Laccase Regulation and Virulence in Cryptococcus

描述(申请人提供)：新生隐球菌是免疫活性和免疫功能受损患者的主要病原体，包括发达国家和发展中国家的艾滋病患者。我们的长期目标是验证毒力因子漆酶的分子调节器影响新生隐球菌毒力的假设。目前提出的假设是，插入突变鉴定出的毒力相关的DEAD-box蛋白Vad1是漆酶和新城疫霉菌毒力的重要调节因子。这是基于以下观察所得。首先，在小鼠模型中，VAD1的缺失会导致毒力丧失，并加速新生葡萄球菌从肺中的清除。其次，差异显示表明，除漆酶外，VAD1的缺失还会导致一些基因转录的改变。最后，尽管保留了漆酶活性，但缺失显示VAD1依赖转录的基因之一PCK1在小鼠模型中显示出减弱的毒力。在特定目标1中，我们将评估Vad1与DEXD/H盒蛋白RCK/p54蛋白亚家族中Vad1的功能保守性。我们计划通过在酵母的RCK/p54突变体中过表达Vad1来测试这一点，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，并对表达亲和力标记的Vad1结构的细胞提取液进行凝胶过滤，以识别和表征可能的Vad1多蛋白复合体。在特定的目标2中，我们将进一步确定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介导的转录抑制因子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。具体地说，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注射小鼠模型评估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在新生隐球菌病理学中的作用提供一种综合的方法，并可能为治疗和预防隐球菌病提供更多的药物开发靶点。

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.