Discovering Histoplasma factors required for initial macrophage interaction

荚膜组织胞浆菌（Histoplasma capsulatum，Hc）是一种二型真菌病原菌，在温度诱导下发生转变 从生长在土壤中的霉菌，到在肺部建立感染的寄生酵母菌， 导致哺乳动物严重的系统性疾病。虽然疾病在免疫功能低下者中最为严重， 丙型肝炎还导致严重的问题，在免疫功能正常的主机。在小鼠和人类中， 组织胞浆菌病证实了巨噬细胞在肺部、全身和全身原发性感染中的关键作用。 传播和解决疾病。从霉菌到酵母菌的转变是发酵过程中的第一个重要步骤。 HC的发病机制分生孢子和菌丝碎片被吸入并萌发为致病酵母形式 负责所有后续步骤和与宿主细胞的相互作用。Hc是一种非常适应 巨噬细胞的寄生虫，在相对中性的膜结合区室中细胞内增殖 博士在巨噬细胞的恶劣环境中调节和存活的能力是Hc的组成部分 发病机制为了充分了解HC感染的过程，我们必须确定以下方面的机制细节： Hc如何成功感染巨噬细胞，因为很少有研究专注于识别 初始HC和巨噬细胞附着所需的粘附因子。该项目的核心目标是 因此，鉴定出Hc所使用的粘附素，这些粘附素是初始巨噬细胞粘附所需的， 随后的毒性。对这些粘附素或粘附素相关因子的分析将揭示以下重要见解： 细胞内寄生虫进入巨噬细胞所需的分子决定簇， 生存在第一个目标中，我们将产生Hc插入突变体库。将筛选突变体， 富集以产生含有巨噬细胞附着所需基因突变的几种候选物。的 第二个目标将确定和表征对粘附至关重要的分子决定因素， 巨噬细胞和HC毒力。我们的目标是定位低结合酵母的插入位点 并通过靶向基因破坏和互补验证特定遗传元件的需求 在体外和体内的毒力。第三个目标是评估预测的粘附素，细胞表面蛋白， 在酵母或分生孢子生长阶段，需要分泌因子附着于巨噬细胞。 这些候选者是基于它们通过粘附素预测程序的鉴定而选择的，并通过 表达数据和转录组学的分析。这种方法将补充我们的公正筛选 并提供了深入了解是否基因产物所需的巨噬细胞附着酵母和分生孢子， 相似或不同。本计画完成后，将可找出Hc所需的分子决定因子 附着到巨噬细胞，并产生一个基本的理解所需的初始相互作用的HC， 成功建立巨噬细胞感染。

Histoplasma capsulatum (Hc), is a dimorphic fungal pathogen that undergoes a temperature-induced transition from a mold that grows in the soil, to a parasitic yeast form that establishes infection in the lung and is capable of causing severe systemic disease in mammals. While disease is most severe in the immunocompromised, Hc also causes serious problems in immunocompetent hosts. In mice and humans, the pathology of histoplasmosis demonstrates the pivotal role of macrophages in primary infection of the lungs, systemic dissemination, and resolution of disease. Transition from mold to yeast is the first essential step in the pathogenesis of Hc. Conidia and mycelial fragments are inhaled and germinate to the pathogenic yeast form that is responsible for all subsequent steps and interactions with host cells. Hc is a remarkably well-adapted parasite of macrophages, proliferating intracellularly in a membrane-bound compartment of relatively neutral pH. The ability to modulate and survive within the hostile environment of the macrophage is integral to Hc pathogenesis. To fully understand the course of Hc infection, we must ascertain mechanistic details regarding how Hc establishes a successful infection of macrophages, as little research has focused on identifying adhesive factors required for the initial Hc and macrophage attachment. The central goal of this project is to therefore identify adhesins employed by Hc that are required for initial macrophage attachment and subsequent virulence. Analysis of these adhesins or adhesin-related factors will reveal important insights into the molecular determinants required by intracellular parasites for macrophage entry and therefore intracellular survival. In the first aim, we will generate a library of Hc insertional mutants. Mutants will be screened and enriched to yield several candidates containing mutations in genes required for macrophage attachment. The second aim will identify and characterize the molecular determinants that are critical for adhering to macrophages and for Hc virulence. Our objective is to map the sites of insertion resulting in low-binding yeasts and through targeted gene disruption and complementation verify the requirement of specific genetic elements for virulence in vitro and in vivo. The third aim will assess whether predicted adhesins, cell surface proteins, or secreted factors are required for attachment to macrophage in either the yeast or conidial phases of growth. These candidates were selected based on their identification by an adhesin prediction program and through the analysis of expression data and transcriptomics. This approach will complement our unbiased screening and provide insight into whether gene products required for macrophage attachment by yeast and conidia are similar or different. The completion of this project will identify molecular determinants required for Hc attachment to macrophages and yield a fundamental understanding of the initial interaction required by Hc for the establishment of a successful infection of macrophages.