Novel Staphylococcal Inhibitors of Neutrophil Granule Enzymes

项目摘要/摘要(项目描述) 人体依靠中性粒细胞来提供对细菌的杀菌先天免疫反应。 病原体。尽管中性粒细胞在血液中以静止状态循环，但它们在体内迅速激活 对一系列生化模式的反应，这些模式意味着潜在病原体的存在或 已经发生了细胞损伤。中性粒细胞的激活导致其形态发生显著变化，并且 触发其胞浆颗粒的动员和分泌。正是这些颗粒包含了关键的 中性粒细胞抗菌库的成分。这些颗粒中最辅助的两种成分 是髓过氧化物酶(MPO)，它能将过氧化氢转化为细胞毒性的次卤酸， 和一系列凝乳酶样丝氨酸蛋白酶(NSPs)，它们可以通过 裂解蛋白质，暴露在其表面或分泌到环境中。合在一起，结合起来 MPO和NSPs的作用构成了中性粒细胞介导的天然防御入侵细菌的基础。 作为宿主/病原体共同进化的结果，革兰氏阳性菌金黄色葡萄球菌 已经开发出一系列强大的小蛋白抑制剂，有效地阻止了许多关键成分 人类的先天免疫反应。在这方面，我们最近鉴定出三种分泌性葡萄球菌 被称为EAP、EapH1和EapH2的蛋白质(表示为“EAP蛋白质”)，它们有效地抑制NSP，以及 新型葡萄球菌对MPO的抑制作用，称为SPIN。通过合作努力，我们已经建立了 在动物感染模型中，EAP蛋白和SPIN都是金黄色葡萄球菌最大毒力所必需的。在这 项目中，我们将使用一系列协同的结晶学和溶液核磁共振方法、物理生物化学 方法和活性分析，以提供这些新型葡萄球菌的详细结构/功能信息 中性粒细胞颗粒酶抑制剂。我们将通过同时进行的两个具体项目来实现这一总体目标 目标。在第一个目标中，我们将研究EAP结构域对NSP选择性的结构基础， 研究蛋白质动力学的变化是否影响EAP/NSP相互作用，并定义结构/活性 EAP结构域蛋白与NSP抑制的关系。第二个目标，我们将确定结构基础 对于Spin/MPO结合，检查Spin在与MPO相互作用时是否经历构象变化， 并确定了通过SPIN介导MPO抑制的生化决定因素。最后，由于NSP和MPO 在许多人类炎症性疾病中，在破坏宿主细胞和组织方面发挥着重要作用 疾病，我们将探索合成肽是否基于EAP蛋白的结构和自旋结合 可以模拟这些葡萄球菌免疫逃避的具有治疗价值的活动 蛋白质。通过完成这项研究计划，我们将为未来的发展奠定基础科学基础 基于我们在这里发现的信息的抗菌和抗炎疗法。

Project Abstract/Summary (Project Description) The human body relies on neutrophils to provide a sterilizing innate immune response against bacterial pathogens. Although neutrophils circulate inside the blood in a quiescent state, they are rapidly activated in reponse to a number of biochemical patterns which signify either that potential pathogens are present or that cellular damage has occured. Activation of neutrophils results in remarkable changes in their morphology, and triggers mobilization and secretion of their cytosolic granules. It is these granules which contain critical components of the neutrophil’s anti-bacterial arsenal. Two of the most abudant components of these granules are the enzyme myeloperoxidase (MPO), which converts hydrogen peroxide into cytotoxic hypohalous acids, and a series of chymotrypsin-like serine proteases (NSPs), which can directly attack the pathogen cell by cleaving proteins that are either exposed on its surface or secreted into the environment. Together, the combined action of MPO and NSPs form the foundation of neutrophil-mediated innate defense against invading bacteria. As a consequence of host/pathogen co-evolution, the Gram-positive bacterium Staphylococcus aureus has developed a powerful array of small protein inhibitors that effectively block many of the critical components of the human innate immune response. In this regard, we recently identified three secreted staphylococcal proteins, called Eap, EapH1, and EapH2 (denoted “EAP proteins”), which potently inhibit NSPs, as well as a novel staphylococccal inhibitior of MPO, called SPIN. Through collaborative efforts, we have established that both EAP proteins and SPIN are required for maximal S. aureus virulence in animal infection models. In this project, we will use a synergistic series of crystallographic and solution NMR methods, physical biochemistry approaches, and activity assays to provide detailed structure/function information on these novel staphylococcal inhibitors of neutrophil granule enzymes. We will accomplish this overall goal through two concurrent Specific Aims. In the first Aim, we will investigate the structural basis for the selectivity of EAP domains toward NSPs, examine whether changes in protein dynamics influence EAP/NSP interactions, and define a structure/activity relationship for NSP inhibition by EAP domain proteins. In the second Aim, we will determine the structural basis for SPIN/MPO binding, examine whether SPIN undergoes changes in conformation upon interaction with MPO, and define the biochemical determinants which mediate MPO inhibition by SPIN. Finally, since NSPs and MPO are known to play signifcant roles in damaging host cells and tissues in a number of human inflammatory diseases, we will explore whether synthetic peptides based upon the structures of EAP proteins and SPIN bound to their targets can mimic the therapeutically-valuable activities of these staphylococcal immune evasion proteins. By completing this research plan, we will lay the basic science foundation for future development of anti-bacterial and anti-inflammatory therapies based upon the information that we uncover here.