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 先天免疫系统最为人所知的是对入侵微生物的基本防御。在人类中， 这种反应需要体液和细胞成分立即协同作用， 分别由补体系统和嗜中性粒细胞代表。有效地杀死微生物，所以- 称为“补体/嗜中性粒细胞轴”的免疫调节是基于一系列高度协调和逐步的分子调节， 识别事件和生化转化，这在其最基本的水平涉及酶。 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） 已经开发了一系列强大的小蛋白抑制剂，可以阻断许多中枢酶的作用， 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， EapH 1和EapH 2（表示为“EAP蛋白”），其有效地抑制三种不同的蛋白酶，称为 中性粒细胞丝氨酸蛋白酶（NSP）是中性粒细胞抗菌武器库的关键组分。在 除此之外，Eap本身也抑制了功能所需的多亚基蛋白酶系统的组装 经典和凝集素补体途径。另外，我们还发现了一种新的葡萄球菌蛋白， 称为“SPIN”，其是嗜中性粒细胞中发现的HOCl生成髓过氧化物酶（MPO）的有效抑制剂。 总的来说，这些S。金黄色葡萄球菌蛋白在体外系统和动物模型中都干扰细菌杀灭。 虽然我们对EAP蛋白和SPIN的初步研究提供了关于结构的重要信息， 这些新型酶抑制剂的功能和机制，许多重要的问题仍然存在。在这个项目中， 我们将采用结构、生物化学、功能和信息学方法的组合来解决这些问题。 问题.在第一系列的研究中，我们将确定S。金黄色 Eap抑制NSP。这将为Eap同源物EapH 1和EapH 2的比较分析提供一种手段， 其被更广泛地表征。我们还将努力定义Eap中的结构决定因素， 允许该蛋白质，而不是EapH 1或EapH 2，抑制除NSP之外的补体系统。在第二 通过一系列的研究，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蛋白中的结构决定因素， 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。我们的最终 通过一系列的研究，我们将利用我们对SPIN和葡萄球菌的广泛的结构和功能数据， 补体抑制剂对建立理解宿主物种的物理基础的范例 毒力蛋白的特异性。通过完成本研究计划，我们将进一步了解两个新的 在宿主/病原体界面发挥作用的酶抑制剂的类别，并奠定了基础科学基础 为将来开发抗菌和抗炎治疗所产生的信息，我们发现。