Modulation of epithelial cell response by P. gingivalis

牙龈卟啉单胞菌对上皮细胞反应的调节

基本信息

批准号：
8242902
负责人：
OZLEM YILMAZ
金额：
$ 37.14万
依托单位：
UNIVERSITY OF FLORIDA
依托单位国家：
美国
项目类别：
财政年份：
2006
资助国家：
美国
起止时间：
2006-03-01 至 2017-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8242902
关键词：
Address Affect Agonist Anaerobic Bacteria Apoptosis Apoptotic Bacteria Biochemical Biological Process CREB1 gene Cell Death Cell Proliferation Cell physiology Cells Cellular Stress Chronic Complex Coupled Data Development Disease Enzymes Epithelial Cells Epithelium Event Fluorescence Generations Genes Gingiva Goals Growth Homologous Gene Human Image Analysis Immune Immune response Immune system Infection Invaded Kinetics Knowledge Life Ligation Mediating Mediator of activation protein Metabolic Metabolic Pathway Mitochondria Molecular Mycobacterium tuberculosis NADPH Oxidase Nucleoside-Diphosphate Kinase Oral Oral cavity Oral mucous membrane structure Organism Outcome Oxidative Stress Oxidative Stress Pathway Pathway interactions Periodontal Diseases Permeability Physiological Play Population Porphyromonas gingivalis Process Production Proteins Pseudomonas aeruginosa Public Health Purinoceptor Reactive Oxygen Species Receptor Signaling Regulation Regulatory Pathway Reporter Research Resolution Role Severities Signal Pathway Signal Transduction Small Interfering RNA Source Stress System Therapeutic Agents Time Tissues Translating cellular imaging cytochrome c cytokine extracellular inhibitor/antagonist insight killings macrophage microbial microorganism mutant novel novel therapeutic intervention oral tissue pathogen receptor response trafficking transcription factor

项目摘要

DESCRIPTION (provided by applicant): Periodontal diseases are prevalent destructive oral polymicrobial infections that remain a significant public health burden. Porphyromonas gingivalis is a gram (-) anaerobe and a major etiological agent in severe forms of the disease. The organism is a successful colonizer of gingival epithelial cells (GECs), which form an initial defense to invading pathogens while serving as primary target cells for opportunistic bacteria. P. gingivalis is a host-adapted pathogen that can survive and replicate in primary GECs, and later spread intercellularly. The infection inhibits GEC death by impacting mitochondrial-apoptotic pathways and suppressing ATP-P2X7 receptor signaling. Ligation of P2X7 receptors with the "danger signal", extracellular ATP (eATP), has recently been shown to result in production of intracellular reactive-oxygen-species (ROS) in macrophages. Although previously known mainly as a toxic agent, ROS are increasingly recognized to play an important physiological role by modulating a variety of key cellular functions including apoptosis, immune responses, and intracellular infections. P. gingivalis secretes an effector, nucleoside diphosphate kinase (Ndk), which scavenges eATP and diminishes P2X7 activity. We have recently found that P. gingivalis modulates cytosolic ROS production and subsequently blocks eATP-induced oxidative stress in primary GECs via secretion of Ndk during infection. However, the Ndk-deficient mutant of P. gingivalis lacks the ability to inactivate eATP-induced oxidative stress and persist intracellularly. The eATP-induced ROS generation appears to be mediated by P2X7 receptor signaling coupled with NADPH oxidase and mitochondrial oxidative stress pathways. The goal of this study is: to define the basic host mechanisms which P. gingivalis utilizes to inhibit eATP-induced cellular oxidative stress and to characterize the role of Ndk in targeting specific host metabolic and regulatory pathways potentially critical for the organism's survival and evasion of eATP- mediated intracellular killing. The kinetics and source of the ROS in primary GECs will be determined by flow-cytofluorimetry, fluorescence cell imaging, and high-resolution Oxygraph. We will identify infection- targeted host molecular circuitries and characterize the spatio-temporal secretion and function of Ndk employing a combination of selective inhibitors, agonists, gene depletion by siRNA, q-PCR, immuno- biochemical and fluorescent protein reporter systems in conjunction with confocal quantitative-image analyses. Finally, we will elucidate manipulation of eATP signaling by P. gingivalis for intracellular growth and survival. These studies will provide a detailed characterization of previously unexplored host molecular networks targeted by Ndk, an effector of P. gingivalis, for persistence in gingival epithelium. The knowledge gained may translate into the development of specific physiological inhibitors that may control or reduce the severity of chronic infections caused by this opportunistic pathogen. PUBLIC HEALTH RELEVANCE: P. gingivalis is a major contributor to severe forms of periodontal diseases, which are among the most common chronic infections affecting the U.S. population. The proposed research aims to define the basic complex molecular events between P. gingivalis and human gingival cells that are infected by this pathogen. These studies will provide valuable data needed to develop specific therapeutic agents to subvert unfavorable interactions with host cells and control the growth and colonization of this microorganism in oral tissues.

