Developing Next-Generation Physiology approaches for human gut microbiome research

项目摘要 人类肠道微生物组领域在单细胞解决的应用方面落后于其他微生物生态学领域。 能够在（接近）原位条件下揭示微生物功能的技术。利用我实验室的专业知识 在处理分类学上、功能上和结构上比肠道更复杂的样本类型时， 通过大多数指标，我建议通过将人类肠道微生物组纳入新时代来改变人类肠道微生物组领域。 单细胞生理生态学研究的一部分。而不是在体内培养肠道微生物组的单个成员 孤立或试图从大量组学方法中推断破坏空间信息的发现，我们将研究 肠道微生物在单细胞分辨率，他们实际上生活，作为空间结构的成员， 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 在使用几种目前最先进的技术从人类胃肠道粘膜活检样本， 在外地没有使用或利用不足。底物类似物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，脂质，或 肽聚糖在特定的物理化学条件下。这些方法也将与荧光相结合 活化细胞分选以分离响应底物修正而改变其代谢活性的细胞。排序 细胞将通过鸟枪宏基因组学进行测序，这将提供活性细胞之间的直接联系， 原位表型和基因型。与稳定同位素结合的非破坏性拉曼显微光谱学 探测，将用于研究生物化学和底物利用的特定成员的人类肠道 微生物组这将使我们能够测试是否在以前的宏基因组学中产生的关于生长底物的预测 培养驱动的研究真正反映了这些微生物如何在人类肠道中生存。最后，我们将开发 新的相关显微镜方法，将整合来自多种成像源的信息， 微生物，它们的化学组成和基因表达活性直接在肠道中的天然方向。 具体来说，我们将采用荧光和电子显微镜，拉曼为基础的化学成像，和能量- 包埋和薄切片粘膜样本的色散x射线光谱法。

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.