Elucidating Perforin-2 mediated killing mechanisms against pathogenic bacteria

描述(申请人提供)：世界卫生组织估计，每年有超过70亿人死于传染病，是年轻人的头号死因。从根本上说，这些病例都源于个人的先天免疫系统无法消除感染，加上传染病病原体利用适应性免疫系统针对特定病原体所需的几天滞后期。尽管对先天免疫系统和获得性免疫系统的了解取得了巨大进展，但宿主与病原体的界面仍然是一个相对未被探索的前沿。到目前为止，在这个界面上研究得最好的一些细菌杀灭机制包括活性氧(ROS)、一氧化氮(NO)和水解酶。尽管有这样的知识体系，但关于这些成分如何相互作用以促进细菌杀灭的确切知识相对较少。最近发现了一个关于这些效应蛋白如何在宿主-病原体界面相互作用的潜在解释，并对巨噬细胞表达的Mpeg1进行了表征。结果表明，该基因编码一个蛋白结构域MACPF，该结构域预测了一个成孔的穿孔素样分子。两个已经被描述的免疫防御的成孔分子是补体的膜攻击复合体(MAC)，它杀死细胞外细菌，以及细胞毒性淋巴细胞的穿孔素-1，它杀死病毒感染的细胞和癌细胞。对Mpeg1的初步研究表明，mpeg1基因编码一种膜相关的成孔蛋白，命名为穿孔素-2(P2)。这些研究进一步表明，P2在所有造血细胞中都有成分表达，在所有细胞中都可以诱导表达。此外，P2似乎是一个重要的组成部分，因为用siRNA击倒P2可以阻止细胞内对病原菌的杀伤。我的目标是确定P2激活的机制，最终导致聚合和细胞内细菌死亡。我假设P2的细胞质结构域是激活和聚合的主要调节因子，细胞质Tail包含几个关键的结构域，经过翻译后修饰后被激活，细胞质Tail将与几个关键的适配蛋白相互作用，调节细胞内的聚合和定位。这项应用的目标是(I)利用功能性细胞内细菌Killig试验确定聚合发生所需的关键细胞质结构域和氨基酸残基；以及(Ii)利用功能损失研究和透射电子显微镜表征促进P2聚合的P2-细胞质结构域相互作用的蛋白质。这项工作的一个引人注目的方面是有可能描述宿主-病原体的界面，并将P2作为先前描述的细菌杀死范例中的常见介体。通过解开触发P2聚合和激活细菌攻击的分子机制，拟议的研究可能为治疗感染的药物干预提供新的靶点。

DESCRIPTION (provided by applicant): The World Health Organization estimates that over seven billion individuals succumb to infectious disease annually and that they are the number one cause of death in the young. Fundamentally, each of these cases stems from an inability of the individual's innate immune system to eliminate infection coupled with the infectious agent capitalizing on the lag period of several days required for the adaptive immune system to prime against the specific pathogen. Despite dramatic advances in the understanding of the innate and adaptive immune systems, the host-pathogen interface is a relatively unexplored frontier. To date, some of the best studied bacterial killing mechanisms at this interface include reactive oxygen species (ROS), nitric oxide (NO), and hydrolases. Notwithstanding this body of knowledge, there is relatively little knowledge of exactly how these components interact with one another to contribute to bacterial killing. A potential explanation for how these effector proteins interact at the host-pathogen interface was recently discovered, with the characterization of macrophage-expressed Mpeg1. It was revealed that this mRNA encodes a protein domain, MACPF, which predicts a pore-forming, perforin-like molecule. Two already described pore-forming molecules of immune defense are the membrane attack complex of complement (MAC) that kills extracellular bacteria, and Perforin-1 of cytotoxic lymphocytes that kills virus-infected and cancer cells. Preliminary studies with Mpeg1 demonstrated that Mpeg-1 cDNA encodes a membrane- associated pore-forming protein, designated Perforin-2 (P2). These studies further characterized P2 as being constitutively expressed in all hematopoietic cells, and inducible in all cells. Furthermore, P2 seems to be a vital component as knockdown of P2 with siRNA blocks intracellular killing of pathogenic bacteria. My goal is to determine the mechanisms of P2 activation, culminating in polymerization and intracellular bacterial killing. I hypothesize that the cytoplasmic domain of P2 is the master regulator of activation and polymerization, that the cytoplasmic tail contains several key domains that undergo post-translational modification to become activated, and that the cytoplasmic tail will interact with several key adaptor proteins to regulate polymerization as well as localization within cells. The objectives of this application are to (i) identify the key cytoplasmic domains and amino acid residues necessary for polymerization to occur utilizing a functional intracellular bacterial killig assay; and (ii) characterize the P2-cytoplasmic domain interacting proteins that promote polymerization of P2 utilizing loss of function studies and transmission electron microscopy. A compelling aspect of this work is the potential to describe the host-pathogen interface with the addition of P2 as a common mediator in the previously described bacterial killing paradigm. By unraveling the molecular mechanisms triggering P2 polymerization and activation of bacterial attack, the proposed studies may provide new targets for drug interventions in the treatment of infections.