Pathobiology of C. neoformans in the Central Nervous System

描述（由申请方提供）：新型隐球菌是人类最常见的危及生命的中枢神经系统感染之一，尽管目前有治疗方法，但发病率和死亡率仍然很高。最近在不列颠哥伦比亚省的温哥华岛爆发的一次疫情表明，隐球菌菌株迅速进化，产生了更强的毒株。本提案的重点是利用分子技术来鉴定、表征和验证C.这对这种胶囊化酵母的毒力复合物很重要。 我们的主要假设是基于一个简单的假设，即在一定的环境压力下，C。新生菌菌株将调节其产生蛋白质的遗传网络/途径，以使它们能够在宿主的不利环境中存活和生长。该提案详细说明了一项调查计划，该计划允许使用新技术进步来进行高度整合但全球性的转录谱筛选，以预测隐球菌病理生物学的弱点。我们的实验室在过去的几年里已经验证了这种整体策略可以成功地使用。我们已经使用cDNA消减技术、差异显示RT-PCR、体内表达技术、基因表达系列分析（SAGE）以及最近的微阵列技术成功地开始鉴定了使C.新形式的病原体通过我们的研究，我们已经确定了以下基因和途径对毒力复合物是重要的：1）编码酶的基因; 2）氧化应激基因; 3）信号传导途径基因; 4）高温生长基因; 5）线粒体和呼吸的影响; 6）减毒无效突变体对宿主免疫的影响。该提案的一个基石是使用微阵列来利用其收集大量数据的能力，并试图通过使用特定的体外和体内条件将我们的理解集中到相关的基因和网络。该筛选设计将通过在相关动物模型中创建用于表型分析的无效突变体与功能研究仔细联系起来。我们的总体科学计划是了解毒力复合物的遗传调控，这将包含对发现中断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。 该项目使用基因组策略来捕获是什么使危及生命的真菌新型隐球菌在免疫功能低下的个体中成为如此有效的病原体。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，用于扩大严重免疫功能低下的人群。

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.