Metatramscriptome of the Oral Microbiome During Periodontal Disease Progression

描述（由申请人提供）：牙周炎是一种由复杂微生物群落协同作用引起的多微生物疾病，导致支持牙齿的组织炎症。牙周炎是一种广泛而严重的健康状况，30%至50%的美国成年人出现中度牙周炎，10%的成年人出现重度牙周炎。本研究计划的目标是原位确定牙周炎在某些部位进展而在其他部位疾病仍然潜伏的发病机制。宏基因组学和元转录组学分析（原位完整复杂细菌群落的基因表达分析）提供了了解牙周感染期间致病性生物膜成分的活性和相对重要性所需的信息。为此，我们提出以下具体目标：确定进展性牙周和非进展性牙周部位微生物群落的患病率和相对丰度的差异。目标2。鉴定在进展性和非进展性牙周部位微生物群落中差异表达的原位基因。我们已经成功地将亚转录组学技术应用于环境样本的研究。评估整个微生物群落表达分析方法的关键步骤是从细菌组合中提取的少量RNA的线性扩增。最终扩增的RNA是原始样品中mRNA相对丰度的直接代表。促进整个细菌群落的超转录组学分析的第二个方面是焦磷酸测序技术现在允许对大量DNA进行测序，避免了与经典技术相关的克隆偏差。目标受试者人群将由20名慢性牙周炎患者组成。之所以选择这一人群，是因为慢性牙周炎是最常见的牙周炎类型，因此，观察到的微生物变化将与很大比例的牙周病患者相关。患者将每月随访6个月，在此期间，他们将接受临床监测，以确定哪些样本将通过宏基因组和亚转录组学分析用于进展和非进展部位的治疗。鉴定致病机制所需的关键基因及其差异表达信息可用于开发早期诊断、治疗、监测和预防的新型靶向方法。此外，潜在的影响超出了牙周炎的研究，因为相同的原理和方法可能适用于其他多微生物疾病。我们相信，我们为这个项目组建的团队（福赛斯研究所与布罗德研究所和哈佛大学公共卫生学院生物统计系的帮助）完全有资格成功实现本申请中提出的目标。

DESCRIPTION (provided by applicant): Periodontitis is a polymicrobial disease caused by the coordinated action of a complex microbial community, which results in inflammation of tissues that support the teeth. Periodontitis is a widespread and serious health condition that occurs in moderate form in 30% to 50% of American adults and in severe form in 10% of adults. The goal of this research program is to identify in situ the molecular mechanisms of pathogenesis by which periodontitis progress in some sites while in others the disease remains latent. Metagenomic and metatranscriptomic analysis (gene expression analysis of entire complex bacterial communities in situ) provides the information required to understand the activity and relative importance of the constituents in the pathogenic biofilm during periodontal infection. To this end we propose the following Specific Aims: Aim 1. Determine differences in prevalence and relative abundance of species in microbial communities from progressing and non-progressing periodontal sites. Aim 2. Identify in situ genes that are differentially expressed in microbial communities from progressing and non-progressing periodontal sites. We have successfully applied metatranscriptomic techniques to the study of environmental samples. The key step of the method to assess expression analysis in whole microbial communities is the linear amplification of small quantities of RNA extracted from the bacterial assemblages. The final amplified RNA is a direct representation of the relative abundance of mRNA in the original sample. The second aspect that facilitates metatranscriptomic analysis of whole bacterial communities is that pyrosequencing techniques allow now to sequence large amounts of DNA avoiding cloning biases associated with classical techniques. The target subject population will consist of 20 chronic periodontitis individuals. This subject population was chosen because chronic periodontitis is the most commonly encountered type of periodontitis and thus, the microbial changes observed will be relevant to a large proportion of subjects with periodontal disease. The patients will be followed monthly for a period of 6 months, during which they will undergo clinical monitoring to determine which samples will be used for compassion of progressing and non-progressing sites by metagenomic and metatranscriptomic analysis. Identification of critical genes that are required for pathogenesis and information about their differential expression can be used to develop novel targeted approaches to early-stage diagnosis, treatment, monitoring and prevention. Moreover, the potential impact extends beyond the study of periodontitis because the same principles and methods potentially can be applied to other polymicrobial diseases. We believe that the team we have assembled for this project (Forsyth Institute with help of Broad Institute and the Department of Biostatistics at Harvard School of Public Health) has all the qualifications to accomplish successfully the goals proposed in the present application. PUBLIC HEALTH RELEVANCE: Periodontitis is a polymicrobial disease caused by complex microbial community, which could result in the loss of teeth. The goal of this project is to understand which organisms are active and what they are doing during periodontitis progression to develop specific treatments against them.