Jennifer T. Smilowitz

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.

Breast Milk Oligosaccharides: Structure-Function Relationships in the Neonate

In addition to providing complete postnatal nutrition, breast milk is a complex biofluid that delivers bioactive components for the growth and development of the intestinal and immune systems. Lactation is a unique opportunity to understand the role of diet in shaping the intestinal environment including the infant microbiome. Of considerable interest is the diversity and abundance of milk glycans that are energetically costly for the mammary gland to produce yet indigestible by infants. Milk glycans comprise free oligosaccharides, glycoproteins, glycopeptides, and glycolipids. Emerging technological advances are enabling more comprehensive, sensitive, and rapid analyses of these different classes of milk glycans. Understanding the impact of inter- and intraindividual glycan diversity on function is an important step toward interventions aimed at improving health and preventing disease. This review discusses the state of technology for glycan analysis and how specific structure-function knowledge is enhancing our understanding of early nutrition in the neonate.

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.

Glycosylation of Human Milk Lactoferrin Exhibits Dynamic Changes During Early Lactation Enhancing Its Role in Pathogenic Bacteria-Host Interactions

Human milk lactoferrin (hmLF) is the most abundant glycoprotein present in human milk and displays a broad range of protective functions in the gut of newborn infants. hmLF is N-glycosylated, but little is known about the lactation stage-related development of the glycosylation phenotype. hmLF glycosylation from milk samples from five donors during the first 10 weeks of lactation was assessed and observed to be more diverse than previously reported. During this period dynamic changes in glycosylation were observed corresponding to a decrease in glycosylation in the second week followed by an increase in total glycosylation as well as higher order fucosylation thereafter. Gene expression analysis was performed in milk somatic cells from a sixth subject. It was found that fucosyltransferase expression increased during entire period, whereas expression of genes for the oligosaccharyl transferase complex decreased in the second week. The effect of hmLF glycosylation was examined for the proteins ability to affect bacterial binding to epithelial cells. hmLF significantly inhibited pathogen adhesion and purified hmLF glycans significantly reduced Salmonella invasion of colonic epithelial cells to levels associated with non-invasive deletion mutants. This study indicates that hmLF glycosylation is tightly regulated by gene expression and that glyco-variation is involved in modulating pathogen association.

Extensive in vivo Human Milk Peptidomics Reveals Specific Proteolysis Yielding Protective Antimicrobial Peptides

Milk is traditionally considered an ideal source of the basic elemental nutrients required by infants. More detailed examination is revealing that milk represents a more functional ensemble of components with benefits to both infants and mothers. A comprehensive peptidomics method was developed and used to analyze human milk yielding an extensive array of protein products present in the fluid. Over 300 milk peptides were identified originating from major and many minor protein components of milk. As expected, the majority of peptides derived from β-casein, however no peptide fragments from the major milk proteins lactoferrin, α-lactalbumin, and secretory immunoglobulin A were identified. Proteolysis in the mammary gland is selective-released peptides were drawn only from specific proteins and typically from only select parts of the parent sequence. A large number of the peptides showed significant sequence overlap with peptides with known antimicrobial or immunomodulatory functions. Antibacterial assays showed the milk peptide mixtures inhibited the growth of Escherichia coli and Staphylococcus aureus . The predigestion of milk proteins and the consequent release of antibacterial peptides may provide a selective advantage through evolution by protecting both the mothers mammary gland and her nursing offspring from infection.

Reconstituted lipoprotein: a versatile class of biologically-inspired nanostructures.

