Discovering Histoplasma factors required for initial macrophage interaction

发现初始巨噬细胞相互作用所需的组织胞浆菌因子

• 批准号: 9243585
• 负责人: WILLIAM E GOLDMAN
• 金额: $ 22.8万
• 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: 2016-12-05 至 2018-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9243585
• 关键词: Adherence; Adhesions; Adhesives; Affect; Agrobacterium; Anabolism; Bacterial Adhesins; Binding; 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; 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; Pathogenicity; Phase; Polysaccharides; Primary Infection; Proliferating; RNA Interference; Regulation; Research; Resolution; Role; Site; Site-Directed Mutagenesis; Soil; Surface; Systemic disease; Temperature; Testing; Virulence; Work; Yeasts; base; forward genetics; fungus; gene product; genetic approach; genetic element; in vivo; insight; macrophage; monolayer; mouse model; mutant; pathogen; programs; receptor; restoration; screening; therapeutic target; transcriptomics; uptake

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.

荚膜组织胞浆菌 (Hc) 是一种二态性真菌病原体，会经历温度诱导的转变 从生长在土壤中的霉菌到在肺部建立感染并能够进行感染的寄生酵母形式 导致哺乳动物严重的全身性疾病。虽然免疫功能低下者的疾病最为严重， Hc 还会给免疫功能正常的宿主带来严重问题。在小鼠和人类中，病理学 组织胞浆菌病证明了巨噬细胞在肺部、全身原发感染中的关键作用 疾病的传播和解决。从霉菌到酵母的转变是第一步 Hc的发病机制。分生孢子和菌丝碎片被吸入并发芽成致病酵母形式 它负责所有后续步骤以及与宿主细胞的相互作用。 Hc 的适应性非常好 巨噬细胞的寄生虫，在相对中性的膜结合区室中在细胞内增殖 酸碱度。巨噬细胞在恶劣环境中调节和生存的能力是 Hc 不可或缺的一部分 发病。为了充分了解 Hc 感染的过程，我们必须确定有关 Hc 感染的机制细节 Hc 如何成功感染巨噬细胞，因为很少有研究关注于识别 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需的，并且 随后的毒力。对这些粘附素或粘附素相关因素的分析将揭示重要的见解 细胞内寄生虫进入巨噬细胞并因此进入细胞内所需的分子决定因素 生存。第一个目标是，我们将生成 Hc 插入突变体文库。突变体将被筛选并 富集以产生几种含有巨噬细胞附着所需基因突变的候选物。这 第二个目标是确定和表征对于坚持至关重要的分子决定因素 巨噬细胞和 Hc 毒力。我们的目标是绘制导致低结合酵母的插入位点 并通过针对性的基因破坏和互补验证特定遗传元件的需求 用于体外和体内毒力。第三个目标将评估预测的粘附素、细胞表面蛋白或 在酵母或分生孢子生长阶段，需要分泌因子来附着到巨噬细胞上。 这些候选人是根据粘附素预测程序的鉴定并通过 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是 相似或不同。该项目的完成将确定 Hc 所需的分子决定因素 附着于巨噬细胞并产生对 Hc 所需的初始相互作用的基本了解 巨噬细胞感染的成功建立。