描述（由申请人提供）：牙周疾病是普遍的破坏性口腔多生物性感染，仍然是巨大的公共卫生负担。牙龈卟啉单胞菌是一种克（ - ）厌氧菌，是严重的疾病形式的主要病因。生物体是牙龈上皮细胞（GEC）的成功菌落，它在作为机会性细菌的主要靶细胞时构成了入侵病原体的初始防御。牙龈疟原虫是一种宿主适应的病原体，可以在原发性GEC中生存并复制，然后间互细胞扩散。感染通过影响线粒体 - 凋亡途径并抑制ATP-P2X7受体信号传导来抑制GEC死亡。最近已显示具有“危险信号”的P2X7受体（EATP）的结扎受体，可在巨噬细胞中产生细胞内活性氧（ROS）。尽管以前主要称为有毒药物，ROS越来越多地通过调节包括细胞凋亡，免疫反应和细胞内感染在内的各种关键细胞功能来发挥重要生理作用。牙龈疟原虫分泌一种效应子，核苷双磷酸激酶（NDK），它去吃掉并减少P2X7活性。我们最近发现，牙龈疟原虫会通过感染期间的NDK分泌来调节胞质ROS的产生，随后通过NDK分泌来阻止EATP诱导的氧化应激。然而，牙龈疟原虫的NDK缺陷突变体缺乏使EATP诱导的氧化应激和细胞内持续存在的能力。 EATP诱导的ROS产生似乎是由P2X7受体信号传导与NADPH氧化酶和线粒体氧化应激途径相连的。这项研究的目的是：定义牙龈疟原虫用来抑制EATP诱导的细胞氧化应激的基本宿主机制，并表征NDK在靶向特定宿主代谢和调节途径中的作用，这可能对生物体的生存和逃避食用PEATP介导的细胞内杀伤至关重要。原发性GEC中ROS的动力学和来源将通过流动性荧光法，荧光细胞成像和高分辨率氧气确定。我们将确定靶向靶向宿主分子电路，并通过选择性抑制剂，激动剂，Q-PCR，免疫化学和膨胀蛋白蛋白蛋白报道器系统的选择性抑制剂，激动剂，基因耗尽的组合来表征NDK的时空分泌和功能。最后，我们将阐明牙龈疟原虫对EATP信号的操纵，以实现细胞内生长和存活。这些研究将为牙龈疟原虫的效应子NDK瞄准的先前未开发的宿主分子网络提供详细的表征，以持续牙龈上皮。获得的知识可能转化为特定生理抑制剂的发展，这些抑制剂可能控制或减少由这种机会性病原体引起的慢性感染的严重程度。公共卫生相关性：牙龈疟原虫是严重形式的牙周疾病的主要因素，这是影响美国人群的最常见的慢性感染之一。拟议的研究旨在定义被该病原体感染的牙龈疟原虫和人牙龈细胞之间的基本复杂分子事件。这些研究将提供有价值的数据，以开发特定的治疗剂，以颠覆与宿主细胞的不利相互作用，并控制口服组织中这种微生物的生长和定殖。