Laccase Regulation and Virulence in Cryptococcus

新生隐球菌是免疫功能低下患者的主要病原体，包括 无论是发达国家还是发展中国家的艾滋病患者。我们的长期目标是测试 假设毒力因子漆酶的分子调节器影响新生隐球菌的毒力。 目前这项提议背后的具体假设是，确定了一种与毒力相关的死盒蛋白Vad1 通过插入突变，是漆酶活性和致病力的重要调节因子。这是基于 以下是观察到的。首先，VAD1基因的缺失会导致新生假单胞菌毒力丧失和加速清除 在小鼠模型中从肺中提取。第二，差异显示表明VAD1的缺失会导致转录改变 除了漆酶外，还有许多基因。最后，显示VAD1依赖的基因之一的缺失 转录，PCK1，在小鼠模型中显示出减弱的毒力，尽管保留了漆酶活性。具体而言 目的1，我们将评估DXD/H box的RCK/p54蛋白亚家族中VAD1的功能保守性 Vad1与之有显著同源性的蛋白质。我们计划通过在RCK/p54中过表达VAD1来测试这一点 酵母突变体，检测Vad1在单倍体结实和交配中的作用，比较Vad1的细胞定位 RCK/p54亚家族成员的蛋白质，并对表达基因的细胞提取液进行凝胶过滤 亲和力标记的Vad1构建用于识别和表征可能的Vad1多蛋白复合体。在具体目标2中， 我们将进一步明确VAD1在漆酶转录中的作用。这将测试我们的假设，即VAD1需要 漆酶的优化表达。我们将分析VAD1介导的转录抑制因子NOT1的降解， Not1在抑制漆酶活性中的作用。在具体目标3中，我们将分析主机在 使用气管内模型的野生型和avadl感染。具体地说，我们将测量真菌CFU、白细胞 每种菌株感染后的募集、细胞因子产生、抗体产生和迟发型变态反应。具体而言 目的4，我们将评估5个M4D7依赖基因在隐球菌毒力中的作用。 静脉注射小鼠模型。完成这些具体目标将提供一种综合的方法来理解 VAD1在新生隐球菌病理学中的作用，可能为临床提供更多的药物开发靶点 治疗和预防隐球菌病。

Cryptococcus neoformans is a major pathogen in immunocompetant 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 affects 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 VAD1results in loss of virulence and accelerated clearance of C. neoformans from lung in mouse models. Second, differential display has shown that deletion of VAD1results 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 VAD1within the RCK/p54 protein subfamily of DEXD/H box proteins to which Vad1 exhibits significant homology. We plan to test this by overexpression of VAD1in an RCK/p54 mutant of yeast, testing the role of VAD1in 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 VAD1in laccase transcription. This will test our hypothesis that VAD1is required for optimal expression of laccase. We will analyze VAD1-mediated degradation of the transcriptional represser, 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 Avadl infections using an intratracheal model. Specifically, we will measure fungal cfu, leukocyte recruitment, cytokine production, antibody production and DTH response after infection with each strains. 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 VAD1in the pathobiology of C. neoformans and may provide additional drug development targetsfor the treatment and prevention of cryptococcosis.