Utilization of Major Fucosylated and Sialylated Human Milk Oligosaccharides by Isolated Human Gut Microbes
TL;DR: Specific Bifidobacteria and Bacteroides differentially digest specific individual HMOS, with the major fucosylated milk oligosaccharides most strongly stimulating key species of mutualist symbionts.
Abstract: Human milk oligosaccharides (HMOS) are not digested in the proximal intestine. In distal intestine, HMOS collectively modify the microbiota, but the response of individual bacteria to individual components of the HMOS is not well defined. Here, each of 25 major isolates of the human intestinal microbiota was fed individual major fucosylated and sialylated HMOS in anaerobic culture. This allowed for an assessment of the influence of specific HMOS on the growth and metabolic products of individual microbiota bacteria. Most Bifidobacteria spp. and Bacteroides spp. grew, induced α-L-fucosidase activity, and produced abundant lactate or short-chain fatty acids (SCFAs) when fed 2'-fucosyllactose (2'-FL), 3-FL, and lactodifucotetraose (LDFT). Lactobacillus delbrueckii ATCC7830, Enterococcus faecalis ATCC19433, and Streptococcus thermophilus ATCC19258 exhibited slight growth, pH reduction, and lactate production when supplemented with 2'-FL or 3-FL, but not LDFT. Supplementation with 3'-sialyllactose (3'-SL) and 6'-SL promoted moderate growth of Bifidobacterium longum JCM7007, 7009, 7010, 7011, 1272, 11347, ATCC15708, Bacteroides vulgatus ATCC8482, and B. thetaiotaomicron ATCC29148; accordingly, these bacteria exhibited greater neuraminidase activity and produced copious lactate, SCFA, or both. Lactobacillus delbrueckii ATCC7830 also consumed 6'-SL. In contrast, Clostridium spp., L. rhamnosus ATCC53103, E. faecalis ATCC29200, Staphylococcus spp., Enterobacter spp., and Escherichia coli K12 did not consume milk oligosaccharides nor produce appreciable acidic fermentation products. Specific Bifidobacteria and Bacteroides differentially digest specific individual HMOS, with the major fucosylated milk oligosaccharides most strongly stimulating key species of mutualist symbionts. This suggests strategies for treating dysbiosis of the microbiota and associated inflammatory disorders.
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TL;DR: Novel hypotheses for how nutrient selection, immune activation and other mechanisms control the biogeography of bacteria in the gut are considered, and the relevance of this spatial heterogeneity to health and disease is discussed.
Abstract: Animals assemble and maintain a diverse but host-specific gut microbial community. In addition to characteristic microbial compositions along the longitudinal axis of the intestines, discrete bacterial communities form in microhabitats, such as the gut lumen, colonic mucus layers and colonic crypts. In this Review, we examine how the spatial distribution of symbiotic bacteria among physical niches in the gut affects the development and maintenance of a resilient microbial ecosystem. We consider novel hypotheses for how nutrient selection, immune activation and other mechanisms control the biogeography of bacteria in the gut, and we discuss the relevance of this spatial heterogeneity to health and disease.
1,637 citations
TL;DR: Mechanistic insight is provided into how milk glycans enrich specific beneficial bacterial populations in infants and clues for enhancing enrichment of bifidobacterial populations in at risk populations - such as premature infants are revealed.
Abstract: Individuals 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. On 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. Infants 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.
297 citations
Cites background from "Utilization of Major Fucosylated an..."
...Two-tailed, unpaired t-test, with an α of 0.05, was used to compare glycan expression in milk between secretor and non-secretor mothers, as well as comparing milk and fecal HMOs received by infants with low amounts of bifidobacteria versus those with high amounts of bifidobacteria, as defined in the Bifidobacterium-specific qPCR section below....
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...In other bifidobacteria such as Bifidobacterium breve, GH29 fucosidases enable the consumption of 2′-fucosylated HMOs [36]....