One of biologys most pervasive nanostructures, the phospholipid membrane, represents an ideal scaffold for a host of nanotechnology applications. Whether engineering biomimetic technologies or designing therapies to interface with the cell, this adaptable membrane can provide the necessary molecular-level control of membrane-anchored proteins, glycopeptides, and glycolipids. If appropriately prepared, these components can replicate in vitro or influence in vivo essential living processes such as signal transduction, mass transport, and chemical or energy conversion. To satisfy these requirements, a lipid-based, synthetic nanoscale architecture with molecular-level tunability is needed. In this regard, discrete lipid particles, including reconstituted high density lipoprotein (HDL), have emerged as a versatile and elegant solution. Structurally diverse, native biological HDLs exist as discoidal lipid bilayers of 5-8 nm diameter and lipid monolayer-coated spheres 10-15 nm in diameter, all belted by a robust scaffolding protein. These supramolecular assemblies can be reconstituted using simple self-assembly methods to incorporate a broad range of amphipathic molecular constituents, natural or artificial, and provide a generic platform for stabilization and transport of amphipathic and hydrophobic elements capable of docking with targets at biological or inorganic surfaces. In conjunction with top-down or bottom-up engineering approaches, synthetic HDL can be designed, arrayed, and manipulated for a host of applications including biochemical analyses and fundamental studies of molecular structure. Also highly biocompatible, these assemblies are suitable for medical diagnostics and therapeutics. The collection of efforts reviewed here focuses on laboratory methods by which synthetic HDLs are produced, the advantages conferred by their nanoscopic dimension, and current and emerging applications.

Nutrigenomics and Personalized Diets: What Will They Mean for Food?

The modern food system feeds six billion people with remarkable diversity, safety, and nutrition. Yet, the current rise in diet-related diseases is compromising health and devaluing many aspects of modern agriculture. Steps to increase the nutritional quality of individual foods will assist in personalizing health and in guiding individuals to achieve superior health. Nutrigenomics is the scientific field of the genetic basis for varying susceptibilities to disease and the diverse responses to foods. Although some of these genetic determinants will be simple and amenable to personal genotyping as the means to predict health, in practice most will not. As a result, genotyping will not be the secret to personalizing diet and health. Human assessment technologies from imaging to proteomics and metabolomics are providing tools to both understand and accurately assess the nutritional phenotype of individuals. The business models are also emerging to bring these assessment capabilities to industrial practice, in which consumers will know more about their personal health and seek personal solutions.

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.

N-Linked Glycan Profiling of Mature Human Milk by High-Performance Microfluidic Chip Liquid Chromatography Time-of-Flight Tandem Mass Spectrometry

N-Linked glycans of skim human milk proteins were determined for three mothers. N-Linked glycans are linked to immune defense, cell growth, and cell-cell adhesion, but their functions in human milk are undetermined. Protein-bound N-linked glycans were released with peptidyl N-glycosidase F (PNGase F), enriched by graphitized carbon chromatography, and analyzed with Chip-TOF MS. To be defined as N-glycans, compounds were required, in all three procedural replicates, to match, within 6 ppm, against a theoretical human N-glycan library and be at least 2-fold higher in abundance in PNGase F-treated than in control samples. Fifty-two N-linked glycan compositions were identified, and 24 were confirmed via tandem mass spectra analysis. Twenty-seven compositions have been found previously in human milk, and 25 are novel compositions. By abundance, 84% of N-glycans were fucosylated and 47% were sialylated. The majority (70%) of total N-glycan abundance was composed of N-glycans found in all three milk samples.

Nutritional lipidomics: Molecular metabolism, analytics, and diagnostics

The field of lipidomics is providing nutritional science a more comprehensive view of lipid intermediates. Lipidomics research takes advantage of the increase in accuracy and sensitivity of mass detection of MS with new bioinformatics toolsets to characterize the structures and abundances of complex lipids. Yet, translating lipidomics to practice via nutritional interventions is still in its infancy. No single instrumentation platform is able to solve the varying analytical challenges of the different molecular lipid species. Biochemical pathways of lipid metabolism remain incomplete and the tools to map lipid compositional data to pathways are still being assembled. Biology itself is dauntingly complex and simply separating biological structures remains a key challenge to lipidomics. Nonetheless, the strategy of combining tandem analytical methods to perform the sensitive, high-throughput, quantitative, and comprehensive analysis of lipid metabolites of very large numbers of molecules is poised to drive the field forward rapidly. Among the next steps for nutrition to understand the changes in structures, compositions, and function of lipid biomolecules in response to diet is to describe their distribution within discrete functional compartments lipoproteins. Additionally, lipidomics must tackle the task of assigning the functions of lipids as signaling molecules, nutrient sensors, and intermediates of metabolic pathways.