Novel carbohydrate binding functions of the CR3 I-domain modulate gonococcal-cervical cell interactions

CR3 I 结构域的新型碳水化合物结合功能调节淋球菌-宫颈细胞相互作用

• 批准号: 10078936
• 负责人: Jennifer L Edwards
• 金额: $ 46.5万
• 依托单位: RESEARCH INST NATIONWIDE CHILDREN'S HOSP
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-01-16 至 2022-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10078936
• 关键词: Adhesions; Affect; Agonist; Bacteria; Bacterial Proteins; Behavior; Binding; Binding Sites; Biological; C3bi; Calorimetry; Carbohydrates; Cell Communication; Cells; Centers for Disease Control and Prevention (U.S.); Cervical; Cervicitis; Cervix Uteri; Cessation of life; Coculture Techniques; Complement; Data; Development; Diagnosis; Disease; Ensure; Epithelial; Epithelial Cells; Event; Female; Fimbriae Proteins; Foundations; Frequencies; Future; Goals; Gonorrhea; HIV Infections; Health; Human; Immune; Immune response; Immune system; Immunity; Immunologic Receptors; In Vitro; Infection; Infertility; Innate Immune Response; Lectin; Ligands; Link; Literature; Macrophage-1 Antigen; Mass Spectrum Analysis; Mediating; Mediator of activation protein; Medical Care Costs; Molecular; Morbidity - disease rate; Mucosal Immunity; Mucous Membrane; Multi-Drug Resistance; Natural Immunity; Nature; Neisseria gonorrhoeae; Outcome; Pathogenesis; Pathway interactions; Pattern recognition receptor; Pelvic Inflammatory Disease; Peptides; Phagocytes; Phase; Pilum; Play; Polysaccharides; Post-Translational Protein Processing; Prevalence; Prevention; Preventive measure; Process; Proteins; Public Health; Recombinants; Reporting; Role; Seminal; Signal Transduction; Structure; Surface; Surface Plasmon Resonance; Testing; Therapeutic; United States; Ursidae Family; Variant; Woman; Work; appendage; base; experience; glycosylation; human disease; human pathogen; improved; innate immune function; innovation; insight; macrophage; microbial; neutrophil; novel; novel strategies; novel therapeutics; pathogen; pathogenic bacteria; prevent; receptor; reproductive tract; response; sugar; trafficking; vaccine development

PROJECT SUMMARY The prevalence of Neisseria gonorrhoeae (Ng), its associated morbidity, and the emergence of untreatable strains, support a critical need for new preventative and therapeutic strategies for this important human- pathogen. Such developments require a complete understanding of the pathobiology of this archetypal, host- adapted pathogen. We show that phase-variable, glycan post-translational modifications on Ng surface appendages, pili (fimbriae), play a vital role in human infection. Complement receptor 3 (CR3) is an important pattern recognition receptor that is the key receptor mediating Ng colonization of human cervical mucosa. CR3 contains an “I-domain” region, which is known as a binding site for protein ligands. However, we found that Ng binding to the I-domain is mediated by the pilin-linked glycan (PLG). This is a seminal finding in innate immunity, as all previous literature ascribe CR3 carbohydrate binding to a separate, so called "lectin" domain. This was also the first demonstration that post-translational modifications made to a bacterial protein modulate pathogenesis. These findings will impact our understanding of microbial pathogenesis and innate immune responses. The goals of the present application are to define the specific contribution of the six, naturally occurring, PLG structures in mediating the Ng-CR3 I-domain interaction and to determine the biological relevance of each of these interactions to infection in females. Guided by strong preliminary data, we hypothesize that variable PLG structures initiate key, but highly divergent, outcomes with CR3 I-domain engagement. We will resolve our hypothesis through two specific aims: Aim 1) Define the effect of variation in pilin glycan structure on direct PLG-I-domain interactions; we will define the specific molecular interactions occurring with the CR3 I-domain for the six natural Ng PLG structures. Aim 2) Define the effect of PLG-I- domain interactions on Ng pathogenesis and CR3 function; we will define targeted epithelial cell responses to CR3 I-domain engagement by Ng that bear different PLG structures and their effect on Ng pathogenesis. Our approach is innovative in using biologically relevant human primary cervical cells, both alone and in co-culture with phagocytes, combined with low passage Ng isolates. This ensures that data obtained are relevant to human processes. By defining the effector functions controlled by I-domain lectin activity on primary human epithelial cells, we will provide critical new information regarding Ng pathogenesis, CR3 function, and the fundamental mechanisms that govern human cervical mucosal immunity. Moreover, we will define the utility of targeting the PLG-CR3 I-domain interaction as a new, and improved, host-targeted approach to treat and/or prevent Ng disease in women. Our studies will very likely impact work on other human pathogens and on innate immunity, broadening the significance of our outcomes. We have extensive experience in defining the molecular mechanisms of Ng pathogenesis. We are ideally placed to do this work in having discovered CR3 as the key receptor for Ng cervical infection and the biosynthetic pathways for pili glycosylation.

