Novel Enzyme Inhibitors in the Immune Evasion Repertoire of Staphylococcus aureus (Equipment Supplement)

金黄色葡萄球菌免疫逃逸的新型酶抑制剂（设备补充）

• 批准号: 10796329
• 负责人: Brian V Geisbrecht
• 金额: $ 7.65万
• 依托单位: KANSAS STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-05-01 至 2026-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10796329
• 关键词: Acids; Address; Adopted; Animal Model; Anti-Bacterial Agents; Anti-Inflammatory Agents; Basic Science; Binding; Biochemical; Complement; Complement Inactivators; Data; Development; Disease; Enzyme Inhibitor Drugs; Enzymes; Equipment; Event; Family; Foundations; Future; Genus staphylococcus; Gram-Positive Bacteria; Heme; Homologous Gene; Human; Hydrogen Peroxide; Immune; Immune Evasion; In Vitro; Individual; Infection; Informatics; Innate Immune Response; Innate Immune System; Invaded; Investigation; Lectin; Microbe; Molecular Conformation; Peptide Hydrolases; Peroxidases; Proteins; Research; Series; Serine Protease; Species Specificity; Staphylococcus aureus; Structure; System; Therapeutic; Virulence; Work; comparative; complement pathway; complement system; cytotoxic; inhibitor; insight; molecular recognition; neutrophil; novel; novel strategies; pathogen; prevent; public health relevance; response; structural determinants

PROJECT SUMMARY The innate immune system is best known as an essential defense against invading microbes. In humans, this response requires an immediate and concerted action by both humoral and cellular components, which are represented by the complement system and neutrophils, respectively. Efficient killing of microbes by this so- called “complement/neutrophil axis” is predicated upon a highly orchestrated and stepwise series of molecular recognition events and biochemical transformations, which at their most fundamental level involve enzymes. As a consequence of host/pathogen co-evolution, the Gram-positive bacterium Staphylococcus aureus has developed a powerful array of small protein inhibitors that block many of the central enzymatic players of the innate immune response. In this regard, we identified three secreted staphylococcal proteins, called Eap, EapH1, and EapH2 (denoted “EAP proteins”), which potently inhibit three different proteases known as Neutrophil Serine Proteases (NSPs) that are critical components of the neutrophil’s anti-bacterial arsenal. In addition to this, Eap itself also inhibits assembly of a multi-subunit protease system that is required for function of the classical and lectin complement pathways. Separately, we also identified a new staphylococcal protein, called “SPIN”, that is a potent inhibitor of the HOCl-generating myeloperoxidase (MPO) found in neutrophils. Collectively, these S. aureus proteins interfere with bacterial killing in both in vitro systems and animal models. While our initial studies on EAP proteins and SPIN have provided important information on the structure, function, and mechanism of these novel enzyme inhibitors, many significant questions still remain. In this project, we will employ a combination of structural, biochemical, functional, and informatics approaches to address these issues. In the first series of investigations, we will determine how the individual repeating domains of S. aureus Eap inhibit NSPs. This will provide a means for comparative analysis to the Eap homologs, EapH1 and EapH2, which are more extensively characterized. We will also work to define the structural determinants within Eap that allow this protein, but not EapH1 or EapH2, to inhibit the complement system in addition to NSPs. In the second series of investigations, we will explore the structural transitions that allow SPIN to adopt an inhibitory conformation upon binding to MPO. We will also define the structural determinants within SPIN proteins that provide an exquisite level of selectivity for MPO when compared to closely related heme peroxidases. In our final series of studies, we will leverage our extensive structural and functional data on SPINs and staphylococcal complement inhibitors toward establishing a paradigm for understanding the physical basis for host species specificity of virulence proteins. By completing this research plan, we will further our understanding of two novel classes of enzyme inhibitors that function at the host/pathogen interface, and lay the basic science foundation for future development of anti-bacterial and anti-inflammatory therapies arising from the information we uncover.

项目摘要 先天免疫系统最著名的是针对入侵微生物的基本防御。在人类中 这种响应需要受到体液和细胞组件的立即和协同的作用，这是 分别由完成系统和中性粒细胞表示。有效地杀死微生物 在高度精心策划和逐步的分子系列中预测称为“补体/中性粒细胞轴” 识别事件和生化转化在其最基本的水平上涉及酶。 由于宿主/病原体共同进化，革兰氏阳性细菌金黄色葡萄球菌 已经开发了强大的小蛋白抑制剂，这些抑制剂阻止了许多中心酶促玩家 先天免疫反应。在这方面，我们确定了三种分泌的葡萄球菌蛋白，称为EAP， EAPH1和EAPH2（表示为“ EAP蛋白”），它们可能抑制三种不同的蛋白 中性粒细胞丝氨酸蛋白酶（NSP）是中性粒细胞抗细菌武器库的关键成分。 除此之外，EAP本身还抑制了功能所需的多生产蛋白酶系统的组装 另外，我们还确定了一种新的葡萄球菌蛋白 称为“自旋”，这是中性粒细胞中发现的HOCL生成髓过氧化物酶（MPO）的潜在抑制剂。 总体而言，这些金黄色葡萄球菌蛋白会干扰细菌在体外系统和动物模型中杀死细菌。 尽管我们对EAP蛋白和SPIN的最初研究提供了有关结构的重要信息，但 这些新型酶抑制剂的功能和机制，仍然存在许多重要的问题。在这个项目中， 我们将采用结构，生化，功能和信息性方法的结合来解决这些方法 问题。在第一个系列投资中，我们将确定金黄色葡萄球菌的单个重复域 EAP抑制NSP。这将为EAP同源物（EAPH1和EAPH2）提供比较分析的手段 更广泛的表征。我们还将努力定义EAP中的结构决定者 除了NSP之外，还要允许该蛋白质（而不是EAPH1或EAPH2）抑制完成系统。在第二个 一系列投资，我们将探索允许旋转采用抑制作用的结构过渡 与MPO结合后的构象。我们还将定义自旋蛋白中的结构确定剂 与密切相关的血红素过氧化物酶相比，MPO的独家选择性水平。在我们的决赛中 一系列研究，我们将利用有关旋转和葡萄球菌的广泛结构和功能数据 补充抑制剂建立范式以理解宿主物种的物理基础 病毒蛋白的特异性。通过完成该研究计划，我们将进一步了解两本小说 在宿主/病原体界面起作用的酶抑制剂类别，并奠定基础科学基础 为了未来开发我们发现的信息引起的抗细菌和抗炎疗法。