Moving Beyond Diversity by Revealing Biological Functions of Uncultured Bacteria

描述（由申请人提供）：许多人类疾病在本质上是多微生物的，因此需要超越传统的一种生物体-一种疾病的概念来寻找解决方案。至关重要的是，需要了解这些社区如何在社区、物种和基因层面上从健康状态演变为疾病状态。虽然许多与健康或疾病状况相关的模型物种已经在体外进行了表征，但很难确定它们在纯培养物中的生理学是否在混合微生物群落存在下在体内得以维持。此外，大多数与宿主相关的微生物仍然是未培养的（估计约占已鉴定微生物的50%），因此除了它们的16 S rDNA序列外，对未培养的物种知之甚少。了解未培养物种的生理学已经成为研究这些多微生物感染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、更详细地了解控制宿主相关微生物群落行为的生理学和生态学原理。这样做，我们也可以揭示目前未培养的成员的功能和生物作用。具体而言，在本申请中，我们将研究口腔微生物群落并寻求：1）通过稳定同位素探测（SIP）鉴定与高和低pH微生物过程高度相关的活性物种; 2）通过元转录组学鉴定在这些活性物种之间表达并共享的基因和主导途径; 3）使用激光捕获显微切割技术研究未培养物种的空间模式和与其他群落成员的关系，以及4）通过从捕获的单细胞进行全基因组扩增，对目前未培养的关键疑似病原体的基因组进行测序。这项研究的成功将大大扩展我们对口腔微生物发病机制的认识，因为它将帮助我们了解未培养物种以及已知病原体的毒力特性，不仅在纯培养物中，而且在多物种牙科生物膜中。我们特别专注于揭示更多关于这些目前未培养的细菌如何有助于健康和疾病过程。目前缺乏这种信息是了解多种微生物疾病的主要障碍。这将对未来龋齿的临床管理产生重大影响，并为表征其他宿主相关社区的物种功能及其相互作用提供全面的方法。 公共卫生相关性：许多广泛传播的人类疾病，如蛀牙（龋齿），是由复杂的微生物群落相互作用引起的（多微生物疾病）。这种复杂性使治疗策略严重复杂化。例如，龋齿在美国和全世界仍然是一个主要的健康问题，在幼儿中的患病率超过50%，在成年人口中增加到约85%。大多数与多种微生物疾病相关的细菌尚未得到很好的研究，因为它们不能在实验室中生长。我们建议使用新的分析方法来研究这些未培养细菌的生物学功能及其在人类健康中的作用。这些方法包括产生基因组和监测这些物种在自然环境中的生理学。这种新方法将提供对疾病进展的更深入了解，并允许随后开发新的治疗方法来对抗这些疾病。

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. PUBLIC HEALTH RELEVANCE: Many widespread human diseases, such as tooth decay (caries), result from the interactions of complex communities of microorganisms (polymicrobial diseases). This complexity severely complicates treatment strategies. Caries, for example, remains a major health issue in the United States and worldwide with a prevalence of more than 50% in young children, increasing to about 85% in the adult population. The majority of bacteria associated with polymicrobial diseases have not been well studied due to the fact that they cannot be grown in the laboratory. We propose to use new analytical methods to investigate the biological functions of these uncultured bacteria and their roles in human health. These approaches include generating the genomes and monitoring the physiology of these species in their natural environment. This new approach will provide a deeper understanding of disease progression and allow for the subsequent development of novel therapeutic approaches to battle these diseases.