项目概要 淋病奈瑟菌 (Ng) 的流行、相关发病率以及无法治疗的疾病的出现 菌株，支持对这一重要人类的新预防和治疗策略的迫切需求 病原。这种发展需要对这种原型宿主的病理学有完整的了解。 适应性病原体。我们证明了 Ng 表面上的相变聚糖翻译后修饰 附属物菌毛（菌毛）在人类感染中发挥着至关重要的作用。补体受体 3 (CR3) 是一个重要的 模式识别受体是介导人宫颈粘膜 Ng 定植的关键受体。 CR3 包含一个“I-结构域”区域，该区域被称为蛋白质配体的结合位点。然而，我们发现 Ng 与 I 结构域的结合是由菌毛蛋白连接聚糖 (PLG) 介导的。这是先天性方面的一个开创性的发现 免疫，因为所有以前的文献都将 CR3 碳水化合物结合到一个单独的所谓“凝集素”结构域上。 这也是首次证明对细菌蛋白质进行翻译后修饰 调节发病机制。这些发现将影响我们对微生物发病机制和先天性的理解。 免疫反应。本申请的目标是定义六者的具体贡献， 天然存在的 PLG 结构介导 Ng-CR3 I 结构域相互作用并确定 这些相互作用与女性感染的生物学相关性。在强有力的初步数据的指导下，我们 假设可变的 PLG 结构引发 CR3 I 结构域的关键但高度不同的结果 订婚。我们将通过两个具体目标来解决我们的假设： 目标 1) 定义变化的影响 菌毛蛋白聚糖结构对直接 PLG-I 结构域相互作用的影响；我们将定义特定的分子相互作用 与六种天然 Ng PLG 结构的 CR3 I 结构域一起出现。目标 2) 定义 PLG-I- 的效果 Ng 发病机制和 CR3 功能的结构域相互作用；我们将定义靶向上皮细胞反应 具有不同 PLG 结构的 Ng 与 CR3 I 结构域的结合及其对 Ng 发病机制的影响。我们的 该方法在使用生物学相关的人类原代宫颈细胞（单独培养和共培养）方面具有创新性 与吞噬细胞结合，与低传代 Ng 分离株结合。这确保了获得的数据与 人类过程。通过定义原代人类 I 域凝集素活性控制的效应器功能 上皮细胞，我们将提供有关 Ng 发病机制、CR3 功能和 控制人类宫颈粘膜免疫的基本机制。此外，我们将定义效用 将 PLG-CR3 I 结构域相互作用作为一种新的、改进的、针对宿主的方法来治疗和/或 预防女性NG病。我们的研究很可能会影响其他人类病原体和 先天免疫，扩大了我们结果的意义。我们在定义 Ng发病机制的分子机制。我们非常适合开展这项工作，因为我们发现了 CR3 Ng 宫颈感染的关键受体和菌毛糖基化的生物合成途径。