A Probe-Enabled Approach for the Selective Isolation and Characterization of Functionally Active Subpopulations in the Gut Microbiome

Abstract

Commensal microorganisms in the mammalian gut play important roles in host health and physiology, but a central challenge remains in achieving a detailed mechanistic understanding of specific microbial contributions to host biochemistry. New function-based approaches are needed that analyze gut microbial function at the molecular level by coupling detection and measurements of in situ biochemical activity with identification of the responsible microbes and enzymes. We developed a platform employing β-glucuronidase selective activity-based probes to detect, isolate, and identify microbial subpopulations in the gut responsible for this xenobiotic metabolism. We find that metabolic activity of gut microbiota can be plastic and that between individuals and during perturbation, phylogenetically disparate populations can provide β-glucuronidase activity. Our work links biochemical activity with molecular-scale resolution without relying on genomic inference.

Who Is Metabolizing What? Discovering Novel Biomolecules in the Microbiome and the Organisms Who Make Them

“…A widespread approach in microbiome research has been to associate different diseases with alterations in the microbiome (dysbiosis), but directionality is often unknown and it is not clear if these changes are causal or simply associative (Olesen and Alm, 2016 ). Fluctuations in community composition do not necessarily indicate changes in community function or metabolic activity (Whidbey et al, 2019 ). In order to be able to design microbiome-modulation based therapies to improve human health, a deeper functional knowledge is required and comprising of (A) complete biochemical characterization of microbiome metabolites, (B) the proteins involved in their production, conversion or transport, (C) the microbial populations responsible for producing, utilizing or otherwise interacting with these molecules, and (D) their effect on host physiology.…”

“…ABPP can also identify viable whole cells in complex samples based on a certain function without needing any information beforehand, which may aid in the study of un-culturable microbes with poorly characterized genomes. ABPP has been coupled with fluorescence-assisted cell sorting (FACS) to identify and isolate single cell population responsible for enzyme activity (Whidbey et al, 2019 ). Hence, combining FACS with ABPP allows for the enrichment of functionally active cells from microbiome with reduced sample complexity.…”

“…ABPs have also been applied to study the microbiome's modification of other host-produced metabolites, such as bile salts (Zhuang et al, 2017 ; Parasar et al, 2019 ), which have implications in the onset of diseases including cholestatic and inflammatory diseases, diabetes, and obesity (Wahlstrom et al, 2016 ). Microbial enzymes such as proteases, hydrolases, and β-glucuronidases have been labeled using various ABPs and applied successfully in investigating the changes in gut microbiome activity in different disease models (Hatzios et al, 2016 ; Mayers et al, 2017 ; Zhuang et al, 2017 ; Parasar et al, 2019 ; Whidbey et al, 2019 ; Jariwala et al, 2020 ). Importantly, these analyses reveal that change in microbial enzyme activity does not faithfully correspond to gene abundance, which reiterates the necessity of function-based analyses such as ABPP for researchers to harness the chemistry of the microbiome.…”

Chemical Reporters for Exploring Microbiology and Microbiota Mechanisms

“…For example, the Wright laboratory has generated a vitamin B 12 ‐based chemical reporter that enables discovery of a light‐sensing B 12 ‐binding transcription regulator that controls folate and ubiquinone biosynthesis . The Wright laboratory has also generated fluorophore‐modified mechanism‐based probe of β‐glucuronidases to label and identify specific microbiota species that contain this activity . Alternatively, the Chang laboratory has generated an azide‐functionalized bile salt hydrolase (BSH) reporter (Figure E) to identify these enzymes from specific cultured microbiota species as well as from fecal and tissue samples .…”

“…[124] The Wright laboratory has also generated fluorophore-modified mechanism-based probe of b-glucuronidases to label and identify specific microbiota speciest hat contain thisa ctivity. [125] Al- Figure 12. Microbiota-derived butyrate inhibits enteric pathogen virulence through site-specific protein acylation.…”