Discovering Histoplasma factors required for initial macrophage interaction

摘要组织胞浆菌(HC)是一种经历温度诱导转变的二态真菌病原体。 从生长在土壤中的霉菌，到寄生的酵母形式，在肺部建立感染并能够 在哺乳动物中引起严重的系统性疾病。虽然疾病在免疫功能受损的人中最为严重， 丙型肝炎还会导致免疫活性宿主的严重问题。在小鼠和人类中，糖尿病的病理学 组织胞浆菌病显示巨噬细胞在肺部原发感染中的关键作用 传播和解决疾病。从霉菌到酵母菌的转变是 丙型肝炎的发病机制。分生孢子和菌丝体碎片被吸入并萌发成致病酵母形式 负责所有后续步骤和与宿主细胞的相互作用。HC是一部适应得非常好的 巨噬细胞的寄生虫，在相对中性的膜结合室内细胞内增殖 PH值。巨噬细胞在恶劣的环境中调节和生存的能力是HC不可或缺的。 发病机制。为了充分了解丙型肝炎病毒感染的过程，我们必须确定关于 丙型肝炎是如何成功感染巨噬细胞的，因为很少有研究集中在确定 最初的HC和巨噬细胞附着所需的黏附因子。这个项目的中心目标是 因此，确定HC使用的粘附素是初始巨噬细胞附着所必需的，并 随之而来的毒力。对这些粘附素或粘附素相关因素的分析将揭示对 细胞内寄生虫进入巨噬细胞从而进入细胞内所需的分子决定因素 生死存亡。在第一个目标中，我们将生成一个HC插入突变体文库。将对突变者进行筛选和 浓缩以产生几个包含巨噬细胞附着所需基因突变的候选基因。这个 第二个目标将确定和表征对坚持以下目标至关重要的分子决定因素 巨噬细胞和HC毒力。我们的目标是定位导致低结合酵母菌的插入位置 并通过有针对性的基因干扰和互补来验证特定遗传元素的要求 在体外和体内的毒力。第三个目标将评估预测的粘附素、细胞表面蛋白或 在酵母或分生孢子的生长阶段，需要分泌因子来附着巨噬细胞。 这些候选人是通过粘附素预测程序根据他们的身份和通过 表达数据和转录组学的分析。这种方法将对我们的无偏见筛选形成补充 并提供了酵母和分生孢子附着巨噬细胞所需的基因产物是否 相似或不同的。该项目的完成将确定HC所需的分子决定因素 附着于巨噬细胞，并对HC所需的初始相互作用有一个基本的了解 建立了成功感染巨噬细胞的方法。

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.