Host Factors in Cryptococcal Infection

描述(申请人提供)：新生隐球菌是一种真菌病原体，会导致免疫功能低下的患者患上严重疾病，每年导致超过60万人死亡。隐球菌病是通过吸入感染颗粒而感染的，这会导致原发肺部感染。新生葡萄球菌是一种兼性胞内致病菌。在最初被肺中的吞噬细胞吞噬后，它可能会溶解或离开这些细胞并扩散 在哺乳动物的整个宿主中，要么在血液中，要么进入额外的吞噬细胞。传播到大脑的后果是致命性脑膜炎。一些新生假单胞菌的毒力因子参与了这种疾病的发病机制，包括保护性黑色素的形成，真菌细胞上广泛的多糖膜的存在，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。尽管宿主吞噬细胞参与了这种疾病，但还没有努力系统地识别和研究对感染过程有重要意义的基因。在初步研究中，我开发和优化了一种高通量成像分析方法，以有效地评估新生葡萄球菌和经RNAi处理的人巨噬细胞样细胞系之间的相互作用。然后，我筛选了宿主基因的一个子集，以确定这些相互作用中的重要因素，方法是使用商业上可用的siRNA文库下调宿主细胞中编码901人激酶和磷酸酶的基因的表达，然后进行相互作用分析。这个筛选确定了8个在宿主：真菌相互作用中具有特定作用的基因；在这个应用中，我建议详细研究其中的两个基因。该项目的长期目标是确定在真菌发病中起重要作用的宿主基因，并发现它们的参与机制。目的1研究两种候选宿主基因在小鼠体外原代细胞和感染模型中的作用。目的2旨在阐明两个感兴趣的寄主基因产物影响寄主真菌相互作用的细胞机制。这些研究将通过确定特定的宿主因素如何影响吞噬细胞与新生葡萄球菌的相互作用来阐明这种重要疾病的发病机制的关键方面。这项工作将增加对隐球菌病的了解，并可能有助于制定治疗或预防战略来抗击这种疾病。

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.