Moving Beyond Diversity by Revealing Biological Functions of Uncultured Bacteria

描述(申请人提供)：许多人类疾病本质上是多菌的，因此需要超越传统的单一有机体-单一疾病的概念来寻找抗击这些疾病的解决方案。最重要的是需要在群落、物种和基因水平上理解这些群落如何从健康状态进化到疾病状态。尽管许多被发现与健康或疾病条件有关的模式物种已经在体外被表征，但很难确定它们在纯培养中的生理学是否在体内保持在混合微生物群落的存在下。此外，大多数寄主相关微生物仍未培养(估计约占已鉴定微生物的50%)，因此除16S rDNA序列外，对未培养物种知之甚少。了解未培养物种的生理学已成为研究这些多菌感染的生态学的主要限制步骤。随着高通量测序、单细胞基因组学以及微生物生态学新工具的出现，我们现在可以更深入、更详细地了解支配宿主相关微生物群落行为的生理和生态原理。通过这样做，我们还可以揭示目前未培养的成员的功能和生物学作用。具体来说，在这一应用中，我们将调查口腔微生物群落，并寻求：1)通过稳定同位素探测确定与高和低pH微生物过程高度相关的活性物种；2)通过元转录识别这些活性物种之间表达和共享的基因和优势途径(S)；3)使用激光捕获显微切割技术调查未培养物种的空间模式和与其他社区成员的关系；4)通过从捕获的单细胞中进行全基因组扩增，对目前尚未培养的关键疑似病原体的基因组进行测序。这项研究的成功将极大地扩展我们对口腔微生物发病机制的认识，因为它将有助于我们了解未培养物种以及已知病原体的毒力特性，不仅在纯培养中，而且在多物种牙科生物膜中也是如此。我们特别关注揭示更多关于这些目前未培养的细菌可能如何对健康和疾病过程做出贡献的信息。目前此类信息的缺乏是理解多菌类疾病的主要障碍。这将对未来龋病的临床管理产生重大影响，并为其他宿主相关群落描述物种的功能及其相互作用提供了一种全面的方法。

DESCRIPTION (provided by applicant): Many human diseases are polymicrobial in nature and therefore require thinking beyond the traditional one organism-one disease concept to find solutions to combating them. Of fundamental importance is the need for an understanding of how these communities evolve from a healthy to a diseased state at a community, species and gene level. Although many model species found associated with healthy or disease conditions have been characterized in vitro, it is difficult to determine if their physiology in pure cultures are maintained in vivo in the presence of a mixed microbial community. In addition, most host-associated microbes remain uncultured (estimated at ~ 50% of those identified) therefore little is known about the uncultured species except for their 16S rDNA sequence. Understanding the physiology of uncultured species has become a major limiting step in studying the ecology of these polymicrobial infections. With the advent of new approaches in high throughput sequencing, single cell genomics as well as new tools in microbial ecology, we can now achieve a deeper and more detailed understanding of the physiological and ecological principals that govern the behavior of host-associated microbial communities. In doing so, we can also reveal the functions and biological role of currently uncultured members. Specifically in this application we will investigate oral microbial communities and seek to; 1) identify the active species, that are highly correlated with high and low pH microbial processes through Stable Isotope Probing (SIP); 2) identify the genes and dominant pathway(s) that are expressed and also shared between these active species through metatranscriptomics; 3) investigate the spatial patterns and relationships of uncultured species with the other community members using Laser Capture Microdissection and 4) sequence the genomes of key suspected pathogens that are currently uncultured, through whole genome amplification from captured single cells. The success of this study would greatly expand our knowledge of oral microbial pathogenesis as it will help us to understand the virulence properties of uncultured species as well as known pathogens, not only in pure culture, but also in multi-species dental biofilms. We are particularly focused on revealing more about how these currently uncultivated bacteria may contribute to heath and disease processes. The present lack of such information presents a major barrier to understanding polymicrobial diseases. This will have a great impact on the future clinical management of dental caries and provide a comprehensive approach for characterizing the function of species and their interactions for other host-associated communities.