Developing Next-Generation Physiology approaches for human gut microbiome research

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.