Culturing of the uncultured: reverse genomics and multispecies consortia in oral

描述(申请人提供)：人类口腔中至少有600种微生物，其中许多是未经培养的，其在人类健康或疾病中的作用尚不清楚。在龈下缝隙中尤其如此，那里的微生物群落通过复杂的食物网相互作用，形成结构化的新陈代谢协会。这些社区可能会导致口腔疾病，如牙周炎，影响大约三分之一的成年人口。最近，利用基于DNA的方法进行的开放式研究揭示了龈下微生物群落结构的复杂性。然而，如果不在了解潜在关键的未培养群体的生理学方面取得进展，治疗以及更重要的是预防牙周炎等疾病将仍然困难。为了解决这一问题，我们提出了一种假设驱动的、有针对性的方法，通过先进的细胞分离和高通量培养技术，选择性地将迄今未培养的与疾病相关的微生物(例如候选的TM7和SR1门)引入研究。我们将使用一种经过验证的、基于免疫学的方法，这种方法已经成功地被证明可以从自然环境中捕获稀有和未培养的微生物。这种方法利用基因组信息来合成由选定生物表达的表面抗原，从而避免了随机的浓缩/培养方法。新的分离株将被单独研究，但更重要的是，与在相同的生态位和健康/疾病状态下发现的潜在代谢物促进剂或合并体的其他物种结合起来研究。将使用微流控系统建立高度可控的微环境，使生物膜培养与实时、先进的成像技术相结合。还将在果蝇中建立活体宿主模型，以模拟生物系统中的复杂相互作用。这些平台将使建立综合比较和功能基因组研究所需的新型口腔微生物区系的微培养成为可能，并将促进我们对牙龈下环境中微生物之间的密切相互作用及其在疾病中的作用的理解。

DESCRIPTION (provided by applicant): The human oral cavity hosts at least 600 microbial species, many of which are uncultivated and whose roles in human health or disease remain unclear. This is especially true in the subgingival crevice where microbial communities interact through complex food webs forming structured metabolic guilds. These communities potentially lead to diseases of the mouth such as periodontitis, which affects approximately one third of the adult population. Recent, open-ended studies utilizing DNA-based methods have shed new light on the complex nature of subgingival microbial community structure. However, without advances in understanding the physiology of potentially key, uncultivated groups, treatment and more importantly, prevention of diseases such as periodontitis will remain difficult. To address this, we propose a hypothesis driven, targeted approach to selectively bring heretofore uncultivated, disease-associated microorganisms (e. g. candidate phyla TM7 and SR1) into study through advanced cell isolation and high-throughput cultivation techniques. We will employ a proven, immunological-based method, which has successfully been shown to capture rare and uncultivated microorganism from natural environments. This method leverages genomic information to synthesize surface antigens expressed by selected organisms thus avoiding a random approach to enrichment/cultivation. Novel isolates will be studied individually but more importantly, in combination with other species potentially serving as metabolite facilitators or syntrophs found in the same niche and health/disease state. Highly controllable microenvironments will be established using microfluidic systems that allow biofilm cultivation in conjunction with real-time, advanced imaging techniques. Model living hosts will also be established in Drosophila to simulate complex interactions in a biological system. These platforms will enable microcultivation of novel oral microbiota needed to establish integrated comparative and functional genomic studies, and will advance our understanding of the intimate interactions between microbes inhabiting the subgingival environment and their role in disease.