Host Factors in Cryptococcal Infection

描述（由申请方提供）：新型隐球菌是一种真菌病原体，可导致免疫功能低下患者发生严重疾病，每年导致超过60万人死亡。隐球菌病通过吸入感染性颗粒而感染，这导致原发性肺部感染。C. neoformans是一种兼性细胞内病原体。在最初被肺中的吞噬细胞吞噬后，它可能溶解或离开这些细胞并扩散 在整个哺乳动物宿主中，在血流中或通过进入额外的吞噬细胞。传播到大脑的后果是致命的脑膜炎。几个C。新形虫的毒力因子与这种疾病的发病机制有关，包括保护性色素黑色素的形成、真菌细胞上广泛的多糖荚膜的存在以及某些蛋白质的分泌。宿主因素也在感染中发挥作用，宿主吞噬细胞与病原体潜伏和传播有关。尽管证明宿主吞噬细胞参与这种疾病，一直没有努力系统地确定和研究基因是重要的感染过程。在初步研究中，我开发并优化了一种高通量成像分析方法，以有效地评估C。neoformans和RNAi处理的人巨噬细胞样细胞系。然后，我筛选了一个宿主基因的子集，以确定在这些相互作用中重要的因素，通过使用市售的siRNA文库下调宿主细胞中编码901种人类激酶和磷酸酶的基因的表达，然后进行相互作用测定。该筛选确定了8个基因在宿主：真菌相互作用中具有特定作用;在本申请中，我建议详细研究其中两个。该项目的广泛，长期的目标是确定宿主基因在真菌发病机制中发挥重要作用，并发现其参与机制。目的一是检测两个候选宿主基因在致病相关模型中的作用，包括小鼠原代细胞和感染模型。目的2是阐明两个宿主基因的产物影响宿主-真菌相互作用的细胞机制。这些研究将阐明这一重要疾病的发病机制的关键方面，通过定义特定的宿主因素如何影响吞噬细胞与C。新人类这项工作将增加对隐球菌病的了解，并可能有助于开发治疗或预防策略来对抗这种疾病。

DESCRIPTION (provided by applicant): Cryptococcus neoformans is a fungal pathogen that causes serious disease in immunocompromised patients and results in an annual death toll of over 600,000. Cryptococcosis is contracted by inhalation of the infectious particle, which leads to a primary pulmonary infection. C. neoformans is a facultative intracellular pathogen. After initial engulfment by phagocytic cells in the lungs it may lyse or exit these cells and disseminate throughout the mammalian host, either in the bloodstream or by entering additional phagocytes. The consequence of dissemination to the brain is lethal meningitis. Several C. neoformans virulence factors have been implicated in the pathogenesis of this disease, including formation of the protective pigment melanin, the presence on fungal cells of an extensive polysaccharide capsule, and secretion of certain proteins. Host factors also play a role in infection, and host phagocytes have been implicated in pathogen latency and dissemination. Despite the demonstration of host phagocytic cell involvement in this disease, there has been no effort to systematically identify and study genes that are significant for the infection process. In preliminary studies, I developed and optimized a high-throughput imaging assay to efficiently evaluate the interactions between C. neoformans and RNAi-treated human macrophage-like cell lines. I then screened a subset of host genes to identify factors important in these interactions, by using commercially available siRNA libraries to down-regulate expression of genes encoding 901 human kinases and phosphatases in the host cells and then performing interaction assays. This screen identified 8 genes as having specific roles in host:fungal interactions; in this application I propose to study two of them in detail. The broad, long-term objectives of this project are to identify host genes that play a significant role in fungal pathogenesis and to discover their mechanisms of involvement. Aim 1 is designed to test the roles of two candidate host genes in models relevant to pathogenesis, including mouse primary cells in vitro and models of infection. Aim 2 is designed to elucidate the cellular mechanisms by which the products of the two host genes of interest influence host:fungal interactions. These studies will elucidate key aspects of pathogenesis of this important disease by defining how specific host factors affect the interaction of phagocytic cells with C. neoformans. This work will increase understanding of cryptococcosis and potentially aid in the development of therapeutic or preventive strategies to fight this disease.