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

• 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
• 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

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）：牙周病是普遍存在的破坏性口腔多微生物感染，仍然是一个重大的公共卫生负担。牙龈卟啉单胞菌是一种革兰氏（-）厌氧菌，是严重形式牙龈卟啉单胞菌的主要病因。该生物是牙龈上皮细胞（GECs）的成功定植者，牙龈上皮细胞形成对入侵病原体的初始防御，同时作为机会性细菌的主要靶细胞。牙龈卟啉卟啉菌是一种适应宿主的病原体，可以在初级gec中生存和复制，然后在细胞间传播。感染通过影响线粒体-凋亡通路和抑制ATP-P2X7受体信号传导抑制GEC死亡。P2X7受体与“危险信号”细胞外ATP （eATP）的连接最近被证明会导致巨噬细胞内细胞内活性氧（ROS）的产生。虽然以前主要被认为是一种有毒物质，但ROS越来越多地被认为通过调节多种关键的细胞功能（包括凋亡、免疫反应和细胞内感染）发挥重要的生理作用。牙龈卟啉卟啉分泌一种效应物，核苷二磷酸激酶（Ndk），它清除eATP并降低P2X7活性。我们最近发现，牙龈假单胞菌在感染期间通过分泌Ndk来调节细胞内ROS的产生，并随后阻断eap诱导的初级gec氧化应激。然而，牙龈卟啉卟啉缺乏症突变体缺乏灭活eap诱导的氧化应激并在细胞内持续存在的能力。eap诱导的ROS生成似乎是通过P2X7受体信号与NADPH氧化酶和线粒体氧化应激途径耦合介导的。本研究的目的是：确定牙龈卟啉卟啉抑制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，这些途径可能对生物体的生存和逃避eATP介导的细胞内杀伤至关重要。原代gec中ROS的动力学和来源将通过流式细胞荧光法、荧光细胞成像和高分辨率氧图测定。我们将确定感染目标宿主分子电路，并采用选择性抑制剂、激动剂、siRNA基因耗尽、q-PCR、免疫生化和荧光蛋白报告系统结合共聚焦定量图像分析来表征Ndk的时空分泌和功能。最后，我们将阐明牙龈卟啉卟啉菌对细胞内生长和生存的eATP信号的操纵。这些研究将提供以前未被探索的宿主分子网络的详细特征，Ndk是牙龈卟啉卟啉的一种效应物，针对牙龈上皮的持久性。所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降低由这种机会性病原体引起的慢性感染的严重程度。