Developing Next-Generation Physiology approaches for human gut microbiome research

项目摘要 人体肠道微生物组领域在单细胞分辨方面的应用落后于其他微生物生态学领域 能够揭示微生物在(接近)原地条件下的功能的技术。通过建立在我的实验室的专业知识 在处理在分类、功能和结构上比Gut更复杂的示例类型时 以大多数指标衡量，我建议通过将人类肠道微生物群领域推向 单细胞生态生理学研究。而不是培养肠道微生物群的单个成员 孤立或试图从破坏空间信息的批量组学方法中推断出发现，我们将研究 肠道微生物在它们实际生活的单细胞分辨率下，作为空间结构和 新陈代谢相互依赖的生态系统。我们将研究微生物的功能活性和代谢多样性 在来自人类胃肠道的粘膜活检样本中使用了几种目前 没有使用或在外地未得到充分利用。底物模拟探测和生物正交标记，结合 荧光原位杂交将用于研究哪些细胞合成新的DNA、蛋白质、脂类或 在特定的物理化学条件下的肽聚糖。这些方法也将与荧光相结合 激活细胞分选，以分离因底物修改而改变代谢活性的细胞。已排序 细胞将通过鸟枪式元基因组学进行测序，这将提供活跃细胞在 原位表型和基因分型。非破坏性拉曼显微光谱学与稳定同位素相结合 探测，将用于研究人类肠道特定成员的生物化学和底物利用 微生物组。这将使我们能够测试关于生长底物的预测是否在之前的元基因组学中产生 培养驱动的研究真正反映了这些微生物是如何在人类肠道中生活的。最后，我们将发展 新的相关显微镜方法将整合来自不同成像来源的信息以进行可视化 微生物、它们的化学成分和基因表达活动直接在肠道中以它们的天然取向进行。 具体地说，我们将使用荧光和电子显微镜，基于拉曼的化学成像，以及能量- 包埋和薄片粘膜样品的色散x射线光谱分析。

Project Summary The human gut microbiome field lags behind other microbial ecology fields in its application of single cell resolved techniques capable of revealing the function of microbes at (close to) in situ conditions. By building on my lab’s expertise in working with sample types that are taxonomically, functionally, and structurally more complex than the gut microbiome by most metrics, I propose to transform the human gut microbiome field by catapulting it into the era of single cell ecophysiology investigations. Rather than growing individual members of the gut microbiome in isolation or trying to extrapolate findings from bulk omics approaches that destroy spatial information, we will study gut microorganisms at single cell resolution where they actually live, as members of spatially structured and metabolically interdependent ecosystems. We will investigate the functional activity and metabolic diversity of microbes in mucosal biopsy samples from the human gastrointestinal tract using several cutting-edge technologies that are currently not used or are underutilized in the field. Substrate analog probing and bioorthogonal labeling, in combination with fluorescence in situ hybridization, will be used to study which cells synthesize new DNA, proteins, lipids, or peptidoglycan under specific physicochemical conditions. These methods will also be combined with fluorescence activated cell sorting to separate cells that change their metabolic activity in response to substrate amendment. Sorted cells will be sequenced via shotgun metagenomics, which will provide a direct link between the active cells’ in situ phenotype and genotype. Non-destructive Raman microspectroscopy, in combination with stable isotope probing, will be used to study the biochemistry and substrate utilization of specific members of the human gut microbiome. This will allow us to test whether predictions about growth substrates generated in previous metagenomics and cultivation driven studies are truly reflective of how these microbes live in the human gut. Last, we will develop novel correlative microscopy approaches that will integrate information from a diversity of imaging sources to visualize microbes, their chemical composition, and gene expression activity directly in their native orientation in the gut. Specifically, we will employ fluorescence and electron microscopy, Raman based chemical imaging, and energy- dispersive x-ray spectroscopy on embedded and thin-sectioned mucosal samples.