌株感染后真菌cfu、白细胞募集、细胞因子产生、抗体产生和DTH反应。在Specific Aim 4中，我们将通过气管内和静脉内小鼠模型评估5个m4d7依赖基因在隐球菌毒力中的作用。这些特定目标的完成将为理解VAD1在新生隐球菌病理生物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靶点。

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.