Rational design of a microbial consortium of mucosal sugar utilizers reduces Clostridiodes difficile colonization

Fátima C Pereira,Thomas Decker,Iva Cobankovic,Craig W Herbold,Kenneth Wasmund,Michael Wagner,Cornelia Vesely,Martin Von Bergen,Barbara Sziranyi,Benedikt Warth,Kang Soo Lee,Nico Jehmlich,David Berry,Roman Stocker

doi:10.1038/s41467-020-18928-1

Abstract

Many intestinal pathogens, including Clostridioides difficile, use mucus-derived sugars as crucial nutrients in the gut. Commensals that compete with pathogens for such nutrients are therefore ecological gatekeepers in healthy guts, and are attractive candidates for therapeutic interventions. Nevertheless, there is a poor understanding of which commensals use mucin-derived sugars in situ as well as their potential to impede pathogen colonization. Here, we identify mouse gut commensals that utilize mucus-derived monosaccharides within complex communities using single-cell stable isotope probing, Raman-activated cell sorting and mini-metagenomics. Sequencing of cell-sorted fractions reveals members of the underexplored family Muribaculaceae as major mucin monosaccharide foragers, followed by members of Lachnospiraceae, Rikenellaceae, and Bacteroidaceae families. Using this information, we assembled a five-member consortium of sialic acid and N-acetylglucosamine utilizers that impedes C. difficile’s access to these mucosal sugars and impairs pathogen colonization in antibiotic-treated mice. Our findings underscore the value of targeted approaches to identify organisms utilizing key nutrients and to rationally design effective probiotic mixtures.

Highlights

Many intestinal pathogens, including Clostridioides difficile, use mucus-derived sugars as crucial nutrients in the gut
Secreted mucin O-glycans have different core structures composed of N-acetylgalactosamine (GalNAc), Nacetylglucosamine (GlcNAc), and galactose residues, which can be further extended with galactose, GlcNAc, GalNAc, fucose or sialic acid (N-acetylneuraminic acid; N-Acetylneuraminic acid (NeuAc) or Neu5Ac) sugar residues, with the latter two frequently occupying terminal positions[12–15] (Fig. 1a)
The lowest concentration values were selected based on reported concentrations of the different monosaccharides in purified hog gastric mucin and mucin gels[36,37] and on the typical O-glycan structure of the large intestine, i.e., sugars such as galactose and GlcNAc are more abundant than are GalNAc or the terminal residues fucose and NeuAc38

Summary

Introduction

Many intestinal pathogens, including Clostridioides difficile, use mucus-derived sugars as crucial nutrients in the gut. We employ deuterium (D) stable isotope probing combined with Raman-activated cell sorting (RACS) and metagenomics to identify organisms that can forage on O-glycan monosaccharides in the mouse gut This approach allows us to non-destructively track microbial activity at the single-cell level in response to a variety of compounds, thereby enabling postmeasurement molecular analysis, and has been successfully employed in the past to identify foragers of host-derived proteins from the mouse gut[34,35]. Applying this recently developed technique, we identified a number of bacterial taxa as generalists in regard to mucosal sugar foraging, being able to utilize all the different O-glycan sugars, as well as taxa that seem to be specialized on only a subset of the available mucosal sugars. Our approach identified a consortium of gut bacteria that contributes to colonization resistance against C. difficile, and further indicates that depletion of mucosal sugars is one of the mechanisms underpinning this resistance

Methods

Results

Conclusion