Germination and Pathogenesis of Cryptococcus Spores

摘要：在美国，侵袭性真菌病的总死亡率约为50%。人 对于艾滋病患者，化疗患者和移植受者是获得危及生命的最高风险。 感染，但许多真菌也导致疾病，在表面上健康的个人。环境酵母 隐球菌是这一趋势的缩影。像许多真菌一样，隐球菌感染发生在吸入 肺，从那里它可以传播到中枢神经系统（CNS）并引起疾病。一旦进入 CNS隐球菌引起真菌性脑膜脑炎，即使使用最先进的药物， 治疗这种高死亡率与其他侵袭性真菌病一致， 迫切需要改进治疗方法。为了开发对抗侵袭性真菌病原体的新策略， 我们必须更好地了解真菌病原体的基本生物学。我们长久以来- 长期的研究目标是了解真菌感染性颗粒的特性以及它们如何引起疾病。在 隐球菌、酵母菌和孢子都可能是人类的传染性颗粒，但对致病性 孢子的潜力在历史上受到技术限制的阻碍。然而最近我们纯化了孢子 到数量上的同质性，足以进行全面的生化、分子和毒力研究。使用 这种新型试剂，我们发现孢子可以在小鼠模型中引起疾病，提供了第一个 孢子在哺乳动物隐球菌病中可作为感染颗粒的证据。的目的 拟议的项目是首次调查真菌孢子从 静止的细胞转化为植物生长的酵母菌（发芽）并感染哺乳动物的肺。我们的中央 假设是通过确定控制孢子萌发的细胞和分子机制， 肺侵袭，我们将确定孢子介导的感染中的关键事件，可以靶向抑制。测试 基于这一假设，我们将实现两个特定的目标：1）识别发生的细胞和分子事件 在孢子萌发过程中和2）确定孢子穿过上皮的机制， 在肺部定植我们将结合联合收割机分子和经典遗传学，基因表达数据，蛋白质 组成数据，以及定量高分辨率发芽测定，以将发芽过程解析为 离散事件同时我们将利用体外组织培养模型，建立一种新的器官型微肺 模型和小鼠鼻内感染模型来确定孢子如何定殖于肺。这些创新 实验将导致发芽途径的深入地图，并深入了解孢子如何侵入 主持人了解与孢子期发生的两个关键事件相关的途径和过程- 介导的疾病（发芽和感染）对这一长期目标做出了重大贡献。 致力于识别新的和不同的分子，细胞和系统的目标，可以利用新的 抗真菌治疗策略以预防和/或治疗隐球菌病和其它致命的人类真菌疾病。

ABSTRACT: In the United States gross mortality from invasive fungal disease is approximately 50%. People with AIDS, chemotherapy patients, and transplant recipients are at highest risk of acquiring life-threatening infections, but many fungi also cause disease in apparently healthy individuals. The environmental yeast Cryptococcus epitomizes this trend. Like many fungi, infection with Cryptococcus occurs when it is inhaled into the lung from which it can disseminate to the central nervous system (CNS) and cause disease. Once in the CNS Cryptococcus causes fungal meningoencephalitis that is fatal ~25% of the time, even with state-of-the-art treatment. This high incidence of mortality is consistent with other invasive fungal diseases and indicative of the dire need for improved therapeutics. To develop new strategies for combating invasive fungal pathogens, it is imperative that we gain a better understanding of the fundamental biology of fungal pathogens. Our long- term research goal is to understand the properties of fungal infectious particles and how they cause disease. In Cryptococcus, both yeast and spores are likely infectious particles of humans, but studies of the pathogenic potential of spores were historically hampered by technical limitations. Recently, however, we purified spores to homogeneity in numbers sufficient for comprehensive biochemical, molecular, and virulence studies. Using this novel reagent, we discovered that spores can cause disease in a mouse model, providing the first evidence that spores can act as infectious particles in mammalian cryptococcosis. The objective of this proposed project is to investigate for the first time the key processes by which fungal spores transition from quiescent cells into vegetatively growing yeast (germinate) and infect the mammalian lung. Our central hypothesis is that by determining the cellular and molecular mechanisms governing spore germination and lung invasion, we will identify key events in spore-mediated infections that can be targeted for inhibition. To test this hypothesis, we will carry out two Specific Aims: 1) identify the cellular and molecular events that occur during germination of spores and 2) determine the mechanism(s) by which spores cross the epithelium to colonize the lung. We will combine molecular and classical genetics, gene expression data, protein composition data, and quantitative high-resolution germination assays to parse the germination process into discrete events. At the same time we will use in vitro tissue culture models, a new organotypic microlung model, and a mouse intranasal model of infection to determine how spores colonize the lung. These innovative experiments will result in an in-depth map of germination pathways and insights into how spores invade the host. Understanding pathways and processes associated with the two pivotal events that occur during spore- mediated disease (germination and infection) makes significant contributions to the long-term objective of this work to identify new and diverse molecular, cellular, and systemic targets that can be exploited for novel antifungal therapeutic strategies to prevent and/or treat cryptococcosis and other fatal human fungal diseases.