Pathobiology of C. neoformans in the Central Nervous System

描述(申请人提供)：新生隐球菌是人类最常见的威胁生命的中枢神经系统感染之一，尽管有现有的治疗方法，但发病率和死亡率仍然很高。最近在不列颠哥伦比亚省温哥华岛爆发的一次疫情表明，隐球菌株已经迅速进化，产生了更强的毒力株。这项建议的重点是使用分子技术来鉴定、表征和验证新生葡萄球菌中的基因，这些基因对这种被包裹的酵母的毒力组合非常重要。 我们的主要假设是基于这样一个简单的假设，即在一定的环境压力下，新生葡萄球菌菌株将调节它们产生蛋白质的遗传网络/途径，使它们能够在宿主的恶劣环境中生存和生长。这项提案详细介绍了一项调查计划，该计划允许利用新的技术进步进行高度整合但全球性的转录图谱筛选，以预测隐球菌病理生物学中的薄弱环节。我们的实验室在过去几年中已经验证了这一总体策略可以成功使用。我们使用了cDNA消减技术、差异显示RT-PCR技术、体内表达技术、基因表达序列分析(SAGE)，以及最近的微阵列技术，成功地开始鉴定使新生葡萄球菌成为一种病原体的基因。通过我们的研究，我们已经确定了以下与毒力组合有关的基因和途径：1)编码酶的基因；2)氧化胁迫基因；3)信号通路基因；4)高温生长基因；5)线粒体和呼吸作用的影响；6)减弱的零突变体对宿主免疫的影响。这一提议的一个基石是利用微阵列收集大量数据的能力，同时试图通过使用特定的体外和体内条件将我们的理解集中在相关的基因和网络上。这一筛选设计将通过在相关动物模型中创建用于表型分析的零突变体，仔细地与功能研究相联系。我们的总体科学计划是了解毒力复合体的遗传调节，这将包含对发现干扰发病机制的基因靶点的强大洞察力，并导致新的治疗策略，如抗真菌药物和疫苗。 项目简介该项目使用基因组策略来捕捉是什么使危及生命的新型隐球菌成为免疫受损个体的有效病原体。根据从这些研究中获得的知识，预计可以确定新的抗真菌靶点，以帮助开发针对这种常见真菌病原体的新药和/或疫苗，用于不断扩大的严重免疫受损人群。

DESCRIPTION (provided by applicant): Cryptococcus neoformans is one of the most common life-threatening central nervous system infections in humans and despite present treatments, morbidity and mortality still remain high. In a recent outbreak on Vancouver Island, British Columbia it was shown that cryptococcal strains have evolved rapidly to produce more virulent strains. The focus of this proposal is to use molecular techniques to identify, characterize, and validate genes in C. neoformans which are important to the virulence composite of this encapsulated yeast. Our primary hypothesis is based around the simple assumption that under certain environmental stresses, C. neoformans strains will regulate their genetic networks/pathways for production of proteins to allow them to survive and grow in the hostile environment of the host. This proposal details a plan of investigations which allows use of new technological advances to perform a highly integrative but global screen of transcriptional profiling in order to predict the weak points in Cryptococcus pathobiology. Our laboratory has over the last few years validated that this overall strategy can be successfully used. We have used cDNA subtraction techniques, differential display RT-PCR, in vivo expression technology, serial analysis of gene expression (SAGE), and most recently microarray technology to successfully begin to identify genes and which make C. neoformans, a pathogen. Through our studies we have identified the following genes and pathways to be important to the virulence composite: 1) genes encoding enzymes; 2) oxidative stress genes; 3) signaling pathway genes; 4) high temperature growth genes; 5) impact of mitochondria and respiration; 6) influence of attenuated null mutants on host immunity. A cornerstone of this proposal is to use microarrays to harness their ability to collect massive data and yet attempt to focus our understanding to relevant genes and networks by using specific in vitro and vivo conditions. This screening design will be carefully linked to functional studies through creation of null mutants for phenotypic analysis in relevant animal models. Our overall scientific plan is to understand the genetic regulation of the virulence composite which will contain a powerful insight into discovery of gene targets that interrupt pathogenesis and lead to new therapeutic strategies such as antifungal drugs and vaccines. PROJECT NARRATIVE This project uses genomic strategies to capture what makes the life-threatening fungus Cryptococcus neoformans such an effective pathogen in immunocompromised individuals. With the knowledge gained from these studies it is anticipated that new antifungal targets can be identified to help develop new drugs and/or vaccines against this common fungal pathogen for an enlarging severely immunocompromised population.