Modulation of epithelial cell response by P. gingivalis

描述(由申请人提供)：牙周病是普遍存在的破坏性口腔多菌感染，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，是该病严重形式的主要病原体。该生物是牙龈上皮细胞(GECs)的成功定殖者，GECs对入侵的病原体形成初步防御，同时也是机会细菌的主要目标细胞。牙龈假单胞菌是一种宿主适应的病原体，可以在原代牙周血管内皮细胞中存活和复制，然后通过细胞间传播。感染通过影响线粒体凋亡途径和抑制ATP-P2X7受体信号通路来抑制GEC的死亡。最近研究表明，P2X7受体与“危险信号”--细胞外ATP(EATP)的结合可导致巨噬细胞产生细胞内的活性氧(ROS)。虽然以前人们主要知道ROS是一种有毒物质，但越来越多的人认识到ROS通过调节多种关键细胞功能发挥着重要的生理作用，包括细胞凋亡、免疫反应和细胞内感染。牙龈假单胞菌分泌一种效应物，核苷二磷酸激酶(NDK)，它可以清除eATP并降低P2X7的活性。我们最近发现，牙龈假单胞菌通过在感染过程中分泌NDK，调节胞浆ROS的产生，从而阻断eATP诱导的原代GECs的氧化应激。然而，牙龈假单胞菌的NDK缺陷突变体缺乏灭活eATP诱导的氧化应激并在细胞内持续存在的能力。EATP诱导的ROS的产生可能是由P2X7受体信号、NADPH氧化酶和线粒体氧化应激途径介导的。本研究的目的是：明确牙龈假单胞菌用来抑制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通路中的作用，这些通路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。ROS在原代GEC中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来确定。我们将结合选择性抑制剂、激动剂、siRNA基因缺失、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，识别以感染为靶点的宿主分子回路，并表征NDK的时空分泌和功能。最后，我们将阐明牙龈假单胞菌对eATP信号的调控，以促进细胞内的生长和存活。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是牙龈假单胞菌的效应者，用于在牙龈上皮细胞中持续存在。所获得的知识可能转化为特定的生理抑制物的开发，可以控制或减轻由这种机会性病原体引起的慢性感染的严重程度。

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.