Pathogenic mechanisms of F. alocis

描述（由申请人提供）：牙周病是人类最常见的细菌感染之一，对医疗保健系统造成重大负担。基于分子的细菌检测技术的最新发展已经确定了许多以前未被认识或未被充分认识的与牙周病变相关的生物体。一种这样的新兴病原体是革兰氏阳性厌氧菌Filifactoralocis。这种微生物是龈下生物膜的组成部分，与健康相比，在牙周病部位的数量增加。F. Alocis产生蛋白酶并能侵入牙龈上皮细胞;然而，关于该生物体的潜在致病性，几乎没有其他的了解。为了利用人类微生物组研究，有必要对新鉴定的生物体进行功能表征。因此，在这项研究中，我们将有助于评估F的致病潜力的过程。alocis在体外和体内。我们的初步数据表明，F。alocis可诱导牙龈上皮细胞凋亡， 与牙周组织稳态的破坏有关。因此，当前建议的第一个目标是开始描述F. alocis诱导的细胞凋亡。我们将集中在MEK-FOXO途径的初步数据表明，F。alocis可以抑制MEK并潜在地激活促凋亡转录因子FOXO。MEK的作用将通过用组成型突变体的基因敲入实验来评估，并且FOXO的相关性通过报告基因测定和siRNA敲减来确定。其次，我们将建立F的体内特征。alocis在小鼠室模型中。F.将建立宿主细胞在体内存活和复制的能力，此外，将测定宿主细胞因子应答、嗜中性粒细胞募集和细胞凋亡。这些研究将产生体外和体内数据，这些数据将开始建立F. alocis，并提供了一个平台，为未来的研究，旨在更全面地了解分子和细胞的致病机制。最终，所获得的知识可以转化为牙周病的新诊断，治疗或预防策略。

DESCRIPTION (provided by applicant): Periodontal diseases are one of the most common bacterial infections of humans and impose a significant burden on the health care system. Recent developments in molecular based bacterial detection technologies have identified a number of previously unrecognized or under-appreciated organisms as associated with periodontal lesions. One such emerging pathogen is the gram-positive anaerobe Filifactor alocis. This organism is a constituent of subgingival biofilms and is present in elevated numbers at site of periodontal disease as compared to health. F. alocis produces proteases and can invade gingival epithelial cells; however, little else is known regarding the potential pathogeniciy of the organism. In order to capitalize on the human microbiome studies it is necessary to functionally characterize the newly identified organisms. Hence, in this study we shall contribute to the process of assessing the pathogenic potential of F. alocis in vitro and in vivo. Our preliminary data indicate that F. alocis can induce apoptosis in gingival epithelial cells an event with relevance to disruption of periodontal tissue homeostasis. The first goal of the current proposal, therefore, is to begin to characterize the mechanistic basis of F. alocis-induced apoptosis. We will focus on the MEK-FOXO pathway as preliminary data show that F. alocis can inhibit MEK and potentially activate the pro-apoptotic transcriptional factor FOXO. The role of MEK will be assessed by gene knockin experiments with constitutively mutants, and the relevance of FOXO determined by reporter assays and siRNA knockdowns. Secondly, we will establish the in vivo characteristics of F. alocis in the mouse chamber model. The ability of F. alocis to survive and replicate in vivo will be established and, in addition, host cytokine responses, neutrophil recruitment and apoptosis will be assayed. These studies will generate both in vitro and in vivo data that will begin to establish the pathogenic credentials of F. alocis and provide a platform for future studies directed toward a more comprehensive understanding of molecular and cellular pathogenic mechanisms. Ultimately, the knowledge gained could be translated into novel diagnostic, therapeutic or preventive strategies for periodontal diseases.