Identifying glycan consumers in human gut microbiota samples using metabolic labeling coupled with fluorescence-activated cell sorting

Abstract

The composition and metabolism of the human gut microbiota are strongly influenced by dietary complex glycans, which cause downstream effects on the physiology and health of hosts. Despite recent advances in our understanding of glycan metabolism by human gut bacteria, we still need methods to link glycans to their consuming bacteria. Here, we use a functional assay to identify and isolate gut bacteria from healthy human volunteers that take up different glycans. The method combines metabolic labeling using fluorescent oligosaccharides with fluorescence-activated cell sorting (FACS), followed by amplicon sequencing or culturomics. Our results demonstrate metabolic labeling in various taxa, such as Prevotella copri, Collinsella aerofaciens and Blautia wexlerae. In vitro validation confirms the ability of most, but not all, labeled species to consume the glycan of interest for growth. In parallel, we show that glycan consumers spanning three major phyla can be isolated from cultures of sorted labeled cells. By linking bacteria to the glycans they consume, this approach increases our basic understanding of glycan metabolism by gut bacteria. Going forward, it could be used to provide insight into the mechanism of prebiotic approaches, where glycans are used to manipulate the gut microbiota composition.

Characterizing the Effect of Amylase Inhibitors on Maltodextrin Metabolism by Gut Bacteria Using Fluorescent Glycan Labeling

“…To determine if metabolic labeling of gut bacteria was possible using a large maltodextrin oligomer, we first applied our previously published pipeline with MAL-F using human stool samples . Fecal samples were collected from 10 healthy unrelated human volunteers under our approved protocol (A04-M27-15B).…”

“…However, we have also observed that bacteria labeled by a fluorescent glycan do not automatically use the glycan for growth, and therefore, validation is required for the low abundant O. formigenes. 51,59 For its part, E. eligens has been reported as unable to grow on amylose or amylopectin, yet the strain ATCC 27750 encodes 6 GH13s. 60 One of these GH13 genes is found neighboring a major facilitator superfamily (MFS) transporter gene, which suggests potential for oligosaccharide uptake.…”

“…Given the microbiome diversity in taxonomy and genes encoded, efficient methods are required to probe gut bacterial metabolism. Fluorescently labeled glycans are a useful and versatile tool that can be applied to investigate glycan uptake and metabolism by bacteria from aquatic, rumen, or human gut origin. , This method is particularly powerful when coupled with fluorescence activated cell sorting (FACS) and sequencing because it can interrogate complex samples in an unbiased fashion. , We have recently shown that gut bacteria actively taking up fluorescent glycans could be recovered from stool samples by FACS and identified by amplicon sequencing . Interestingly, not all bacteria taking up fluorescent glycans were able to grow in vitro on that glycan.…”

“… 50 , 51 This method is particularly powerful when coupled with fluorescence activated cell sorting (FACS) and sequencing because it can interrogate complex samples in an unbiased fashion. 48 , 51 We have recently shown that gut bacteria actively taking up fluorescent glycans could be recovered from stool samples by FACS and identified by amplicon sequencing. 51 Interestingly, not all bacteria taking up fluorescent glycans were able to grow in vitro on that glycan.…”

Recent Progress in Human Milk Oligosaccharides and Its Antiviral Efficacy

“…The intestinal microbiota is vital to the human body as it assists in digestion, releases essential nutrients, normalizes metabolism, participates in the immune system, and protects against various diseases. 80 Advances in genetic and analytical tools and methods have revealed that the gut microbiota are complex and diverse and may be associated with certain diseases. For example, the composition of the gut microbiota affects mucosal integrity, GI tract development, pathogen invasion, and nutrient absorption and assimilation.…”