Culturing Uncultivatable Gut Microorganisms

描述（由申请人提供）：大多数肠道微生物仍然不可培养，必须克服这一重大障碍，以了解微生物组在人类健康中的作用。该项目的目标是开发一种高通量的方法来培养以前无法培养的细菌。 我们以前对来自外部环境的不可培养的微生物的研究已经取得了一些进展：（1）通过原位培养可以培养大量的否则不可培养的细菌。当将微生物置于扩散室中，然后将其返回到它们的自然环境中时，大部分原本不可培养的微生物将生长;（2）从一个室到另一个室的再接种产生可在体外合成培养基上生长的驯化变体;（3）许多不可培养的物种将在与来自相同环境的可培养生物体共培养的合成培养基上生长;（4）我们最近发现了第一个不可培养细菌的生长促进因子。一个分析驱动的纯化导致铁载体的鉴定为必需的因素所产生的辅助生物，触发不可培养的细菌从海洋沉积物的增长。 我们发现，生长共培养可用于从肠道植物群获得不可培养的生物体。在这个项目中，我们将开发一种高通量的共培养方法，以便从肠道微生物组中获得大量以前无法培养的微生物。将代表主要分类组的24个可培养肠道物种的组排列在微量滴定板中，并将携带具有0.2 - 5 m孔膜的插入物的平台置于威尔斯孔中。以这种方式，每个孔将被分成用给定的可培养物种接种的底部和通过膜的孔与其连接的顶部。然后将通过细胞分选仪分离来自人粪便样品的悬浮液，并将单个细胞沉积在每个孔的上室中。孵育后，将收集孔的两个部分的材料，并分别检测两种微生物的生长和共培养。这将导致不可培养的物种和它们的助手的隔离。16 S rRNA基因序列测定将鉴定这些微生物。将对来自各种分类组的至少10个不可培养分离株进行全基因组测序。基因组测序将为所提出的从微生物组中获得新的不可培养物种的方法提供最终验证。将通过生物测定指导的纯化从相应辅助微生物的上清液中分离生长因子。新化合物的结构将被确定。然后将单独和组合地检查生长因子使得能够体外培养不可培养的微生物的能力。我们开发的工具和方法可能会导致许多肠道细菌的培养，并将帮助我们了解肠道微生物组在健康和疾病中的作用。 公共卫生相关性：大多数肠道细菌是不可培养的，在实验室条件下不生长，原因不明。我们发现，这些生物中有许多依赖于邻近的可培养物种生长。在这个项目中，我们将开发一种方法，通过将它们与正确的辅助物种配对，大规模分离以前无法培养的微生物，这将使它们的基因组测序成为可能，并详细研究它们在健康和疾病中的作用。

DESCRIPTION (provided by applicant): The majority of gut microbes remain uncultivatable, and this significant obstacle must be overcome to understand the role of the microbiome in human health. The goal of this project is to develop a high-throughput method to grow previously uncultivatable bacteria. Our previous work with uncultivatable microorganisms from the external environment has lead to a number of advances: (1) it is possible to cultivate a substantial number of otherwise uncultivatable bacteria by growing them in situ. When microorganisms are placed into a diffusion chamber which is then returned to their natural environment, a substantial proportion of otherwise uncultivatable microorganisms will grow; (2) reinoculation from chamber to chamber produces domesticated variants that can grow on synthetic media in vitro; (3) many uncultivatable species will grow on synthetic media in co-culture with a cultivable organism from the same environment; (4) we recently discovered the first growth promoting factors for uncultivatable bacteria. An assay-driven purification lead to the identification of siderophores as essential factors produced by helper organisms that trigger growth of uncultivatable bacteria from marine sediment. We find that growth co-culture can be used to obtain uncultivatable organisms from the gut flora. In this project, we will develop a high-throughput approach to co-culture in order to obtain a large collection of previously uncultivatable microorganisms from the gut microbiome. A panel of 24 cultivable gut species representing the main taxonomic groups will be arrayed in a microtiter plate and a platform carrying inserts with a 0.2 5m pore membrane will be placed in the wells. In this manner, each well will be separated into a bottom section inoculated with a given cultivable species and a top section connected with it through pores of the membrane. A suspension from a human fecal sample will then be separated by a cell sorter, and individual cells will be deposited in the upper chamber of each well. After incubation, material from both parts of a well will be collected and tested for growth of the two organisms separately and in co-culture. This will lead to the isolation of uncultivatable species and their helpers. 16S rRNA gene sequence determination will then identify these microorganisms. Whole genome sequencing will be performed for at least ten of the uncultivatable isolates from a variety of taxonomic groups. The genome sequencing will provide an ultimate validation of the proposed approach to obtain novel uncultivatable species from the microbiome. Growth factors will be isolated from the supernatant of corresponding helper organisms by bioassay-guided purification. Structures of the new compounds will be determined. The growth factors will then be examined, individually and in combination, for their ability to enable in vitro cultivation of uncultivatable microorganisms. The tools and approaches we develop are likely to lead to the cultivation of many gut bacteria, and will help us understand the role of the gut microbiome in health and disease. PUBLIC HEALTH RELEVANCE: The majority of gut bacteria are uncultivatable, and do not grow under laboratory conditions for unknown reasons. We find that many of these organisms depend on neighboring, cultivable species for growth. In this project, we will develop a method for large-scale isolation of previously uncultivatable microorganisms by pairing them with the correct helper species, which will enable their genome sequencing, and detailed study of their role in health and disease.