Novel Enzyme Inhibitors in the Innate Immune Evasion Repertoire of Staphylococci

项目摘要 最广为人知的是，先天免疫系统是抵御入侵微生物的重要防御系统。在人类身上， 这种反应需要体液和细胞成分立即采取协调一致的行动，这两个部分是 分别以补体系统和中性粒细胞为代表。通过这种方法有效地杀灭微生物- 所谓的补体/中性粒细胞轴是基于一系列高度协调和循序渐进的分子。 识别事件和生化转化，在其最基本的层面上涉及酶。 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，可以阻止许多核心的酶作用于 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(表示为“EAP蛋白”)，它们能有效地抑制三种不同的蛋白酶，即 中性粒细胞丝氨酸蛋白酶(NSP)是中性粒细胞抗菌库的关键成分。在……里面 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 经典的和凝集素互补的途径。另外，我们还鉴定了一种新的葡萄球菌蛋白， 它被称为“Spin”，是一种在中性粒细胞中发现的产生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在体外系统和动物模型中都会干扰细菌的杀灭。 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， 关于这些新型酶抑制剂的功能和作用机制，仍有许多重要的问题。在这个项目中， 我们将采用结构、生化、功能和信息学方法的组合来解决这些问题 问题。在第一系列研究中，我们将确定金黄色葡萄球菌的单个重复结构域是如何 EAP对NSP有抑制作用。这将提供对EAP同系物、EapH1和EapH2进行比较分析的手段， 它们有更广泛的特征。我们还将努力定义EAP中的结构性决定因素 除了NSP外，还允许该蛋白质抑制补体系统，而不是EapH1或EapH2。在第二个 一系列的研究，我们将探索允许自旋采用抑制性的结构转变 与MPO结合后的构象。我们还将定义自旋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，MPO具有极高的选择性。在我们的决赛中 在一系列研究中，我们将利用我们关于旋转和葡萄球菌的广泛的结构和功能数据 补体抑制剂有助于建立理解寄主物种的物理基础的范例 毒力蛋白的特异性。通过完成这一研究计划，我们将加深对两部小说的理解 在宿主/病原体界面起作用并奠定基础科学基础的酶抑制剂的类别 关于未来抗菌和抗炎疗法的发展，根据我们发现的信息。

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.