New Technologies to Identify Novel Periodontal Pathogens

描述（由申请人提供）：牙周病影响了35%的美国成年人，并导致骨和牙齿脱落。美国纳税人每年的治疗费用高达44亿美元。研究表明牙周病和全身疾病有关。超过一半的口腔类群尚未在体外培养。关于这些分类群中哪些是常见的和众多的-因此与牙周健康和疾病相关-在知识上存在差距。我们的长期目标是揭开新的牙周病原体和确定他们使用牙周破坏的机制。本提案的总体目标是确定与牙周健康或疾病相关的突出龈下分类群，这些分类群目前未被培养或可培养但目前未被识别。最近出现了关于牙周炎中未培养/未识别分类群的报道。我们的中心假设是龈下生物膜中未培养/未识别的分类群的一个子集是致病的。许多口腔未培养/未识别的分类群是短暂的，由食物或液体带入，与牙周健康或疾病几乎没有关系。因此，我们的目标不是恢复任何“可驯化的”未培养的龈下分离菌；在多种复合体的生长未栽培分类群中都不是。我们的目标是在牙周健康或疾病中确定数字上突出的未培养/未识别的分类群，并在纯培养中恢复它们，作为确定它们在牙周健康或疾病中的作用的第一步。这一建议的基本原理是消除以瞬态分类群为代表的“分心”，并集中精力寻找新的突出的牙龈下细菌。我们将使用创新技术来量化大量生物膜样品中广泛的不可培养/未识别的分类群。我们将通过以下具体目标来检验中心假设并实现本应用的目标：1)我们将确定在牙周健康和疾病中突出的未培养/未识别的细菌。这将通过筛选来自16名牙周健康和16名牙周炎受试者的320个个体龈下生物膜样本来实现。我们将采用我们开发的一种创新技术，rna寡核苷酸定量技术（ROQT）。它基于16s rRNA靶向寡核苷酸探针，将确定140个未培养/未识别的物种/类群/克隆中哪些在龈下生物膜中是常见的和众多的。一旦确定了突出的分类群，2)我们将使用适当的探针在纯培养中分离可培养但尚未被识别的突出分类群。我们将在15种条件下对这些分类群进行生长。这一目标将区分在正常实验室条件下可以培养的分类群。这项研究具有重要的生物学意义，因为目前被认为是未开发/未识别的分类群将被分离，表征和适当分类。这对于毒力、抗原性和功能基因组学研究以及生长因子和信号分子的研究至关重要。这项研究的结果将通过导致疾病预防、诊断和治疗的致病机制和新靶点的鉴定来支持NIDCR的使命。

DESCRIPTION (provided by applicant): Periodontal diseases affect 35% of US adults and lead to bone and tooth loss. Treatment costs reach U$ 4.4 billion per year to American taxpayers. Studies have linked periodontal diseases and systemic conditions. Over half of the oral taxa have not been cultivated in vitro. There is a gap in knowledge regarding which of those taxa are common and numerous-and therefore relevant - in periodontal health and disease. Our long term goal is to unveil novel periodontal pathogens and determine the mechanisms they use for periodontal breakdown. The overall objective of this proposal is to identify prominent subgingival taxa associated with periodontal health or disease that are currently uncultivated or cultivable but currently unrecognized. Reports of uncultivated/ unrecognized taxa present in periodontitis have recently emerged. Our central hypothesis is that a subset of the uncultivated/unrecognized taxa in subgingival biofilms is pathogenic. Many oral uncultivated/unrecognized taxa are transients and brought in by food or liquids and have little, if any, relevance to periodontal health or disease. Thus, we do not aim at recovering just any "domesticateable" uncultivated subgingival isolate; neither at growing uncultivated taxa in multispecies complex. We aim at identifying numerically prominent uncultivated/unrecognized taxa in periodontal health or disease and recover them in pure culture as a first step in determining their role in periodontal health or disease. The rationale of this proposal is to eliminate the "distraction" represented by transient taxa and focus the search for novel prominent subgingival bacteria. We will use innovative technologies to quantify a wide range of uncultivable/unrecognized taxa in large numbers of biofilm samples. We will test the central hypothesis and accomplish the objective of this application by pursuing the following Specific Aims. 1) We will identify prominent uncultivated/unrecognized bacteria in periodontal health and disease. This will be achieved by screening 320 individual subgingival biofilm samples from 16 periodontally healthy and 16 periodontitis subjects. We will employ an innovative technique developed by us, the RNA-Oligonucleotide Quantification Technique (ROQT). It is based on 16s rRNA targeted oligonucleotide probes and it will determine which of 140 uncultivated/unrecognized species/taxa/ clones are common and numerous in subgingival biofilms. Once prominent taxa are defined, 2) we will use the appropriate probes to isolate in pure culture, cultivable but as yet unrecognized prominent taxa. We will use 15 conditions for growth of those taxa. This aim will distinguish taxa that can be cultivated under normal laboratory conditions. This research is significant because biologically meaningful taxa currently considered to be uncultivated/unrecognized will be isolated, characterized and properly classified. This is crucial for virulence, antigenicity and functional genomics studies and investigation of growth factors and signaling molecules. Results from this study will support NIDCR's mission by leading to the identification of pathogenic mechanisms and novel targets for disease prevention, diagnosis and therapy. PUBLIC HEALTH RELEVANCE: The proposed research is relevant of public health because, despite increasing knowledge of the development of periodontal diseases, these infections affect more than 30% of US adults and are more prevalent among minorities. These diseases have been linked to systemic conditions and their treatment costs billions annually. This project is relevant to NIDCR's mission because understanding of the role of uncultivated/unrecognized subgingival bacteria can provide new targets for prevention, diagnosis and treatment of oral infections.