Culturing Uncultivatable Gut Microorganisms

描述（由申请人提供）：大多数肠道微生物仍然是不可培养的，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方法来培养以前不可培养的细菌。我们之前对来自外部环境的不可培养微生物的研究已经取得了许多进展：(1)通过原位培养可以培养大量其他不可培养的细菌。当微生物被放入扩散室，然后被放回其自然环境时，将生长出相当大比例的其他不可培养的微生物；(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异体；(3)许多不可培养的物种将在合成培养基上与来自同一环境的可培养生物共培养；(4)我们最近首次发现了不可培养细菌的生长促进因子。测定驱动的纯化鉴定出铁载体是由辅助生物产生的基本因子，这些辅助生物触发了海洋沉积物中不可培养细菌的生长。我们发现生长共培养可用于从肠道菌群中获得不可培养的微生物。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不可培养的微生物。将代表主要分类群的24种可培养肠道物种的面板排列在微滴盘中，并在孔中放置带有0.2 m孔膜的插入物的平台。以这种方式，每个孔将被分成接种了给定可培养物种的底部部分和通过膜孔与之连接的顶部部分。从人类粪便样本中提取的悬浮液将被细胞分选器分离，单个细胞将被放置在每个孔的上腔中。孵育后，将从井的两个部分收集材料，并分别测试两种生物的生长和共同培养。这将导致不可栽培的物种和它们的帮手被隔离。16S rRNA基因序列测定将鉴定这些微生物。将对来自不同分类群的至少10个不可培养的分离株进行全基因组测序。基因组测序将为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。生长因子将从相应的辅助生物的上清液中通过生物测定引导纯化分离出来。新化合物的结构将被确定。然后，将对生长因子进行单独和组合检查，以确定它们是否能够在体外培养不可培养的微生物。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群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.