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Illuminating Functional Networks and Keystone Species in the Gut

Periodic Reporting for period 2 - FunKeyGut (Illuminating Functional Networks and Keystone Species in the Gut)

Reporting period: 2018-10-01 to 2020-03-31

We live in an intimate symbiosis with our gut microbiota, which provides us services such as vitamin production, breakdown of dietary compounds, and immune training. Sequencing-based approaches that have been applied to catalogue the gut microbiota have revealed intriguing discoveries associating the microbiome with diet and disease. The next outstanding challenge is to unravel the many activities and interactions that define gut microbiota function.

The gut microbiota is a diverse community of cooperating and competing microbes. These interactions form a network that links organisms with each other and their environment. Interactions in such a “functional network” are based partially, though not exclusively, on food webs. Certain “keystone species”, such as Rumonicoccus bromii, are thought to play a major role in these networks. Though some evidence exists for the presence of keystone species, their identity and activity remains largely unknown. As keystone species are vital to networks they are ideal targets for manipulating the gut microbiota to improve metabolic health and protect against enteropathogen infection.

Given the complexity of the gut microbiota, networks can only be elucidated directly in the native community. This project aims to identify functional networks and keystone species in the human gut using novel approaches that are uniquely and ideally suited for studying microbial activity in complex communities. Using state-of-the-art methods such as stable isotope labeling, Raman microspectroscopy, and secondary ion mass spectrometry (NanoSIMS) we will illuminate functional networks in situ. This will allow us to identify what factors shape gut microbiota activity, reveal important food webs, and ultimately use network knowledge to target the microbiota with prebiotic/probiotic treatments rationally designed to promote health.
Thus-far we have established abundance-based co-occurrence networks on publicly-available sequencing datasets of the human gut microbiota to determine putative keystone taxa, genes, and gene transcripts. These keystones will be experimentally-tested in the course of the project. We have also established routines for function-based sorting of microbial cells stimulated by specific incubation conditions. This sorting is based on heavy water incubations followed by microfluidic-based Raman microspectroscopy and sorting, as well as bioorthogonal non-canonical amino acid tagging (BONCAT) and sorting with fluorescence-activated cell sorting. These tools allow us to perform downstream genomics/metagenomics as well as targeted cultivation of microbes of interest. We have applied this approach to a variety of questions involving keystone species involved in degradation of prebiotic compounds as well as other complex carbon compounds and mucosal glycans.
The development of a Raman-based cultivation workflow for strict anaerobes is major progress beyond the state-of-the-art. We expect that this approach will allow us to directly isolate keystone species that may be potential probiotic species to be administered to re-establish a healthy gut microbiota.
Example of a functional network