CRYPTOCOCCAL INTERACTIONS WITH HOST CELLS

描述（由申请人提供）：新型隐球菌是一种机会性病原体，可导致艾滋病或其他免疫功能低下患者发生危及生命的疾病；肺外隐球菌感染是一种艾滋病定义疾病。我们研究的长期目标是了解隐球菌病的发病机制。本应用集中在这一过程的一个基本部分，这种兼性细胞内病原体与哺乳动物宿主的吞噬细胞的相互作用。这些相互作用会对宿主和酵母菌造成不同程度的损害，但所涉及的机制尚不清楚，这限制了我们影响有利于宿主的解决方案的能力。我们建议结合前向遗传学、高通量筛选试验和重点随访研究，以确定该病原体成功粘附并进入宿主细胞所需的隐球菌因子。目的1是利用插入诱变和自动筛选来识别这些过程中的突变体。目的二是确定选定的基因产物影响病原体粘附和进入相互作用的机制。这些研究将采用细胞粘附、进入、细胞内存活和生长的测定；序列恢复、分析和基因删除的分子技术；表型评估包括形态学研究、生化分析和毒力分析。结果将使用严格的统计方法和适当的控制进行评估，并将在我们在隐球菌基础生物学方面的专业知识的背景下进行解释。拟议的研究将阐明致病机制的一个关键方面，决定生物体的生存和传播以及宿主限制疾病的能力。这项工作将为重要病原体的研究开辟新的领域，并开发可应用于真菌生物学其他领域的实验工具。公共卫生相关性：这项研究与公共卫生高度相关，因为所研究的生物体会导致严重的人类疾病，而目前的治疗方法还不够。新型隐球菌与宿主细胞的相互作用是其致病能力的基础，因此了解和抑制这些过程可能会促进隐球菌感染的治疗。此外，这项工作将有助于基础科学知识，这将提供对病原微生物以及其他生物学领域的见解。

DESCRIPTION (provided by applicant): Cryptococcus neoformans is an opportunistic pathogen that is responsible for life-threatening disease in patients with AIDS or other conditions of immunocompromise; extrapulmonary cryptococcal infection is an AIDS-defining illness. The long-term goal of our studies is to understand the pathogenesis of cryptococcosis. This application focuses on a fundamental part of this process, the interactions of this facultative intracellular pathogen with the phagocytic cells of its mammalian host. These interactions resolve with varying levels of damage to the host and yeast, but the mechanisms involved are poorly understood, limiting our ability to influence resolution in favor of the host. We propose to combine forward genetics, high-throughput screening assays, and focused follow-up studies to determine the cryptococcal factors that are required for this pathogen to successfully adhere to and enter host cells. Aim I is to identify mutants in these processes, using insertional mutagenesis and automated screening. Aim II is to determine the mechanisms by which selected gene products influence pathogen adherence and entry interactions. These studies will employ assays of cell adhesion, entry, and intracellular survival and growth; molecular techniques for sequence recovery, analysis, and gene deletion; and phenotypic assessment including morphological studies, biochemical analysis, and virulence assays. Results will be assessed using rigorous statistical methods and appropriate controls, and will be interpreted in the context of our expertise in the fundamental biology of Cryptococcus. The proposed studies will elucidate a crucial aspect of pathogenesis that determines the survival and dissemination of the organism and the host's ability to limit disease. This work will additionally open new areas for investigation of an important pathogen and develop experiment tools that can be applied to other areas of fungal biology. PUBLIC HEALTH RELEVANCE: This research is highly relevant to public health because the organism under study causes serious human illness for which current therapies are not adequate. The interactions of Cryptococcus neoformans with host cells are fundamental to its ability to cause disease, so understanding and inhibiting these processes may advance treatment of cryptococcal infection. Further, this work will contribute to basic science knowledge, which will provide insights into pathogenic microbes as well as other areas of biology.