Sarah M. Totten

Sialylated Milk Oligosaccharides Promote Microbiota-Dependent Growth in Models of Infant Undernutrition

Identifying interventions that more effectively promote healthy growth of children with undernutrition is a pressing global health goal. Analysis of human milk oligosaccharides (HMOs) from 6-month-postpartum mothers in two Malawian birth cohorts revealed that sialylated HMOs are significantly less abundant in those with severely stunted infants. To explore this association, we colonized young germ-free mice with a consortium of bacterial strains cultured from the fecal microbiota of a 6-month-old stunted Malawian infant and fed recipient animals a prototypic Malawian diet with or without purified sialylated bovine milk oligosaccharides (S-BMO). S-BMO produced a microbiota-dependent augmentation of lean body mass gain, changed bone morphology, and altered liver, muscle, and brain metabolism in ways indicative of a greater ability to utilize nutrients for anabolism. These effects were also documented in gnotobiotic piglets using the same consortium and Malawian diet. These preclinical models indicate a causal, microbiota-dependent relationship between S-BMO and growth promotion.

Maternal fucosyltransferase 2 status affects the gut bifidobacterial communities of breastfed infants.

BackgroundIndividuals with inactive alleles of the fucosyltransferase 2 gene (FUT2; termed the ‘secretor’ gene) are common in many populations. Some members of the genus Bifidobacterium, common infant gut commensals, are known to consume 2′-fucosylated glycans found in the breast milk of secretor mothers. We investigated the effects of maternal secretor status on the developing infant microbiota with a special emphasis on bifidobacterial species abundance.ResultsOn average, bifidobacteria were established earlier and more often in infants fed by secretor mothers than in infants fed by non-secretor mothers. In secretor-fed infants, the relative abundance of the Bifidobacterium longum group was most strongly correlated with high percentages of the order Bifidobacteriales. Conversely, in non-secretor-fed infants, Bifidobacterium breve was positively correlated with Bifidobacteriales, while the B. longum group was negatively correlated. A higher percentage of bifidobacteria isolated from secretor-fed infants consumed 2′-fucosyllactose. Infant feces with high levels of bifidobacteria had lower milk oligosaccharide levels in the feces and higher amounts of lactate. Furthermore, feces containing different bifidobacterial species possessed differing amounts of oligosaccharides, suggesting differential consumption in situ.ConclusionsInfants fed by non-secretor mothers are delayed in the establishment of a bifidobacteria-laden microbiota. This delay may be due to difficulties in the infant acquiring a species of bifidobacteria able to consume the specific milk oligosaccharides delivered by the mother. This work provides mechanistic insight into how milk glycans enrich specific beneficial bacterial populations in infants and reveals clues for enhancing enrichment of bifidobacterial populations in at risk populations - such as premature infants.

Variation in consumption of human milk oligosaccharides by infant gut-associated strains of Bifidobacterium breve.

ABSTRACT Human milk contains a high concentration of complex oligosaccharides that influence the composition of the intestinal microbiota in breast-fed infants. Previous studies have indicated that select species such as Bifidobacterium longum subsp. infantis and Bifidobacterium bifidum can utilize human milk oligosaccharides (HMO) in vitro as the sole carbon source, while the relatively few B. longum subsp. longum and Bifidobacterium breve isolates tested appear less adapted to these substrates. Considering the high frequency at which B. breve is isolated from breast-fed infant feces, we postulated that some B. breve strains can more vigorously consume HMO and thus are enriched in the breast-fed infant gastrointestinal tract. To examine this, a number of B. breve isolates from breast-fed infant feces were characterized for the presence of different glycosyl hydrolases that participate in HMO utilization, as well as by their ability to grow on HMO or specific HMO species such as lacto-N-tetraose (LNT) and fucosyllactose. All B. breve strains showed high levels of growth on LNT and lacto-N-neotetraose (LNnT), and, in general, growth on total HMO was moderate for most of the strains, with several strain differences. Growth and consumption of fucosylated HMO were strain dependent, mostly in isolates possessing a glycosyl hydrolase family 29 α-fucosidase. Glycoprofiling of the spent supernatant after HMO fermentation by select strains revealed that all B. breve strains can utilize sialylated HMO to a certain extent, especially sialyl-lacto-N-tetraose. Interestingly, this specific oligosaccharide was depleted before neutral LNT by strain SC95. In aggregate, this work indicates that the HMO consumption phenotype in B. breve is variable; however, some strains display specific adaptations to these substrates, enabling more vigorous consumption of fucosylated and sialylated HMO. These results provide a rationale for the predominance of this species in breast-fed infant feces and contribute to a more accurate picture of the ecology of the developing infant intestinal microbiota.

