Investigating Host Microbiota Relationships Through Functional Metagenomics.

Elisabeth Laville,Josette Perrier,Emna Bouhajja,Jeremy Esque,Bernard Henrissat,Gabrielle Potocki-Veronese,Eric Di Pasquale,Nada Bejar,Elodie Drula,Alexandra S Tauzin,Stephane Berdah,Patrick Robe,Marion Leclerc,Marc Maresca

doi:10.3389/fmicb.2019.01286

Abstract

The human Intestinal mucus is formed by glycoproteins, the O- and N-linked glycans which constitute a crucial source of carbon for commensal gut bacteria, especially when deprived of dietary glycans of plant origin. In recent years, a dozen carbohydrate-active enzymes from cultivated mucin degraders have been characterized. But yet, considering the fact that uncultured species predominate in the human gut microbiota, these biochemical data are far from exhaustive. In this study, we used functional metagenomics to identify new metabolic pathways in uncultured bacteria involved in harvesting mucin glycans. First, we performed a high-throughput screening of a fosmid metagenomic library constructed from the ileum mucosa microbiota using chromogenic substrates. The screening resulted in the isolation of 124 clones producing activities crucial in the degradation of human O- and N-glycans, namely sialidases, β-D-N-acetyl-glucosaminidase, β-D-N-acetyl-galactosaminidase, and/or β-D-mannosidase. Thirteen of these clones were selected based on their diversified functional profiles and were further analyzed on a secondary screening. This step consisted of lectin binding assays to demonstrate the ability of the clones to degrade human intestinal mucus. In total, the structural modification of several mucin motifs, sialylated mucin ones in particular, was evidenced for nine clones. Sequencing their metagenomic loci highlighted complex catabolic pathways involving the complementary functions of glycan sensing, transport, hydrolysis, deacetylation, and deamination, which were sometimes associated with amino acid metabolism machinery. These loci are assigned to several Bacteroides and Feacalibacterium species highly prevalent and abundant in the gut microbiome and explain the metabolic flexibility of gut bacteria feeding both on dietary and human glycans.

Highlights

The human intestinal tract harbors a dense commensal microbial community whose interactions with the host are of paramount importance for its health and well-being, for the development of immunity and protection against invasion by pathogens
The 20,000 clones of the E. coli fosmid library constructed from the ileum mucosa microbiota, covering in total 0.7 Gb of metagenomic sequence, were screened for most the α- and β-glycosidase activities required for the degradation of N- and O-glycans
Α-L-fucosidases, β-D-Nacetyl-glucosaminidases, β-D-N-acetyl-galactosaminidases, α-D-neuraminic-acid hydrolases, α-D- and β-D-mannosidases were searched for using chromogenic reagents (5-Bromo-4chloro-3-indoxyl-glycosyl, known as X-glycosyl substrates) in a rich solid medium on which the metagenomic clones had been gridded

Summary

Introduction

The human intestinal tract harbors a dense commensal microbial community whose interactions with the host are of paramount importance for its health and well-being, for the development of immunity and protection against invasion by pathogens. One of the best-known functions of the microbiota is its contribution to the digestion of dietary fibers, derived mainly from plant cell walls. Dietary fibers, consisting of polysaccharides of great structural diversity, represent the main energy source for the microbiota. The majority of gut bacterial species possess an enzymatic arsenal in the form of carbohydrate-active enzymes (CAZymes; Lombard et al, 2014) used to depolymerize these fibers. In addition to dietary fibers, intestinal microorganisms use glycans associated with human glycoproteins as a source of energy in particular mucin glycans that are secreted by the goblet cells of the intestinal epithelium. The enzymatic machinery of these bacteria allows mucins to be recycled

Methods

Results

Conclusion