Molecular Analysis of Trypanosome Infection

锥虫感染的分子分析

• 批准号: 7289529
• 负责人: Fernando Villalta
• 金额: $ 36.63万
• 依托单位: MEHARRY MEDICAL COLLEGE
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-01 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7289529
• 关键词: Adhesions; Affect; Americas; Amino Acids; Animals; Area; Binding; Blood; Cardiac; Cardiovascular system; Cells; Chagas Disease; Chimera organism; Chronic; Computational Biology; Computer Simulation; Data; Deletion Mutation; Disease; Event; Extracellular Domain; Extracellular Matrix; Functional disorder; Gastrointestinal tract structure; Genes; Goals; Heart Diseases; Human; In Vitro; Individual; Infection; Interruption; Intervention; Knock-out; Knowledge; LOX gene; Laminin; Lectin; Ligands; Low Density Lipoprotein Receptor; Mammalian Cell; Mediating; Modeling; Molecular; Molecular Analysis; Morbidity - disease rate; Mus; Mutate; Outcome; Parasites; Pathogenesis; Pathology; Peptides; Preventive; Process; Protein-Lysine 6-Oxidase; Proteins; RNA Interference; Range; Rate; Receptor Cell; Regulation; Reporting; Research; Research Personnel; Role; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Structure; Structure-Activity Relationship; Surface; Testing; Therapeutic Intervention; Time; Tissues; Transcriptional Activation; Transgenic Organisms; Trypanosoma; Trypanosoma cruzi; Up-Regulation; Work; base; heart function; human morbidity; in vivo; innovation; laminin-1; mortality; mouse model; novel; novel strategies; oxidized low density lipoprotein; receptor; receptor density; response; tool

DESCRIPTION (provided by applicant): Trypanosoma cruzi is a blood and tissue parasite that affects millions of individuals causing significant human morbidity and mortality. Basic understanding of the role of the critical surface molecules that participate in the first step of T. cruzi infection may provide novel targets for therapy. Our long-range goal is to understand the molecular mechanisms that allow T. cruzi to infect mammalian cells and cause disease, so that specific molecular intervention strategies can be developed against T. cruzi infection. The objective of this application is to identify which host cell receptors mediate T. cruzi binding to mammalian cells, when receptor-mediated signaling pathways contribute to infection. The hypothesis of this application is that T. cruzi gp83, a ligand molecule that the parasite uses to attach to mammalian cells, binds to the lectin-like oxidized low-density lipoprotein receptor (LOX-1) of mammalian host cells to mediate trypanosome attachment, thereby activating signaling events leading to initial infection and pathogenesis. We have formulated this hypothesis based on strong preliminary results, showing that interruption of LOX-1 gene by gene trapping or silencing LOX-1 gene by RNAi inhibits T. cruzi binding to cells and infection, whereas over-expression of LOX-1 in cells causes over-attachment of trypanosomes to cells and increased infection. Supporting also this hypothesis are our findings showing that gp83 specifically binds to LOX-1 and that we have identified critical regions on gp83 and LOX-1 that interact with each other to apparently mediate T. cruzi attachment leading to parasite entry. We will test our central hypothesis by pursuing the following specific aims: (1) to determine the structural-function relationships of the LOX-1-gp83 interaction; (2) to determine the LOX-1- dependent signaling pathways that mediate trypanosome gp83-dependent infection; and (3) to determine the in vivo role of the LOX-1 gene in the process of T. cruzi infection and pathogenesis using a novel LOX-1 knock out mouse model and a novel transgenic mouse model over-expressing LOX-1. Mutations, deletions and substitutions in critical regions of LOX-1 and gp83 will be performed to identify the motifs in each interacting molecule involved in T. cruzi binding to cells. Computational modeling of the trypanosome gp83 and the interacting peptide loop from the extracellular domain of LOX-1 will be determined. The mechanism underlying the novel gp83-LOX-1 signal transduction pathway leading to regulation of laminin ?-1 expression and infection will be elucidated using LOX-1(-/-) cells and LOX(+/+) cells. We will use our novel mouse models that over-express LOX-1 or that are null for LOX-1 expression to provide definitive in vivo tools to understand the other complementary mechanisms that contribute to trypanosome infection (in the LOX-1 null animals) and the mechanisms by which LOX-1 mediates infection (in the over-expressing animals).

描述（由申请人提供）：克氏锥虫是一种血液和组织寄生虫，影响数百万人，导致严重的人类发病率和死亡率。对参与克氏锥虫感染第一步的关键表面分子的作用的基本了解可能为治疗提供新的靶点。我们的长期目标是了解克氏锥虫感染哺乳动物细胞并引起疾病的分子机制，以便针对克氏锥虫感染制定特定的分子干预策略。本申请的目的是确定当受体介导的信号通路导致感染时，哪些宿主细胞受体介导克氏锥虫与哺乳动物细胞的结合。该应用的假设是，T. cruzi gp83（寄生虫用来附着在哺乳动物细胞上的配体分子）与哺乳动物宿主细胞的凝集素样氧化低密度脂蛋白受体（LOX-1）结合，介导锥虫附着，从而激活导致初始感染和发病机制的信号传导事件。我们基于强有力的初步结果提出了这一假设，表明通过基因捕获中断LOX-1基因或通过RNAi沉默LOX-1基因会抑制克氏锥虫与细胞的结合和感染，而细胞中LOX-1的过度表达会导致锥虫对细胞的过度附着并增加感染。我们的研究结果也支持这一假设，表明 gp83 与 LOX-1 特异性结合，并且我们已经确定了 gp83 和 LOX-1 上的关键区域，它们相互作用，明显介导克氏锥虫附着，导致寄生虫进入。 我们将通过追求以下具体目标来检验我们的中心假设：（1）确定LOX-1-gp83相互作用的结构-功能关系； (2)确定介导锥虫gp83依赖性感染的LOX-1依赖性信号通路； (3)利用新型LOX-1敲除小鼠模型和新型过表达LOX-1转基因小鼠模型，确定LOX-1基因在克氏锥虫感染和发病过程中的体内作用。将在 LOX-1 和 gp83 的关键区域进行突变、缺失和取代，以鉴定参与克氏锥虫与细胞结合的每个相互作用分子中的基序。将确定锥虫 gp83 和 LOX-1 胞外域相互作用肽环的计算模型。将使用LOX-1(-/-)细胞和LOX(+/+)细胞阐明导致调节层粘连蛋白α-1表达和感染的新型gp83-LOX-1信号转导途径的潜在机制。我们将使用过度表达 LOX-1 或 LOX-1 表达无效的新型小鼠模型来提供明确的体内工具，以了解有助于锥虫感染的其他补充机制（在 LOX-1 无效动物中）以及 LOX-1 介导感染的机制（在过度表达动物中）。