Pathogenic mechanisms of F. alocis

描述(申请人提供)：牙周病是人类最常见的细菌感染之一，给医疗保健系统带来了巨大的负担。基于分子的细菌检测技术的最新发展已经确定了一些以前未被认识或被低估的生物与牙周病变有关。其中一种新出现的病原体是革兰氏阳性厌氧菌纤毛因子。这种微生物是龈下生物膜的一个组成部分，与健康相比，在牙周病部位的数量较多。金黄色葡萄球菌能产生蛋白水解酶，并能侵袭牙龈上皮细胞；然而，关于该菌潜在的致病机理，人们知之甚少。为了利用人类微生物组研究，有必要确定新发现的生物的功能特征。因此，在这项研究中，我们将有助于评估阿洛克斯体内外致病潜力的过程。我们的初步数据表明，金黄色葡萄球菌可以诱导牙龈上皮细胞的凋亡。 与牙周组织动态平衡的破坏有关。因此，当前提案的第一个目标是开始描述白曲霉诱导的细胞凋亡的机制基础。我们将重点关注MEK-FOXO途径，因为初步数据表明，F.alocis可以抑制MEK，并潜在地激活促凋亡转录因子FOXO。MEK的作用将通过结构性突变的基因敲击实验来评估，FOXO的相关性将通过报告分析和siRNA击倒来确定。其次，我们将在小鼠小室模型中建立阿罗氏假丝酵母菌的体内特性。将建立华支睾吸虫在体内存活和复制的能力，并将检测宿主细胞因子反应、中性粒细胞募集和细胞凋亡。这些研究将产生体外和体内的数据，开始建立弗洛克斯菌的致病凭证，并为未来旨在更全面地了解分子和细胞致病机制的研究提供平台。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.