Functional genomics of C.neoformans pathogenesis

新生隐球菌是一种机会性真菌病原体，对免疫功能低下的患者有重要的临床影响。特别是，艾滋病患者极易患上隐球菌病。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安全有效地治疗。在过去的十年里，人们利用分子生物学、遗传学、免疫学和生物化学的方法对新生葡萄球菌的发病机制进行了深入的研究。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。由于最近的两个进展，有机会使用大规模筛查方法有效地测试新生葡萄球菌基因对发病机制的贡献。首先，我们开发了一种新生假单胞菌的遗传筛选，可以从大量插入突变中鉴定出毒力突变。这种筛选提高了识别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的需要-从而降低了成本，最大限度地减少了对动物的使用，并加快了结果。其次，目前正在进行一项基因组计划，目标是对新生葡萄球菌的整个基因组进行测序。该项目将为确定新生隐球菌绝大多数潜在的开放阅读框提供原始数据。每个开放阅读框架对发病机制都有潜在的重要作用。我们建议开发技术来快速构建新生葡萄球菌开放阅读框中的靶向插入，并估计我们可以干扰新生葡萄球菌大约6%的开放阅读框。该项目将重点研究对生物发生和细胞壁维持至关重要的基因。每个插入将被标记一个唯一的序列，这将允许在大量突变体库中识别该突变体。将在小鼠模型中筛选大量突变体的生长情况，并在这一竞争性试验中快速识别影响毒力的基因突变。我们将为该项目产生的特定插入突变体库为开发新的抗真菌靶标、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.