Parasite dissemination in Toxoplasmic encephalitis

 描述(申请人提供)：弓形虫是一种机会性寄生虫，感染全球三分之一的人口，是导致中枢神经系统感染并发艾滋病的最常见原因之一。弓形虫在中枢神经系统建立慢性感染，艾滋病毒感染造成的免疫损害会导致寄生虫重新激活和致命性脑炎。这项建议解决了临床艾滋病期间寄生虫发病机制知之甚少的一个方面--弓形虫是如何通过大脑迁移的？有证据表明，弓形虫感染迁徙的白细胞，通过一种“特洛伊木马”机制，促进了重要器官如大脑的定植。尤其是单核细胞是高度能动的细胞，也是弓形虫感染的目标。我们强有力的初步数据表明，弓形虫感染 人类单核细胞诱导了一种过度迁移的表型，并失调了被称为整合素的关键黏附分子的功能。该提案的目的是确定弓形虫是如何诱导单核细胞运动并通过大脑传播的。中心假设是，弓形虫通过利用感染细胞的迁移潜力在组织中传播。提出了两个目的来检验这一假设：1)确定弓形虫如何促进受感染细胞的迁移，2)确定弓形虫如何通过受感染的大脑传播。在第一个目标中，我们将研究从健康捐赠者和HIV患者感染的原代人单核细胞中整合素信号和焦点黏附形成的调节。我们将通过使用表达FAK的突变变体的单核细胞来确定FAK的作用，这些突变的FAK是结构性活跃的或非磷酸化的(无效)。我们还将确定支架分子他林和帕西林与FAK的相互作用，以及它们在迁移过程中在受感染的单核细胞中的组装和拆解动力学。在第二个目标中，我们将使用活体双光子成像和弓形虫感染的动物模型来确定被感染的髓系细胞类型(S)，并加强寄生虫通过小鼠大脑的传播。表达荧光标记的单核细胞或小胶质细胞的转基因小鼠将感染弓形虫。它们在实质中的迁移动态将在急性感染和慢性感染小鼠中确定，这些小鼠由于免疫抑制而接受寄生虫重新激活。所有必要的试剂都在实验室或商业上可以买到，所有的技术，包括生化和迁移分析、全内反射荧光(TIRF)显微镜和活体成像，目前都由我们的研究团队使用。这项研究的意义在于，了解弓形虫的传播可能允许这一过程的治疗靶点，以减轻艾滋病期间的疾病。这项提案具有创新性，因为它应用了多学科方法和新工具来确定介导弓形虫在体外和体内传播的细胞和分子机制，这最终将揭示艾滋病毒/艾滋病患者寄生虫致病的一个关键方面。

 DESCRIPTION (provided by applicant): Toxoplasma gondii is an opportunistic parasite that infects one-third of the global population and is among the most frequent causes of central nervous system (CNS) infection complicating AIDS. T. gondii establishes a chronic infection in the CNS, and immune compromise due to HIV infection leads to parasite reactivation and fatal encephalitis. This proposal addresses a poorly understood aspect of parasite pathogenesis during clinical AIDS - how does T. gondii migrate through the brain? Evidence suggests that T. gondii infection of migratory leukocytes facilitates the colonization of vital organs such as the brain via a 'Trojan horse' mechanism. Monocytes in particular are highly motile cells and a target for T. gondii infection. Our strong preliminary data demonstrate that T. gondii infection of human monocytes induces a hypermigratory phenotype and dysregulates the function of key adhesion molecules called integrins. The objective of this proposal is to define how T. gondii induces monocyte motility and disseminates through the brain. The central hypothesis is that T. gondii spreads through tissues by co-opting the migratory potential of infected cells. Two aims are proposed to test the hypothesis: 1) Determine how T. gondii enhances the migration of infected cells, and 2) Define how T. gondii disseminates through the infected brain. In the first aim, we will investigate the regulation of integrin signaling and focal adhesion formation in infected primary human monocytes from healthy donors and from HIV patients. We will determine the role of focal adhesion kinase (FAK) by using monocytes expressing mutant variants of FAK that are constitutively active or non-phosphorylatable (inactive). We will also determine the interactions of the scaffolding molecules talin and paxillin with FAK and the dynamics of their assembly and disassembly in infected monocytes during migration. In the second aim, we will use intravital two-photon imaging and an animal model for T. gondii infection to define the myeloid cell type(s) that are infected and potentiate parasite spread through the mouse brain. Transgenic mice that express fluorescently labeled monocytes or microglia will be infected with T. gondii. The dynamics of their migration through the parenchyma will be determined during acute infection and in chronically infected mice undergoing parasite reactivation due to immune suppression. All of the necessary reagents are on hand in the lab or commercially available, and all the techniques, including biochemical and migration assays, total internal reflection fluorescence (TIRF) microscopy, and intravital imaging, are currently employed by our research team. The significance of this research is that understanding T. gondii dissemination may allow for therapeutic targeting of this process to mitigate disease during AIDS. The proposal is innovative because it applies a multidisciplinary approach and novel tools to identify the cellular and molecular mechanisms that mediate T. gondii dissemination in vitro and in vivo, which will ultimately shed light on a key aspect of parasite pathogenesis in HIV/AIDS patients.