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）是一种二型真菌病原体，其经历温度诱导的转变，从土壤中生长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性疾病。虽然疾病是最严重的免疫功能低下，HC也会导致严重的问题，免疫功能正常的主机。在小鼠和人类中，组织胞浆菌病的病理学证实了巨噬细胞在肺部原发性感染、全身性传播和疾病消退中的关键作用。从霉菌到酵母菌的转化是HC发病的第一个重要步骤。分生孢子和菌丝体片段被吸入并萌发为致病酵母形式，其负责所有后续步骤和与宿主细胞的相互作用。Hc是一种非常适应的巨噬细胞寄生虫，在相对中性pH的膜结合区室中在细胞内增殖。在巨噬细胞的恶劣环境中调节和生存的能力是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，我们必须确定关于HC如何建立成功感染巨噬细胞的机制细节，因为很少有研究集中在确定初始HC和巨噬细胞附着所需的粘附因子上。因此，本项目的中心目标是鉴定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的毒力所必需的。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巨噬细胞进入，因此细胞内生存所需的分子决定因素的重要见解。在第一个目标中，我将产生Hc插入突变体库。将筛选和富集突变体以产生在巨噬细胞附着所需的基因中含有突变的几种候选物。在第二个目标，我将确定和表征的分子决定因素，是至关重要的粘附巨噬细胞和HC的毒力。我的目标是绘制插入位点，从而产生低结合酵母，并通过靶向基因破坏和互补，验证体外和体内毒力对特定遗传元件的要求。在第三个目标，我将采取候选基因的方法来评估是否预测的粘附素，细胞表面蛋白，或分泌的因素所需的附着到巨噬细胞在酵母或分生孢子生长阶段。基于它们在酵母或分生孢子生长期的转录上调和/或预测编码细胞表面或分泌因子来选择这些候选物。这种方法将补充无偏筛选方法，并提供洞察所需的酵母和分生孢子巨噬细胞附着的基因产物是相似还是不同。该项目的完成将确定HC附着到巨噬细胞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基本的了解。