New Technologies to Identify Novel Periodontal Pathogens

描述(由申请人提供)：牙周病影响35%的美国成年人，并导致骨骼和牙齿脱落。美国纳税人每年的治疗费用高达44亿美元。研究已经将牙周病和全身疾病联系起来。超过一半的口腔类群尚未进行体外培养。关于这些分类群中的哪些在牙周健康和疾病中是常见的和众多的，因此是相关的，在知识上存在差距。我们的长期目标是揭示新的牙周病原体，并确定它们用于牙周破裂的机制。这项建议的总体目标是确定与牙周健康或疾病相关的突出的牙周下分类群，这些分类群目前未培养或可培养但目前未被识别。最近出现了牙周炎中存在的未培养/未识别的分类群的报告。我们的中心假设是，龈下生物膜中未培养/未被识别的类群的子集是致病的。许多口腔未培养/未识别的类群是暂时的，由食物或液体带入，与牙周健康或疾病几乎没有相关性。因此，我们的目标不是仅仅恢复任何“可驯化”的未培养的龈下分离株；也不是要在多物种复合体中培养未培养的类群。我们的目标是鉴定在牙周健康或疾病中数量显著的未培养/未识别的类群，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。这项建议的基本原理是消除暂时分类群所代表的“分心”，并将重点放在寻找新的突出的龈下细菌上。我们将使用创新技术来量化大量生物膜样本中广泛的不可培养/未识别的分类群。我们将检验中心假设，并通过追求以下具体目标来实现本应用的目标。1)我们将确定牙周健康和疾病中突出的未培养/未识别的细菌。这将通过从16名牙周健康和16名牙周炎受试者中筛选320个单独的龈下生物膜样本来实现。我们将采用我们开发的一项创新技术--RNA-寡核苷酸量化技术(ROQT)。它基于16S rRNA靶向寡核苷酸探针，它将确定140个未培养/未识别的物种/分类/克隆中哪些是常见的和大量存在于龈下生物膜中。一旦确定了突出分类群，2)我们将使用适当的探针在纯培养中分离可培养但尚未识别的突出分类群。我们将使用15个条件来生长这些分类群。这一目的将区分在正常实验室条件下可以培养的分类群。这项研究具有重要意义，因为目前被认为是未培养/未被识别的具有生物学意义的类群将被分离、表征和适当分类。这对毒力、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。这项研究的结果将有助于确定致病机制和疾病预防、诊断和治疗的新目标，从而支持NIDCR的使命。 公共卫生相关性：拟议的研究与公共卫生相关，因为尽管人们对牙周病的发展有越来越多的了解，但这些感染影响了超过30%的美国成年人，而且在少数族裔中更普遍。这些疾病与全身疾病有关，每年的治疗费用高达数十亿美元。该项目与NIDCR的使命相关，因为了解未培养/未被识别的龈下细菌的作用可以为口腔感染的预防、诊断和治疗提供新的目标。

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.