Carlito B. Lebrilla

The genome sequence of Bifidobacterium longum subsp. infantis reveals adaptations for milk utilization within the infant microbiome

Following birth, the breast-fed infant gastrointestinal tract is rapidly colonized by a microbial consortium often dominated by bifidobacteria. Accordingly, the complete genome sequence of Bifidobacterium longum subsp. infantis ATCC15697 reflects a competitive nutrient-utilization strategy targeting milk-borne molecules which lack a nutritive value to the neonate. Several chromosomal loci reflect potential adaptation to the infant host including a 43 kbp cluster encoding catabolic genes, extracellular solute binding proteins and permeases predicted to be active on milk oligosaccharides. An examination of in vivo metabolism has detected the hallmarks of milk oligosaccharide utilization via the central fermentative pathway using metabolomic and proteomic approaches. Finally, conservation of gene clusters in multiple isolates corroborates the genomic mechanism underlying milk utilization for this infant-associated phylotype.

Human milk glycobiome and its impact on the infant gastrointestinal microbiota

Human milk contains an unexpected abundance and diversity of complex oligosaccharides apparently indigestible by the developing infant and instead targeted to its cognate gastrointestinal microbiota. Recent advances in mass spectrometry-based tools have provided a view of the oligosaccharide structures produced in milk across stages of lactation and among human mothers. One postulated function for these oligosaccharides is to enrich a specific “healthy” microbiota containing bifidobacteria, a genus commonly observed in the feces of breast-fed infants. Isolated culture studies indeed show selective growth of infant-borne bifidobacteria on milk oligosaccharides or core components therein. Parallel glycoprofiling documented that numerous Bifidobacterium longum subsp. infantis strains preferentially consume small mass oligosaccharides that are abundant early in the lactation cycle. Genome sequencing of numerous B. longum subsp. infantis strains shows a bias toward genes required to use mammalian-derived carbohydrates by comparison with adult-borne bifidobacteria. This intriguing strategy of mammalian lactation to selectively nourish genetically compatible bacteria in infants with a complex array of free oligosaccharides serves as a model of how to influence the human supraorganismal system, which includes the gastrointestinal microbiota.

Consumption of Human Milk Oligosaccharides by Gut-related Microbes

Human milk contains large amounts of complex oligosaccharides that putatively modulate the intestinal microbiota of breast-fed infants by acting as decoy binding sites for pathogens and as prebiotics for enrichment of beneficial bacteria. Several bifidobacterial species have been shown to grow well on human milk oligosaccharides. However, few data exist on other bacterial species. This work examined 16 bacterial strains belonging to 10 different genera for growth on human milk oligosaccharides. For this propose, a chemically defined medium, ZMB1, was used, which allows vigorous growth of a number of gut-related microorganisms in a fashion similar to complex media. Interestingly, Bifidobacterium longum subsp. infantis, Bacteroides fragilis , and Bacteroides vulgatus strains were able to metabolize milk oligosaccharides with high efficiency, whereas Enterococcus , Streptococcus , Veillonella , Eubacterium , Clostridium , and Escherichia coli strains grew less well or not at all. Mass spectrometry-based glycoprofiling of the oligosaccharide consumption behavior revealed a specific preference for fucosylated oligosaccharides by Bi. longum subsp. infantis and Ba. vulgatus. This work expands the current knowledge of human milk oligosaccharide consumption by gut microbes, revealing bacteroides as avid consumers of this substrate. These results provide insight on how human milk oligosaccharides shape the infant intestinal microbiota.

In Vitro Fermentation of Breast Milk Oligosaccharides by Bifidobacterium infantis and Lactobacillus gasseri

ABSTRACT It has been proposed that human milk oligosaccharides (HMO) function as a prebiotic for bifidobacteria, yet this activity has not been adequately investigated. In this study, Bifidobacterium infantis was shown to ferment purified HMO as a sole carbon source, while another gut commensal, Lactobacillus gasseri, did not ferment HMO. Our results support the hypothesis that HMO selectively amplify bacterial populations in the infant intestine.

Glycomics and disease markers

Glycomics is the comprehensive study of all glycans expressed in biological systems. The biosynthesis of glycan relies on a number of highly competitive processes involving glycosyl transferases. Glycosylation is therefore highly sensitive to the biochemical environment and has been implicated in many diseases including cancer. Recently, interest in profiling the glycome has increased owing to the potential of glycans for disease markers. In this regard, mass spectrometry is emerging as a powerful technique for profiling the glycome. Global glycan profiling of human serum based on mass spectrometry has already led to several potentially promising markers for several types of cancer and diseases.

