ena-STUDY-INSA Toulouse-25-11-2018-16:03:33:171-973
Investigating host microbiota relationships by activity based metagenomic
The intestinal mucus is formed by human glycoproteins, the O- and N-linked glycans of which constitute a crucial source of carbon for commensal gut bacteria, especially in case of deprivation of dietary glycans of plant origin. These last years, few dozens of carbohydrate active enzymes from cultivated mucin degraders have been characterized, but these biochemical data are far from being exhaustive, considering the fact that uncultured species dominate in the human gut microbiota. In this study, we used functional metagenomics to identify novel metabolic pathways involved in mucin-glycan harvesting by uncultured bacteria. First, the high-throughput screening of a fosmid metagenomic library constructed from the ileum mucosa microbiota with chromogenic substrates allowed us to isolate 124 clones producing sialidases, ß-D-N-acetyl-glucosaminidases, ß-D-N-acetyl-galactosaminidases, and/or ß-D-mannosidases, activities which are crucial to deconstruct human O-and N-glycans. Then, a second screening step, based on lectin binding assays, was applied to 13 of these clones selected for their diversified functional profiles, in order to demonstrate their ability to degrade human intestinal mucus. In total, structural modification of several mucin motives, especially the sialylated ones, was evidenced for 9 clones. Sequencing of their metagenomic loci highlighted complex catabolic pathways involving complementary functions of glycan sensing, transport, hydrolysis, deacetylation and deamination, sometimes associated to amino acid metabolisation machineries. These loci, assigned to several Bacteroides and Feacalibacterium species, are highly prevalent and abundant in the gut microbiome, and explain the metabolic flexibility of gut bacteria feeding both on dietary and human glycans.
The intestinal mucus is formed by human glycoproteins, the O- and N-linked glycans of which constitute a crucial source of carbon for commensal gut bacteria, especially in case of deprivation of dietary glycans of plant origin. These last years, few dozens of carbohydrate active enzymes from cultivated mucin degraders have been characterized, but these biochemical data are far from being exhaustive, considering the fact that uncultured species dominate in the human gut microbiota. In this study, we used functional metagenomics to identify novel metabolic pathways involved in mucin-glycan harvesting by uncultured bacteria. First, the high-throughput screening of a fosmid metagenomic library constructed from the ileum mucosa microbiota with chromogenic substrates allowed us to isolate 124 clones producing sialidases, ß-D-N-acetyl-glucosaminidases, ß-D-N-acetyl-galactosaminidases, and/or ß-D-mannosidases, activities which are crucial to deconstruct human O-and N-glycans. Then, a second screening step, based on lectin binding assays, was applied to 13 of these clones selected for their diversified functional profiles, in order to demonstrate their ability to degrade human intestinal mucus. In total, structural modification of several mucin motives, especially the sialylated ones, was evidenced for 9 clones. Sequencing of their metagenomic loci highlighted complex catabolic pathways involving complementary functions of glycan sensing, transport, hydrolysis, deacetylation and deamination, sometimes associated to amino acid metabolisation machineries. These loci, assigned to several Bacteroides and Feacalibacterium species, are highly prevalent and abundant in the gut microbiome, and explain the metabolic flexibility of gut bacteria feeding both on dietary and human glycans.