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%。人民 艾滋病患者、化疗患者和移植受者感染危及生命的风险最高。 感染，但许多真菌也会在表面上健康的人身上致病。环境酵母菌 隐球菌就是这一趋势的缩影。像许多真菌一样，当隐球菌被吸入体内时，就会感染它 它可以从肺传播到中枢神经系统(CNS)并引起疾病。一次在 中枢神经系统隐球菌会导致真菌性脑膜脑炎，即使是在最先进的情况下，也有25%的几率是致命的 治疗。这种高死亡率与其他侵袭性真菌疾病是一致的，并表明 对改进疗法的迫切需求。为了开发抗击侵袭性真菌病原体的新策略，它 当务之急是我们更好地了解真菌病原体的基本生物学。我们的长- 学期研究目标是了解真菌感染颗粒的特性以及它们是如何致病的。在……里面 隐球菌，酵母和孢子都很可能是人类的感染性颗粒，但对致病菌的研究 孢子的潜力历来受到技术限制的阻碍。然而，最近我们提纯了孢子 数量上的同质性足以进行全面的生化、分子和毒力研究。vbl.使用 这种新型试剂，我们发现孢子可以在小鼠模型中致病，提供了第一个 有证据表明，在哺乳动物隐球菌病中，孢子可以作为具有感染性的颗粒。这样做的目的是 拟议的项目是首次调查真菌孢子从 静止的细胞转化为营养生长的酵母(萌发)，并感染哺乳动物的肺。我们的中央 假说是通过确定控制孢子萌发和生长的细胞和分子机制 对于肺部侵袭，我们将确定孢子介导性感染中的关键事件，这些事件可以被靶向抑制。为了测试 在这一假设下，我们将实现两个具体目标：1)识别发生的细胞和分子事件 在孢子萌发过程中，2)确定孢子穿过上皮进入上皮的机制(S) 侵占肺脏。我们将结合分子和经典遗传学、基因表达数据、蛋白质 成分数据，以及定量的高分辨率发芽分析，以解析发芽过程 离散事件。同时我们将使用体外组织培养模型，一种新的器官型微肺 模型，以及小鼠鼻腔感染模型，以确定孢子如何在肺部定植。这些创新之处 实验将产生一张深入的萌发路径图，并深入了解孢子是如何入侵 主持人。了解与孢子期间发生的两个关键事件相关的途径和过程- 介导性疾病(发芽和感染)对这一长期目标做出了重大贡献 努力确定新的和多样化的分子、细胞和系统靶点，可用于开发新的 预防和/或治疗隐球菌病和其他致命的人类真菌疾病的抗真菌治疗战略。

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.