Elucidating the Mechanistic Basis of H. capsulatum-Macrophage Interactions

阐明荚膜梭菌-巨噬细胞相互作用的机制基础

• 批准号: 9121108
• 负责人: CHELSEA BOYD
• 金额: $ 5.61万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-08-26 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9121108
• 关键词: Adherence; Adhesions; Adhesives; Affect; Agrobacterium; Anabolism; Bacterial Adhesins; Binding; Biological; Breathing; Candidate Disease Gene; Cell Surface Proteins; Cell surface; Cells; Cellular biology; Complement; Data; Disease; Environment; Event; Gene Proteins; Gene Targeting; Genes; Genetic; Genetic Screening; Goals; Growth; Histoplasma capsulatum; Histoplasmosis; Human Pathology; ITGB2 gene; Immunocompetent; Immunocompromised Host; In Vitro; Individual; Infection; Insertional Mutagenesis; Intracellular Membranes; Libraries; Ligands; Lung; Mammals; Maps; Mediating; Membrane; Modification; Molds; Molecular; Molecular Chaperones; Mus; Mutation; Parasites; Pathogenesis; Phase; Polysaccharides; Primary Infection; Process; Proliferating; RNA Interference; Regulation; Research; Resolution; Role; Site; Site-Directed Mutagenesis; Soil; Surface; Systemic disease; Temperature; Testing; Up-Regulation; Virulence; Work; Yeasts; base; forward genetics; fungus; gene product; genetic element; in vivo; insight; interest; macrophage; monolayer; mouse model; mutant; pathogen; public health relevance; receptor; restoration; screening; therapeutic target; transcriptomics; uptake

 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.

 描述（由申请人提供）：荚膜组织胞浆菌 (Hc) 是一种二态性真菌病原体，它经历温度诱导的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引起哺乳动物中的严重全身性疾病。虽然疾病在免疫功能低下者中最为严重，但 Hc 也会在免疫功能正常的宿主中引起严重问题，组织胞浆菌病的病理学表明其起着关键作用。巨噬细胞在肺部原发性感染、全身传播和疾病消退中的作用 分生孢子和菌丝碎片被吸入并发芽成致病性酵母形式。 Hc 是一种非常适应的巨噬细胞寄生虫，能够在相对中性的 pH 值的膜结合区室中进行细胞内增殖。巨噬细胞的恶劣环境是 Hc 发病机制不可或缺的一部分，为了充分了解 Hc 感染的过程，我们必须确定 Hc 如何成功感染巨噬细胞的机制细节，因为很少有研究关注于确定初始 Hc 和 Hc 所需的粘附因子。因此，该项目的中心目标是确定 Hc 所使用的粘附素，这些粘附素是初始巨噬细胞粘附和随后分析这些粘附素或粘附素相关因子所需的。重要的是揭示细胞内寄生虫进入巨噬细胞所需的分子决定因素，因此细胞内生存在第一个目标中，我将筛选和富集 Hc 插入突变体库，以产生包含所需基因突变的几个候选者。在第二个目标中，我将识别和表征对于粘附巨噬细胞和 Hc 毒力至关重要的分子决定因素，我的目标是绘制插入位点，从而产生低结合。酵母，并通过有针对性的基因破坏和互补，验证体外和体内毒力的特定遗传元件的要求。在第三个目标中，我将采用候选基因方法来评估预测的粘附素、细胞表面蛋白或分泌因子是否是正确的。这些候选物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。公正的筛选方法，并深入了解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。该项目的完成将确定 Hc 附着于巨噬细胞所需的分子决定因素，并对 Hc 所需的初始相互作用有一个基本的了解。建立成功的巨噬细胞感染。