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。我们的长期目标是检验毒力因子漆酶的分子调节因子影响新生隐球菌毒力的假说。本研究提出的假设是，通过插入突变鉴定出的一种与毒性相关的DEAD盒蛋白Vad 1是C.新人类这是基于以下意见。首先，VAD 1的缺失导致C.小鼠模型中的肺新生儿。第二，差异显示表明，VAD 1的缺失导致除了漆酶之外的许多基因的转录改变。最后，删除的基因之一，VAD 1依赖的转录，PCK 1，在小鼠模型中表现出减弱的毒力，尽管保留漆酶活性。在特定目标1中，我们将评估VAD 1在DEXD/H盒蛋白的RCK/p54蛋白亚家族中的功能保守性，其中VAD 1与DEXD/H盒蛋白具有显著的同源性。我们计划通过在酵母的RCK/p54突变体中过表达VAD 1来测试这一点，测试VAD 1在单倍体结实和交配中的作用，比较VAD 1蛋白与RCK/p54亚家族成员的细胞定位，并对表达亲和标记的VAD 1构建体的细胞提取物进行凝胶过滤，以识别和表征可能的VAD 1多蛋白复合物。在具体目标2中，我们将进一步确定VAD 1在漆酶转录中的作用。这将验证我们的假设，即VAD 1是漆酶的最佳表达所必需的。我们将分析VAD 1介导的转录抑制因子NOT 1的降解，以及NOT 1在抑制漆酶活性中的作用。在具体目标3中，我们将使用内分泌模型分析野生型和delta-vadl感染之间的宿主反应。具体而言，我们将测量真菌cfu，白细胞募集，细胞因子的产生，抗体的产生和DTH反应后，感染每种菌株。在具体目标4中，我们将使用肠道内和静脉内小鼠模型评估5个M4 D 7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VAD 1在C.新型隐球菌，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.