Novel Enzyme Inhibitors in the Innate Immune Evasion Repertoire of Staphylococci

葡萄球菌先天免疫逃避库中的新型酶抑制剂

• 批准号: 10576908
• 负责人: Brian V Geisbrecht
• 金额: $ 36.33万
• 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/10576908
• 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; 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 ABSTRACT 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 先天免疫系统被认为是抵御微生物入侵的重要防御手段。在人类中， 这种反应需要体液和细胞成分立即采取协调一致的行动，这些成分是 分别由补体系统和中性粒细胞代表。通过这种方式有效杀灭微生物 称为“补体/中性粒细胞轴”的基础是高度协调和逐步的一系列分子 识别事件和生化转化，其最基本的水平涉及酶。 由于宿主/病原体共同进化，革兰氏阳性菌金黄色葡萄球菌 开发了一系列强大的小蛋白质抑制剂，可以阻断许多核心酶的作用 先天免疫反应。在这方面，我们鉴定了三种分泌型葡萄球菌蛋白，称为 Eap， EapH1 和 EapH2（称为“EAP 蛋白”），可有效抑制三种不同的蛋白酶 中性粒细胞丝氨酸蛋白酶 (NSP) 是中性粒细胞抗菌库的关键组成部分。在 除此之外，Eap 本身还抑制功能所需的多亚基蛋白酶系统的组装 经典补体途径和凝集素补体途径。另外，我们还鉴定了一种新的葡萄球菌蛋白， 称为“SPIN”，是中性粒细胞中产生 HOCl 的髓过氧化物酶 (MPO) 的有效抑制剂。 总的来说，这些金黄色葡萄球菌蛋白会干扰体外系统和动物模型中的细菌杀灭。 虽然我们对 EAP 蛋白和 SPIN 的初步研究提供了有关结构的重要信息， 这些新型酶抑制剂的功能和机制，仍然存在许多重大问题。在这个项目中， 我们将结合使用结构、生化、功能和信息学方法来解决这些问题 问题。在第一个系列的研究中，我们将确定金黄色葡萄球菌的各个重复结构域如何 Eap 抑制 NSP。这将为 Eap 同源物 EapH1 和 EapH2 的比较分析提供方法， 具有更广泛的特征。我们还将努力定义 Eap 内的结构决定因素 除了 NSP 之外，还允许该蛋白（但不包括 EapH1 或 EapH2）抑制补体系统。在第二个 通过一系列研究，我们将探索允许 SPIN 采用抑制性的结构转变 与 MPO 结合后的构象。我们还将定义 SPIN 蛋白内的结构决定因素 与密切相关的血红素过氧化物酶相比，它对 MPO 具有极高的选择性。在我们的决赛中 系列研究中，我们将利用关于 SPIN 和葡萄球菌的广泛结构和功能数据 补体抑制剂有助于建立理解宿主物种物理基础的范例 毒力蛋白的特异性。通过完成这个研究计划，我们将进一步了解两种新颖的 在宿主/病原体界面发挥作用的酶抑制剂类别，并奠定基础科学基础 从我们发现的信息中得出抗菌和抗炎疗法的未来发展。