Label-free absolute quantitation of oligosaccharides using multiple reaction monitoring

An absolute quantitation method for measuring free human milk oligosaccharides (HMOs) in milk samples was developed using multiple reaction monitoring (MRM). To obtain the best sensitivity, the instrument conditions were optimized to reduce the source and postsource fragmentation prior to the quadrupole transmission. Fragmentation spectra of HMOs using collision-induced dissociation were studied to obtain the best characteristic fragments. At least two MRM transitions were used to quantify and identify each structure in the same run. The fragment ions corresponded to the production of singly charged mono-, di-, and trisaccharide fragments. The sensitivity and accuracy of the quantitation using MRM were determined, with the detection limit in the femtomole level and the calibration range spanning over 5 orders of magnitude. Seven commercial HMO standards were used to create calibration curves and were used to determine a universal response for all HMOs. The universal response factor was used to estimate absolute amounts of other structures and the total oligosaccharide content in milk. The quantitation method was applied to 20 human milk samples to determine the variations in HMO concentrations from women classified as secretors and nonsecretors, a phenotype that can be identified by the concentration of 2′-fucosylation in their milk.

Glyco-Analytical Multispecific Proteolysis (Glyco-AMP): A Simple Method for Detailed and Quantitative Glycoproteomic Characterization

Despite recent advances, site-specific profiling of protein glycosylation remains a significant analytical challenge for conventional proteomic methodology. To alleviate the issue, we propose glyco-analytical multispecific proteolysis (Glyco-AMP) as a strategy for glycoproteomic characterization. Glyco-AMP consists of rapid, in-solution digestion of an analyte glycoprotein (or glycoprotein mixture) by a multispecific protease (or protease cocktail). Resulting glycopeptides are chromatographically separated by isomer-specific porous graphitized carbon nano-LC, quantified by high-resolution MS, and structurally elucidated by MS/MS. To demonstrate the consistency and customizability of Glyco-AMP methodology, the glyco-analytical performances of multispecific proteases subtilisin, pronase, and proteinase K were characterized in terms of quantitative accuracy, sensitivity, and digestion kinetics. Glyco-AMP was shown be effective on glycoprotein mixtures as well as glycoproteins with multiple glycosylation sites, providing detailed, quantitative, site- and structure-specific information about protein glycosylation.

Human Milk Secretory Immunoglobulin A and Lactoferrin N-Glycans Are Altered in Women with Gestational Diabetes Mellitus

Very little is known about the effects of gestational diabetes mellitus (GDM) on lactation and milk components. Recent reports suggested that hyperglycemia during pregnancy was associated with altered breast milk immune factors. Human milk oligosaccharides (HMOs) and N-glycans of milk immune-modulatory proteins are implicated in modulation of infant immunity. The objective of the current study was to evaluate the effect of GDM on HMO and protein-conjugated glycan profiles in breast milk. Milk was collected at 2 wk postpartum from women diagnosed with (n = 8) or without (n = 16) GDM at week 24-28 in pregnancy. Milk was analyzed for HMO abundances, protein concentrations, and N-glycan abundances of lactoferrin and secretory immunoglobulin A (sIgA). HMOs and N-glycans were analyzed by mass spectrometry and milk lactoferrin and sIgA concentrations were analyzed by the Bradford assay. The data were analyzed using multivariate modeling confirmed with univariate statistics to determine differences between milk of women with compared with women without GDM. There were no differences in HMOs between milk from women with vs. without GDM. Milk from women with GDM compared with those without GDM was 63.6% lower in sIgA protein (P < 0.05), 45% higher in lactoferrin total N-glycans (P < 0.0001), 36-72% higher in lactoferrin fucose and sialic acid N-glycans (P < 0.01), and 32-43% lower in sIgA total, mannose, fucose, and sialic acid N-glycans (P < 0.05). GDM did not alter breast milk free oligosaccharide abundances but decreased total protein and glycosylation of sIgA and increased glycosylation of lactoferrin in transitional milk. The results suggest that maternal glucose dysregulation during pregnancy has lasting consequences that may influence the innate immune protective functions of breast milk.

Rapid-throughput glycomics applied to human milk oligosaccharide profiling for large human studies

AbstractGlycomic analysis is the comprehensive determination of glycan (oligosaccharide) structures with quantitative information in a biological sample. Rapid-throughput glycomics is complicated due to the lack of a template, which has greatly facilitated analysis in the field of proteomics. Furthermore, the large similarities in structures make fragmentation spectra (as obtained in electron impact ionization and tandem mass spectrometry) less definitive for identification as it has been in metabolomics. In this study, we develop a concept of rapid-throughput glycomics on human milk oligosaccharides, which have proven to be an important bioactive component of breast milk, providing the infant with protection against pathogenic infection and supporting the establishment of a healthy microbiota. To better understand the relationship between diverse oligosaccharides structures and their biological function as anti-pathogenic and prebiotic compounds, large human studies are needed, which necessitate rapid- to high-throughput analytical platforms. Herein, a complete glycomics methodology is presented, evaluating the most effective human milk oligosaccharide (HMO) extraction protocols, the linearity and reproducibility of the nano-liquid chromatography chip time-of-flight mass spectrometry (nano-LC chip-TOF MS) method, and the efficacy of newly developed, in-house software for chromatographic peak alignment that allows for rapid data analysis. High instrument stability and retention time reproducibility, together with the successful automated alignment of hundreds of features in hundreds of milk samples, allow for the use of an HMO library for rapid assignment of fully annotated structures. Graphical Abstractᅟ

