Novel Staphylococcal Inhibitors of Neutrophil Granule Enzymes

新型葡萄球菌中性粒细胞颗粒酶抑制剂

• 批准号: 9906231
• 负责人: Brian V Geisbrecht
• 金额: $ 28.88万
• 依托单位: KANSAS STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-04-01 至 2022-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9906231
• 关键词: Acids; Acute; Adult Respiratory Distress Syndrome; Affinity; Animals; Anti-Bacterial Agents; Anti-Inflammatory Agents; Area; Arterial Fatty Streak; Atherosclerosis; Azurophilic Granule; Bacteria; Basic Science; Binding; Biochemical; Biochemistry; Biological Assay; Blood; Cardiovascular system; Cells; Chronic; Chronic Obstructive Airway Disease; Cleaved cell; Collaborations; Complex; Cytoplasmic Granules; Cytosol; Data; Development; Digestion; Disease; Environment; Enzymes; Equilibrium; Event; Evolution; Extracellular Matrix; Family; Fostering; Foundations; Future; Genus staphylococcus; Goals; Gram-Positive Bacteria; Health; Heart; Human; Human body; Hydrogen Peroxide; Immune; Immune Evasion; Infection; Inflammation; Inflammatory; Innate Immune Response; Innate Immune System; Invaded; Laboratories; Lead; Length; Leukocyte Elastase; Leukocytes; Life; Low-Density Lipoproteins; Mediating; Methods; Modeling; Molecular Conformation; Morphology; Mutagenesis; Neutrophil Activation; Pathologic; Pattern; Peptide Hydrolases; Periodicity; Peroxidases; Play; Prevention strategy; Process; Property; Protease Inhibitor; Protein Dynamics; Proteins; Pulmonary Emphysema; Research; Respiratory Burst; Respiratory System; Rheumatoid Arthritis; Role; Sequence Homology; Series; Serine Protease; Serine Proteinase Inhibitors; Site; Staphylococcal Infections; Staphylococcus aureus; Stimulus; Structure; Structure-Activity Relationship; Surface; System; Tertiary Protein Structure; Testing; Therapeutic; Thrombus; Tissues; Virulence; Work; base; cell injury; chymotrypsin; cytotoxic; design; extracellular; healing; heme a; human disease; inhibitor/antagonist; nanomolar; neutrophil; novel; novel strategies; oxidative damage; pathogen; pathogenic bacteria; prevent; public health relevance; response; structured data; synthetic peptide; therapeutic target

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.

项目摘要/概要（项目描述） 人体依赖于中性粒细胞来提供针对细菌的杀菌先天免疫应答。 病原体虽然中性粒细胞在血液中以静止状态循环，但它们在血液中被迅速激活。 对许多生化模式作出反应，这些模式表明存在潜在的病原体， 已经发生细胞损伤。中性粒细胞的活化导致其形态的显著变化， 触发其胞质颗粒的动员和分泌。正是这些颗粒含有关键的 嗜中性粒细胞的抗菌成分。这些颗粒中最丰富的两种成分 是髓过氧化物酶（MPO），它将过氧化氢转化为细胞毒性的次卤酸， 以及一系列糜蛋白酶样丝氨酸蛋白酶（NSP），其可以通过以下方式直接攻击病原体细胞： 切割暴露在其表面或分泌到环境中的蛋白质。合在一起， MPO和NSP的作用形成了嗜中性粒细胞介导的针对入侵细菌的先天防御的基础。 作为宿主/病原体共同进化的结果，革兰氏阳性细菌金黄色葡萄球菌（Staphylococcus aureus） 已经开发出一系列强大的小蛋白抑制剂，可以有效地阻断许多关键成分， 人类先天免疫反应的一部分。在这方面，我们最近确定了三个分泌型葡萄球菌 蛋白，称为Eap、EapH 1和EapH 2（表示为“EAP蛋白”），其有效抑制NSP，以及 一种新的MPO葡萄球菌抑制剂SPIN。通过合作努力，我们已经确定， 最大S需要EAP蛋白和SPIN。在动物感染模型中的金黄色葡萄球菌毒力。在这 项目，我们将使用一系列的晶体学和溶液核磁共振方法，物理生物化学 方法和活性测定，以提供这些新的葡萄球菌的详细结构/功能信息， 中性粒细胞颗粒酶的抑制剂。我们将通过两个并行的具体目标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的选择性的结构基础， 检查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结构域蛋白抑制NSP的关系。在第二个目标中，我们将确定结构基础 对于SPIN/MPO结合，检查SPIN与MPO相互作用后是否发生构象变化， 并确定了SPIN介导MPO抑制的生化决定簇。最后，由于NSP和MPO 已知在许多人类炎性疾病中， 疾病，我们将探讨是否合成肽的基础上的结构EAP蛋白和SPIN结合 可以模拟这些葡萄球菌免疫逃避的有治疗价值的活性， proteins.通过本研究计划的完成，将为今后的发展奠定基础科学基础。 抗菌和抗炎疗法的基础上，我们发现这里的信息。

项目成果

The Staphylococcus aureus Extracellular Adherence Protein Eap Is a DNA Binding Protein Capable of Blocking Neutrophil Extracellular Trap Formation.

• DOI: 10.3389/fcimb.2018.00235
• 发表时间: 2018
• 期刊: Frontiers in cellular and infection microbiology
• 影响因子: 5.7
• 作者: Eisenbeis J;Saffarzadeh M;Peisker H;Jung P;Thewes N;Preissner KT;Herrmann M;Molle V;Geisbrecht BV;Jacobs K;Bischoff M
• 通讯作者: Bischoff M