RESUMEN
Human milk oligosaccharides (HMOs) promote the development of the neonatal intestinal, immune, and nervous systems and has recently received considerable attention. Here we investigated how the maternal diet affects HMO biosynthesis and how any diet-induced HMO alterations influence the infant gut microbiome and immunity. Using capillary electrophoresis and MS-based analyses, we extracted and measured HMOs from breast milk samples and then correlated their levels with results from validated 24-h diet recall surveys and breast milk fatty acids. We found that fruit intake and unsaturated fatty acids in breast milk were positively correlated with an increased absolute abundance of numerous HMOs, including 16 sulfonated HMOs we identified here in humans for the first time. The diet-derived monosaccharide 5-N-glycolyl-neuraminic acid (Neu5Gc) was unambiguously detected in all samples. To gain insights into the potential impact of Neu5Gc on the infant microbiome, we used a constrained ordination approach and identified correlations between Neu5Gc levels and Bacteroides spp. in infant stool. However, Neu5Gc was not associated with marked changes in infant immune markers, in contrast with sulfonated HMOs, whose expression correlated with suppression of two major Th2 cytokines, IL-10 and IL-13. The findings of our work highlight the importance of maternal diet for HMO biosynthesis and provide as yet unexplored targets for future studies investigating interactions between HMOs and the intestinal microbiome and immunity in infants.
Asunto(s)
Microbioma Gastrointestinal/efectos de los fármacos , Leche Humana/metabolismo , Oligosacáridos/farmacología , Ácidos Sulfónicos/química , Bacteroides/efectos de los fármacos , Bacteroides/aislamiento & purificación , Secuencia de Carbohidratos , Dieta , Electroforesis Capilar , Ácidos Grasos Insaturados/metabolismo , Heces/microbiología , Humanos , Lactante , Recién Nacido , Interleucina-10/metabolismo , Interleucina-13/metabolismo , Espectrometría de Masas , Ácidos Neuramínicos/química , Ácidos Neuramínicos/metabolismo , Ácidos Neuramínicos/farmacología , Oligosacáridos/análisis , Ácidos Sulfónicos/metabolismo , Células Th2/citología , Células Th2/efectos de los fármacos , Células Th2/inmunología , Células Th2/metabolismoRESUMEN
Clostridium perfringens is a Gram-positive opportunistic pathogen that is the principal etiological agent of necrotic enteritis (NE) in poultry. The ability of C. perfringens to incite NE depends upon its ability to penetrate the protective mucus barrier within the small intestine, which is largely composed of heavily glycosylated proteins called mucins. Mucins are decorated by N- and O-linked glycans that serve both as a formidable gel-like barrier against invading pathogens and as a rich carbon source for mucolytic bacteria. The composition of avian O-linked glycans is markedly different from mucins in other vertebrates, being enriched in sulfated monosaccharides and N-acetyl-d-neuraminic acid (Neu5Ac, sialic acid). These modifications increase the overall negative charge of mucins and are believed to impede colonization by enteric pathogens. The mechanism by which C. perfringens penetrates the poultry intestinal mucus layer during NE is still unknown. However, the CAZome (i.e., the total collection of proteins encoded within a genome active on carbohydrates) of C. perfringens strain CP1 encodes several putative and known enzymes with activities consistent with the modification of mucin. To further investigate this relationship, O-glycans from Gallus gallus domesticus mucus were extracted from the small intestine and characterized using gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry. Chicken mucin monosaccharides included l-fucose (Fuc), d-mannose (Man), d-galactose (Gal), N-acetyl-d-galactosamine (GalNAc), N-acetyl-d-glucosamine (GlcNAc), and Neu5Ac (sialic acid). Using these monosaccharides as sole carbon sources, we showed that C. perfringens CP1 grew on Neu5Ac, Man, Gal, and GlcNAc but not on Fuc and GalNAc. We also demonstrated C. perfringens grew on different native-state preparations of intestinal mucins and mucus including porcine mucins, chicken mucus, and chicken mucins. Finally, anaerobic incubation of chicken mucin O-glycans with C. perfringens and subsequent analysis of the glycans revealed that there was preferential removal of Neu5Ac. These observations are discussed in the context of the predicted metabolic potential of C. perfringens CP1 and the mucolytic enzymes encoded within its CAZome.
Asunto(s)
Pollos/microbiología , Clostridium perfringens/fisiología , Mucinas/química , Polisacáridos/química , Animales , Intestino Delgado/metabolismo , Intestino Delgado/microbiologíaRESUMEN
Bovine milk oligosaccharides (BMOs), like their analogues in human milk, have important prebiotic functions. Environmental factors have previously been linked to variation in BMO structures, and thus to test the hypothesis that the bovine diet may lead to these changes in relative BMO abundances, a rapid capillary electrophoresis (CE)-based work flow was developed to profile the BMOs extracted from the milk of cows fed distinctly different diets. Over the first week of lactation, few significant differences were observed between the different diet groups, with the dominant changes being clearly linked to lactation period. CE analyses indicated the presence of ten unusually anionic BMOs, which were predicted to be phosphorylated and sulfated species. Nine unique sulfated BMOs were detected by high-resolution accurate mass spectrometry, none of which have been previously described in bovine milk. The biosynthesis of these was in direct competition with 3'-sialyllactose, the most abundant BMO in bovine milk.