A novel gene cluster allows preferential utilization of fucosylated milk oligosaccharides in Bifidobacterium longum subsp. longum SC596

The infant intestinal microbiota is often colonized by two subspecies of Bifidobacterium longum: subsp. infantis (B. infantis) and subsp. longum (B. longum). Competitive growth of B. infantis in the neonate intestine has been linked to the utilization of human milk oligosaccharides (HMO). However, little is known how B. longum consumes HMO. In this study, infant-borne B. longum strains exhibited varying HMO growth phenotypes. While all strains efficiently utilized lacto-N-tetraose, certain strains additionally metabolized fucosylated HMO. B. longum SC596 grew vigorously on HMO, and glycoprofiling revealed a preference for consumption of fucosylated HMO. Transcriptomes of SC596 during early-stage growth on HMO were more similar to growth on fucosyllactose, transiting later to a pattern similar to growth on neutral HMO. B. longum SC596 contains a novel gene cluster devoted to the utilization of fucosylated HMO, including genes for import of fucosylated molecules, fucose metabolism and two α-fucosidases. This cluster showed a modular induction during early growth on HMO and fucosyllactose. This work clarifies the genomic and physiological variation of infant-borne B. longum to HMO consumption, which resembles B. infantis. The capability to preferentially consume fucosylated HMO suggests a competitive advantage for these unique B. longum strains in the breast-fed infant gut.

Identification of oligosaccharides in feces of breast-fed infants and their correlation with the gut microbial community

Glycans in breast milk are abundant and found as either free oligosaccharides or conjugated to proteins and lipids. Free human milk oligosaccharides (HMOs) function as prebiotics by stimulating the growth of beneficial bacteria while preventing the binding of harmful bacteria to intestinal epithelial cells. Bacteria have adapted to the glycan-rich environment of the gut by developing enzymes that catabolize glycans. The decrease in HMOs and the increase in glycan digestion products give indications of the active enzymes in the microbial population. In this study, we quantitated the disappearance of intact HMOs and characterized the glycan digestion products in the gut that are produced by the action of microbial enzymes on HMOs and glycoconjugates from breast milk. Oligosaccharides from fecal samples of exclusively breast-fed infants were extracted and profiled using nanoLC-MS. Intact HMOs were found in the fecal samples, additionally, other oligosaccharides were found corresponding to degraded HMOs and non-HMO based compounds. The latter compounds were fragments of N-glycans released through the cleavage of the linkage to the asparagine residue and through cleavage of the chitobiose core of the N-glycan. Marker gene sequencing of the fecal samples revealed bifidobacteria as the dominant inhabitants of the infant gastrointestinal tracts. A glycosidase from Bifidobacterium longum subsp. longum was then expressed to digest HMOs in vitro, which showed that the digested oligosaccharides in feces corresponded to the action of glycosidases on HMOs. Similar expression of endoglycosidases also showed that N-glycans were released by bacterial enzymes. Although bifidobacteria may dominate the gut, it is possible that specific minority species are also responsible for the major products observed in feces. Nonetheless, the enzymatic activity correlated well with the known glycosidases in the respective bacteria, suggesting a direct relationship between microbial abundances and catabolic activity.

Parallel Comparison of N-Linked Glycopeptide Enrichment Techniques Reveals Extensive Glycoproteomic Analysis of Plasma Enabled by SAX-ERLIC

Protein glycosylation is of increasing interest due to its important roles in protein function and aberrant expression with disease. Characterizing protein glycosylation remains analytically challenging due to its low abundance, ion suppression issues, and microheterogeneity at glycosylation sites, especially in complex samples such as human plasma. In this study, the utility of three common N-linked glycopeptide enrichment techniques is compared using human plasma. By analysis on an LTQ-Orbitrap Elite mass spectrometer, electrostatic repulsion hydrophilic interaction liquid chromatography using strong anion exchange solid-phase extraction (SAX-ERLIC) provided the most extensive N-linked glycopeptide enrichment when compared with multilectin affinity chromatography (M-LAC) and Sepharose-HILIC enrichments. SAX-ERLIC enrichment yielded 191 unique glycoforms across 72 glycosylation sites from 48 glycoproteins, which is more than double that detected using other enrichment techniques. The greatest glycoform diversity was observed in SAX-ERLIC enrichment, with no apparent bias toward specific glycan types. SAX-ERLIC enrichments were additionally analyzed by an Orbitrap Fusion Lumos mass spectrometer to maximize glycopeptide identifications for a more comprehensive assessment of protein glycosylation. In these experiments, 829 unique glycoforms were identified across 208 glycosylation sites from 95 plasma glycoproteins, a significant improvement from the initial method comparison and one of the most extensive site-specific glycosylation analysis in immunodepleted human plasma to date. Data are available via ProteomeXchange with identifier PXD005655.