Elucidating the Mechanistic Basis of H. capsulatum-Macrophage Interactions

 描述(由申请人提供)：组织胞浆菌(HC)，是一种二相性真菌病原体，在温度诱导下从生长在土壤中的霉菌转变为寄生酵母形式，在肺部建立感染，并能够在哺乳动物中引起严重的全身疾病。虽然疾病在免疫功能低下的人中最严重，但丙型肝炎也会导致免疫能力强的宿主出现严重问题。在小鼠和人类中，组织胞浆菌病的病理学表明巨噬细胞在肺部原发感染、全身性传播和疾病解决中起着关键作用。从霉菌到酵母菌的转变是HC发病的第一个基本步骤。分生孢子和菌丝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是一种适应能力很强的巨噬细胞寄生虫，在pH相对中性的膜结合室内细胞内增殖。巨噬细胞在恶劣的环境中调节和生存的能力是HC发病所不可或缺的。为了充分了解HC感染的过程，我们必须确定HC如何建立成功的巨噬细胞感染的机制细节，因为很少有研究集中于确定HC最初与巨噬细胞附着所需的黏附因子。因此，该项目的中心目标是确定HC使用的粘附素，这些粘附素是初始巨噬细胞附着和随后的毒力所必需的。对这些粘附素或粘附素相关因子的分析将揭示细胞内寄生虫进入巨噬细胞并因此在细胞内存活所需的分子决定因素。在第一个目标中，我将生成一个HC插入突变体的文库。将对突变体进行筛选和浓缩，以产生几个包含巨噬细胞附着所需基因突变的候选基因。在第二个目标中，我将识别和表征对巨噬细胞黏附和HC毒力至关重要的分子决定因素。我的目标是定位插入位置，导致低结合酵母菌，并通过有针对性的基因中断和互补，验证特定的遗传元件在体外和体内对毒力的要求。在第三个目标中，我将采用候选基因方法来评估在酵母或分生孢子生长阶段是否需要预测的粘附素、细胞表面蛋白或分泌因子来附着到巨噬细胞上。这些候选基因是根据它们在酵母或分生孢子生长阶段的转录上调和/或编码细胞表面或分泌因子的预测来选择的。这种方法将补充无偏筛选方法，并提供对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洞察。该项目的完成将确定HC与巨噬细胞附着所需的分子决定因素，并对HC成功感染巨噬细胞所需的初始相互作用有一个基本的了解。

 DESCRIPTION (provided by applicant): 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 I 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. In the second aim I will identify and characterize the molecular determinants that are critical for adhering to macrophages and for Hc virulence. My 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. In the third aim I will take a candidate gene approach to assess whether predicted adhesins, cell surface proteins, or secreted factors are required for attachment to macrophages in either the yeast or conidial phases of growth. These candidates were selected based on their transcriptional upregulation in the yeast or conidial phases of growth and/or prediction to encode cell surface or secreted factors. This approach will complement the unbiased screening approach 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.