Host Responses to Mycobacterium Infection in Zebrafish

描述(申请人提供)：结核病是全世界传染病死亡的主要原因，由于艾滋病毒流行，其后果越来越严重。现有的卡介苗疫苗效果微乎其微。感染和疾病是由细胞内的结核分枝杆菌引起的。面对复杂的免疫反应，细菌可以无限期地存在于肉芽肿、高度分化的巨噬细胞和其他免疫细胞的紧密聚集中。与大多数传染病一样，宿主免疫的不同方面对结核感染和疾病的影响尚不清楚。为了更好地了解宿主对结核感染的反应，我们将利用这样一个事实，即结核杆菌的近亲海洋分枝杆菌是斑马鱼的天然病原体，导致一种类似结核病的疾病。斑马鱼是基因易驯化的脊椎动物，被用作疾病和发育的模型。我们利用斑马鱼胚胎的光学透明性来实时观察胚胎巨噬细胞对海洋分枝杆菌的感染。这种感染在许多方面与成人结核病相似。我们将利用斑马鱼-M、海豚感染模型来研究宿主免疫基因在感染中的作用。我们将使用整体原位杂交来确定哪些被认为在结核病中重要的宿主免疫标记物在发育中的胚胎中表达。我们将确定它们的表达在感染过程中是否发生了变化。我们将使用吗啡技术使这些基因失活，并通过实时可视化确定它们何时以及如何在感染中发挥作用。我们将确定它们在感染的不同方面所起的作用，从早期巨噬细胞对海洋分枝杆菌的反应到肉芽肿的形成。在单个宿主基因功能失活的情况下，实时观察斑马鱼胚胎感染的能力将使我们能够以前所未有的细节研究宿主免疫系统的作用。拟议中的实验将内在地产生关于各种免疫基因在斑马鱼发育中所起作用的新信息。

DESCRIPTION (provided by applicant): Tuberculosis is the leading cause of death from infectious diseases worldwide, with increasing consequences due to the HIV epidemic. The existing vaccine, BCG, has marginal efficacy. Infection and disease are caused by the intracellular bacterial pathogen Mycobacterium tuberculosis. In the face of a complex immune response, the bacteria can persist indefinitely in granulomas, tight aggregates of highly differentiated macrophages and other immune cells. As with most infectious diseases, the impact of the different aspects of host immunity on tuberculous infection and disease are not well understood. To better understand the host responses to tuberculous infections, we will exploit the fact that Mycobacterium marinum, a close relative of M. tuberculosis is a natural pathogen of zebrafish, causing a tuberculosis-like disease. Zebrafish are genetically tractable vertebrates that are used as models of disease and development. We have exploited the optical transparency of the zebrafish embryo to visualize M. marinum infection of embryonic macrophages in real time. The infection parallels adult tuberculosis in many ways. We will use the zebrafish-M, marinum infection model to study the role of host immune genes in infection. We will use whole mount in situ hybridizations to determine which host immune markers thought to be important in tuberculosis, are expressed in the developing embryo. We will determine if their expression is changed during infection. We will inactivate these genes using morpholino technology and determine by real time visualization when and how they act in infection. We will determine their role for the different facets of infection, from early macrophage migration in response to M. marinum to granuloma formation. The ability to visualize infection in real time in zebrafish embryos with functional inactivation of individual host genes will allow us to study the role of the host immune system in unprecedented detail. The proposed experiments will inherently yield new information about the role of various immune genes in zebrafish development.