Bacteroides in the Infant Gut Consume Milk Oligosaccharides via Mucus-Utilization Pathways

Newborns are colonized with an intestinal microbiota shortly after birth, but the factors governing the retention and abundance of specific microbial lineages are unknown. Nursing infants consume human milk oligosaccharides (HMOs) that pass undigested to the distal gut, where they may be digested by microbes. We determined that the prominent neonate gut residents, Bacteroides thetaiotaomicron and Bacteroides fragilis, induce the same genes during HMO consumption that are used to harvest host mucus glycans, which are structurally similar to HMOs. Lacto-N-neotetraose, a specific HMO component, selects for HMO-adapted species such as Bifidobacterium infantis, which cannot use mucus, and provides a selective advantage to B. infantis in vivo when biassociated with B. thetaiotaomicron in the gnotobiotic mouse gut. This indicates that the complex oligosaccharide mixture within HMOs attracts both mutualistic mucus-adapted species and HMO-adapted bifidobacteria to the infant intestine that likely facilitate both milk and future solid food digestion.

A Serum Glycomics Approach to Breast Cancer Biomarkers

Because the glycosylation of proteins is known to change in tumor cells during the development of breast cancer, a glycomics approach is used here to find relevant biomarkers of breast cancer. These glycosylation changes are known to correlate with increasing tumor burden and poor prognosis. Current antibody-based immunochemical tests for cancer biomarkers of ovarian (CA125), breast (CA27.29 or CA15-3), pancreatic, gastric, colonic, and carcinoma (CA19-9) target highly glycosylated mucin proteins. However, these tests lack the specificity and sensitivity for use in early detection. This glycomics approach to find glycan biomarkers of breast cancer involves chemically cleaving oligosaccharides (glycans) from glycosylated proteins that are shed or secreted by breast cancer tumor cell lines. The resulting free glycan species are analyzed by MALDI-FT-ICR MS. Further structural analysis of the glycans can be performed in FTMS through the use of tandem mass spectrometry with infrared multiphoton dissociation. Glycan profiles were generated for each cell line and compared. These methods were then used to analyze sera obtained from a mouse model of breast cancer and a small number of serum samples obtained from human patients diagnosed with breast cancer or patients with no known history of breast cancer. In addition to the glycosylation changes detected in mice as mouse mammary tumors developed, glycosylation profiles were found to be sufficiently different to distinguish patients with cancer from those without. Although the small number of patient samples analyzed so far is inadequate to make any legitimate claims at this time, these promising but very preliminary results suggest that glycan profiles may contain distinct glycan biomarkers that may correspond to glycan “signatures of cancer.”

Development of an Annotated Library of Neutral Human Milk Oligosaccharides

Human milk oligosaccharides (HMOs) perform a number of functions including serving as prebiotics to stimulate the growth of beneficial intestinal bacteria, as receptor analogues to inhibit binding of pathogens, and as substances that promote postnatal brain development. There is further evidence that HMOs participate in modulating the human immune system. Because the absorption, catabolism, and biological function of oligosaccharides (OS) have strong correlations with their structures, structure elucidation is key to advancing this research. Oligosaccharides are produced by competing enzymes that provide the large structural diversity and heterogeneity that characterizes this class of compounds. Unlike the proteome, there is no template for oligosaccharides, making it difficult to rapidly identify oligosaccharide structures. In this research, annotation of the neutral free oligosaccharides in milk is performed to develop a database for the rapid identification of oligosaccharide structures. Our strategy incorporates high performance nanoflow liquid chromatography and mass spectrometry for characterizing HMO structures. HPLC-Chip/TOF MS provides a sensitive and quantitative method for sample profiling. The reproducible retention time and accurate mass can be used to rapidly identify the OS structures in HMO samples. A library with 45 neutral OS structures has been constructed. The structures include information regarding the epitopes such as Lewis type, as well as information regarding the secretor status.

Ion-molecule reactions as probes of gas-phase structures of peptides and proteins

A review with over 100 references describes the recent applications of ion-molecule reactions to the study of gas-phase protonated peptides and proteins. The topic is focused specifically on the proton transfer and hydrogen-deuterium exchange reactions of amino acids, peptides, and proteins. A brief background is given of the various methods used for assigning proton affinities and gas-phase basicities. The methods used for measuring the kinetics of deuterium incorporation of charged ion in the presence of a background pressure of deuterating reagents are also described. Ion-molecule reactions are used to determine, among other things, the gas-phase basicities and proton affinities of amino acids, peptides, and proteins, the sites of protonation, intra- and intermolecular interactions, and conformational differences and changes in gas-phase ionic species. Singly charged and multiply charged ions are both covered.

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.