Longitudinal analysis of human milk oligosaccharides and mucin-2-glycans in infants reveals enrichment of sulfated and glucuronic acid-bearing HMOs

In infants, the acquisition of an adult-like gastrointestinal (GI) microbiome begins at birth and fully develops by toddlerhood. This colonisation is influenced by the diet, which, in breast-fed infants, contains high concentrations of soluble human milk oligosaccharides (HMOs), prebiotics that bear structural resemblance to the glycans that decorate Mucin-2 (MUC2). MUC2 is the first line of defense between the microbiota and the underlying mucosal tissues; it also provides a nutrient-rich habitat for GI commensal microbes. The research described herein builds on two earlier findings: the ability to sample MUC2 adhering to the surface of stool samples and the discovery of a class of sulfated HMOs within human milk. Studies have shown that de-sulfation of MUC2-borne glycans is a key to their metabolism by GI microbes in vitro and, if sulfated glycans are otherwise metabolically-resistant, two hypotheses follow. First, in contrast with major prebiotic HMOs, sulfated HMOs will be enriched in the infant GI tract; second, high abundances of glyco-epitopes like sulfation in HMOs will deflect the microbiome's metabolic activity away from MUC2, leading to detectable changes in the host glycome. We show that both HMOs and MUC2 could be non-invasively sampled from the same infant stool samples. Capillary electrophoresis analyses of paired milk/stool samples demonstrated significant enrichment of sulfate-containing HMOs in stool. High-resolution mass spectrometry (HRMS) was used to detect numerous sulfated HMOs, including a new class concurrently containing glucuronic acid (GlcA) residues. Infant MUC2-derived glycans were also analyzed by HRMS; this is the first reported semi-quantitative longitudinal study and provides a benchmark for future research.