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

项目摘要 淋病奈瑟菌（Ng）的流行率、相关发病率以及无法治愈的 菌株，支持新的预防和治疗策略的迫切需要，这一重要的人类- 病原体这种发展需要对这种原型宿主的病理生物学有全面的了解- 适应性病原体我们发现，相可变，聚糖翻译后修饰的Ng表面 附属物皮利（菌毛）在人类感染中起着至关重要的作用。补体受体3（CR 3）是一种重要的 模式识别受体，其是介导人宫颈粘膜的Ng定殖的关键受体。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结构域的结合由菌毛蛋白连接聚糖（PLG）介导。这是一个开创性的发现， 免疫性，因为所有先前文献将CR 3碳水化合物结合到单独的所谓“凝集素”结构域。 这也是首次证明细菌蛋白质的翻译后修饰 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 免疫反应。本申请的目标是定义六个方面的具体贡献， 天然存在的PLG结构介导Ng-CR 3 I-结构域相互作用，并确定 这些相互作用中的每一种与女性感染的生物学相关性。根据初步数据，我们 假设可变PLG结构启动关键但高度不同的CR 3 I结构域结果 订婚我们将通过两个具体的目标来解决我们的假设：目标1）定义变异的影响， 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 对于六种天然Ng PLG结构，存在CR 3 I结构域。目的2）确定PLG-I的作用- 结构域相互作用对Ng发病机制和CR 3功能的影响;我们将定义靶向上皮细胞对 CR 3 I-结构域与具有不同PLG结构的Ng的结合及其对Ng发病机制的影响。我们 一种创新的方法是使用生物学相关的人原代宫颈细胞，单独和共培养 与低传代Ng分离物组合。这确保了所获得的数据与 人的过程。通过定义由I-结构域凝集素活性控制的效应器功能， 上皮细胞，我们将提供关于Ng发病机制，CR 3功能和细胞增殖的重要新信息。 控制人类宫颈粘膜免疫的基本机制。此外，我们将定义效用 靶向PLG-CR 3 I-结构域相互作用作为一种新的，改进的，靶向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的研究， 先天免疫，扩大了我们研究结果的重要性。我们在定义 Ng发病机制的分子机制。我们非常适合做这项工作，因为我们发现了CR 3， Ng宫颈感染的关键受体和皮利糖基化的生物合成途径。

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.