Laccase Regulation and Virulence in Cryptococcus

新型隐球菌是免疫功能正常和免疫功能低下患者的主要病原体， 无论是在发达国家还是发展中国家，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说。 本建议背后的具体假设是，鉴定出与毒性相关的死亡盒蛋白Vad 1， 通过插入突变，是一个重要的漆酶和毒力的调节因子。新人类这是基于 以下意见。首先，VAD 1的缺失导致C.新生 从小鼠模型的肺中分离出来。第二，差异显示表明，VAD 1的缺失导致转录改变 除了漆酶之外还有很多基因。最后，缺失显示VAD 1依赖性的基因之一， 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 目的1：研究VAD 1在DEXD/H box的RCK/p54蛋白亚家族中的功能保守性 与Vad 1具有显著同源性的蛋白质。我们计划通过在RCK/p54细胞中过表达VAD 1来验证这一点。 突变体的细胞定位，检测VAD 1在单倍体结实和交配中的作用，比较VAD 1在单倍体结实和交配中的细胞定位 蛋白质与RCK/p54亚家族成员的结合，并对表达RCK/p54亚家族成员的细胞的细胞提取物进行凝胶过滤。 亲和标记的Vad 1构建体，以鉴定和表征可能的Vad 1多蛋白复合物。在具体目标2中， 我们将进一步明确VAD 1在漆酶转录中的作用。这将检验我们的假设，即VAD 1是 漆酶的优化表达。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，我们将分析 野生型和Avadl感染。具体来说，我们将测量真菌cfu，白细胞 各菌株感染后的募集、细胞因子产生、抗体产生和DTH应答。在特定 目的4，我们将使用一种内分泌和免疫荧光技术评估5个M4 D 7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个综合的方法来理解 VAD 1在C.并可能提供额外的药物开发目标， 隐球菌病的治疗和预防。

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.