The role of surface-bound ectonucleotidase CD73 in modulation of Porphyromonas gingivalis infection in gingival epithelial cells

PROJEST摘要/摘要 危险信号分子胞外ATP(EATP)由受感染的细胞释放，并被代谢为 其中一个步骤是由CD73催化的，CD73是一种表面结合的酶，它可以 将一磷酸腺苷(AMP)转化为腺苷。CD73对AMP的这种水解率是不可逆转的- 腺苷信号的限制步骤，通常被称为从促炎到免疫的“开关” (EATP)到抗炎(腺苷)介体。牙龈卟啉单胞菌是一种重要的致病菌 与严重的牙周病相关，并可以使用多种机制成功地入侵、复制 并在人类牙龈上皮细胞(GECs)内和通过GECs传播，GECs是最初防御的主要手臂 在粘膜表面。尽管在包括癌症在内的各种病理状态中的重要性与日俱增， 炎症性疾病，最近的细胞感染，CD73在调节宿主免疫反应和 口腔中的细菌感染还不是很清楚。我们的实验室此前显示，牙龈假单胞菌 能抑制NADPH氧化酶2产生eATP介导的活性氧(ROS)，而细菌 在GEC中，腺苷2a受体偶联的腺苷信号调节和促进生长。我们的小说 初步研究结果还显示，牙龈假单胞菌在GEC中的CD73表达显著增加。 此外，CD73介导的信号特异性地影响牙龈假单胞菌的细胞内存活，并被抑制 ROS的产生，并抑制白介素6(IL-6)的基因表达，其添加导致下降 牙龈假单胞菌在牙周血管内皮细胞中的水平。因此，我们的主要假设是牙龈假单胞菌可以选择性地 调节宿主CD73介导的免疫信号通路促进丰富的细胞内生长和生存 在牙龈上皮细胞中。我们将通过完成以下具体目标来检验我们的假设。特定的 目的1通过进一步研究，确定CD73在牙周炎假单胞菌细胞内感染中的调控作用。 支持牙龈假单胞菌/CD73偶联信号在GEC中的功能重要性。特定目标2将 阐明CD73调控牙龈假单胞菌细胞内生长的部分细胞和分子机制 和存活，特别是通过干扰促炎症的IL-6信号。这两个目标都将使用主要 GECS模型系统，从功能上剖析机制并定义分子事件。因此，这是 提案将表征口腔细菌感染的新型生理相关调节剂(S)和 揭示宿主CD73/牙龈假单胞菌/IL-6轴之间的多向串扰对牙周炎的调控 宿主细胞内牙龈假单胞菌感染。这项研究的结果将从根本上提供新的 从分子水平了解牙龈假单胞菌在口腔黏膜中的持续机制 有助于未来开发有效的治疗方法来控制牙龈假单胞菌相关感染。这 研究金还将提供口腔微生物学/免疫学方面的培训，并将促进受训人员的发展。 成为一位独特且急需的口腔健康学术临床医生/科学家。

PROJEST SUMMARY/ABSTRACT Danger signal molecule extracellular ATP (eATP) is released by infected cells and becomes metabolized to adenosine by multiple enzymatic steps, one of which is catalyzed by CD73, a surface-bound enzyme that converts adenosine monophosphate (AMP) to adenosine. This AMP hydrolysis by CD73 is an irreversible rate- limiting step in adenosine signaling that is often referred to as “an immunological switch” from pro-inflammatory (eATP) to anti-inflammatory (adenosine) mediator. Porphyromonas gingivalis is a keystone pathogen strongly associated with severe periodontal disease and can use multiple mechanisms to successfully invade, replicate, and disseminate within and through the human gingival epithelial cells (GECs), a major arm of initial defenses in mucosal surfaces. Despite the growing significance in various pathological states including cancer, inflammatory diseases, and recently cellular infection, the role of CD73 for regulating host immune response and bacterial infection in the oral cavity is not well understood. Our laboratory previously showed that P. gingivalis can inhibit eATP-mediated reactive oxygen species (ROS) generation by NADPH oxidase 2 and that the bacterial growth is modulated and enhanced by adenosine 2a receptor-coupled adenosine signaling in GECs. Our novel preliminary findings also revealed significantly increased CD73 expression with P. gingivalis in GECs. Furthermore, CD73-mediated signaling specifically impacted on the P. gingivalis intracellular survival, inhibited ROS generation, and dampened the gene expression of interleukin-6 (IL-6) whose addition led to decreased levels of P. gingivalis in GECs. Thus, our overarching hypothesis is that P. gingivalis can selectively regulate host CD73-mediated immune signaling pathways for affluent intracellular growth and survival in the gingival epithelium. We will test our hypothesis through completion of the following specific aims. Specific Aim 1 will establish the regulatory role of CD73 for P. gingivalis intracellular infection by further investigating functional importance supporting the P. gingivalis/CD73 coupled signaling in the GECs. Specific Aim 2 will elucidate select cellular and molecular mechanisms by which CD73 modulates P. gingivalis intracellular growth and survival, specifically through interfering with pro-inflammatory IL-6 signaling. Both aims will use primary GECs model system to functionally dissect out mechanisms and define the molecular events. Hence, this proposal will characterize novel physiologically relevant modulator(s) of oral bacterial infection and reveal the multidirectional cross-talk between host CD73/P. gingivalis/IL-6 axis for the modulation of intracellular P. gingivalis infection in the host cells. The results of this study will provide fundamentally novel molecular understanding of P. gingivalis persistence mechanisms in the oral mucosa and may ultimately contribute to future development of effective therapeutics for controlling P. gingivalis associated infections. This fellowship will also provide training in oral microbiology/immunology and will foster the development of the trainee into a unique and critically needed oral health academic clinician/scientist.