Molecular Analysis of Trypanosome Infection

描述(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。对参与旋毛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鲁兹毛滴虫感染哺乳动物细胞并导致疾病的分子机制，以便开发针对克鲁兹毛滴虫感染的特定分子干预策略。这项应用的目的是确定当受体介导的信号通路有助于感染时，哪些宿主细胞受体介导了克氏锥虫与哺乳动物细胞的结合。这种应用的假设是，克氏锥虫用来附着在哺乳动物细胞上的配体分子gp83与哺乳动物宿主细胞的凝集素样氧化型低密度脂蛋白受体(LOX-1)结合，介导锥虫附着，从而激活导致初始感染和发病的信号事件。我们基于强大的初步结果提出了这一假说，表明通过RNAi捕获或沉默LOX-1基因来阻断LOX-1基因可以抑制克氏锥虫与细胞的结合和感染，而LOX-1在细胞中的过表达会导致锥虫过度附着在细胞上并增加感染。我们的发现也支持这一假设，即gp83与LOX-1特异性结合，我们已经确定gp83和LOX-1上的关键区域相互作用，显然介导了克氏锥虫的附着导致寄生虫进入。 我们将通过追求以下特定目标来验证我们的中心假设：(1)确定LOX-1-gp83相互作用的结构与功能关系；(2)确定介导锥虫gp83依赖感染的LOX-1依赖的信号通路；以及(3)利用新的LOX-1敲除小鼠模型和高表达LOX-1的新型转基因小鼠模型，确定LOX-1基因在体内在克氏锥虫感染和发病过程中的作用。将进行LOX-1和gp83关键区域的突变、缺失和替换，以确定参与T.ruzi与细胞结合的每个相互作用分子中的基序。锥虫gp83和LOX-1胞外区相互作用肽环的计算模型将被确定。通过LOX-1(-/-)细胞和LOX(+/+)细胞阐明新的gp83-LOX-1信号转导通路调控层粘连蛋白？-1表达和感染的机制。我们将使用我们的新的LOX-1过度表达或LOX-1表达缺失的小鼠模型来提供明确的活体工具，以了解导致锥虫感染的其他补充机制(在LOX-1缺失的动物中)以及LOX-1介导感染的机制(在过度表达的动物中)。

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).