Functional genomics of C.neoformans pathogenesis

新型隐球菌是一种机会致病真菌，对免疫功能低下的患者有重要的临床影响。 特别是艾滋病患者特别容易感染隐球菌病。 最常见的临床表现是肺隐球菌病和脑膜脑炎。 目前的抗真菌药物不足以提供安全有效的治疗。 近十年来，对C.利用分子生物学、遗传学、免疫学和生物化学方法对新生儿进行了深入研究。功能基因组学有可能加速我们对复杂生物体发病机制的理解。由于最近的两项进展，存在有效测试C.使用大规模筛选方法对新生儿基因进行致病性研究。 首先，我们开发了一种C的遗传筛查。新变型，其允许从大量插入突变体中鉴定毒力突变体。 这种筛选提高了鉴定改变致病性的基因的效率和速度，并消除了在动物模型中单独测试每个突变体的需要-从而降低了成本，最大限度地减少了动物的使用，并加速了结果。 第二，一个基因组计划，目标是对C.目前正在进行中。 该项目将提供原始数据，用于识别C中的绝大多数潜在开放阅读框架。新人类 每个开放阅读框对发病机制都有潜在的重要性。我们建议开发技术，以快速构建有针对性的插入在C。neoformans打开阅读框架，并估计我们可以破坏C的大约6%的开放阅读框架。新人类 该项目将重点关注对细胞壁的生物发生和维持重要的基因。 每个插入将用独特的序列标记，这将允许在大量突变体中鉴定该突变体。将在小鼠模型中筛选大量突变体的生长情况，并将快速鉴定在该竞争性试验中影响毒力的基因突变。 我们将为该项目产生的特异性插入突变体库为开发新的抗真菌靶点、研究发病机制和宿主反应提供了宝贵的资源。

Cryptococcus neoformans is an opportunistic fungal pathogen that has significant clinical impact on immunocompromised patients. In particular, patients with AIDS are exquisitely vulnerable to cryptococcosis. The most common clinical presentations are pulmonary cryptococcosis and meningoencephalitis. Current antifungal agents are inadequate for safe and effective therapy. Over the past decade, the pathogenesis of C. neoformans has been intensively studied using molecular biology, genetic, immunological and biochemical approaches. Functional genomics has the potential to accelerate our understanding of pathogenesis in complex organisms. Because of two recent advances, the opportunity exists to efficiently test the contribution of C. neoformans genes to pathogenesis using a mass screening approach. First, we have developed a genetic screen for C. neoformans that allows identification of virulence mutants from a large pool of insertion mutants. This screen increases the efficiency and rapidity of identification of genes which alter the pathogenicity and eliminates the need to test each and every mutant individually in an animal model - thus reducing cost, minimizing the use of animals, and accelerating results. Second, a genome project with the goal of sequencing the entire genome of C. neoformans is currently underway. This project will provide the raw data for identification of the vast majority of potential open reading frames in C. neoformans. Each open reading frame is potentially important for pathogenesis. We propose to develop technology to rapidly construct targeted insertions in C. neoformans open reading frames and estimate that we can disrupt approximately 6 percent of the open reading frames from C. neoformans. This project will focus on genes important for biogenesis and maintenance of the cell wall. Each insertion will be tagged with a unique sequence that will allow identification of that mutant within a large pool of mutants. Large groups of mutants will be screened for growth in a mouse model, and mutations in genes that affect virulence in this competitive assay will be rapidly identified. The bank of specific insertional mutants that we will generate for this project represents a valuable resource for the development of novel antifungal targets studies of pathogenesis and host response.