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...The strongest corollaries of input milk matched with a High-BifqPCR stool were high absolute amounts of non-fucosylated neutral HMOs, high absolute amounts of α(1-2)-fucosylated HMOs, and low relative amounts of α(1-3/4)-fucosylated HMOs, perhaps reflecting competitiveness with 2′-fucosylation....
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...Noting the previous differences in fecal glycoprofiles, we hypothesized that secretor mothers would enrich their infants in bifidobacteria that are able to consume 2′-fucosylated HMOs....
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...Glycoprofiling by nano-LC chip TOF (time of flight) mass spectrometry Both milk and fecal HMOs were analyzed using an Agilent nano-LC Chip time-of-flight mass spectrometer (Agilent, Santa Clara, CA, USA), as described previously [32,90]....
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TL;DR: Bifidobacterium longum subspecies infantis (B. infantis) is unique among gut bacteria in its prodigious capacity to digest and consume any human milk oligosaccharide structure as discussed by the authors.
Abstract: Oligosaccharides are abundant in human milk. Production of these highly diverse structures requires significant energy expenditure by the mother and yet these human milk oligosaccharides offer no direct nutritive value to her infant. A primary function of human milk oligosaccharides is to shape the infant’s intestinal microbiota with life-long consequences. Bifidobacterium longum subspecies infantis (B. infantis) is unique among gut bacteria in its prodigious capacity to digest and consume any human milk oligosaccharide structure, the result of a large repertoire of bacterial genes encoding an array of glycosidases and oligosaccharide transporters not found in other bacterial species. In vitro, B. infantis grows better than other bacterial strains in the presence of human milk oligosaccharides, displays anti-inflammatory activity in premature intestinal cells, and decreases intestinal permeability. In premature infants, B. infantis given in combination with human milk increases B. infantis and decreases Enterobacteriaceae in the feces. Probiotics containing B. infantis decrease the risk of necrotizing enterocolitis in premature infants. Colonization with B. infantis is also associated with increased vaccine responses. Probiotic organisms have historically been selected based on ease of production and stability. The advantages of B. infantis, selected through coevolution with human milk glycans, present an opportunity for focused manipulation of the infant intestinal microbiota.
289 citations
Cites background from "Utilization of Major Fucosylated an..."
...Among multiple microbial species studied, only two genera, Bifidobacterium and Bacteroides, are able to comprehensively utilize HMOs as a primary food source (Table 1) (8,9)....
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TL;DR: These studies indicate that milk oligosaccharides support normal microbial communities and behavioral responses during stressor exposure, potentially through effects on the gut microbiota-brain axis.
Abstract: There are extensive bidirectional interactions between the gut microbiota and the central nervous system (CNS), and studies demonstrate that stressor exposure significantly alters gut microbiota community structure. We tested whether oligosaccharides naturally found in high levels in human milk, which have been reported to impact brain development and enhance the growth of beneficial commensal microbes, would prevent stressor-induced alterations in gut microbial community composition and attenuate stressor-induced anxiety-like behavior. Mice were fed standard laboratory diet, or laboratory diet containing the human milk oligosaccharides 3′Sialyllactose (3′SL) or 6′Sialyllactose (6′SL) for 2 weeks prior to being exposed to either a social disruption stressor or a non-stressed control condition. Stressor exposure significantly changed the structure of the colonic mucosa-associated microbiota in control mice, as indicated by changes in beta diversity. The stressor resulted in anxiety-like behavior in both the light/dark preference and open field tests in control mice. This effect was associated with a reduction in immature neurons in the dentate gyrus as indicated by doublecortin (DCX) immunostaining. These effects were not evident in mice fed milk oligosaccharides; stressor exposure did not significantly change microbial community structure in mice fed 3′SL or 6′SL. In addition, 3′SL and 6′SL helped maintain normal behavior on tests of anxiety-like behavior and normal numbers of DCX+ immature neurons. These studies indicate that milk oligosaccharides support normal microbial communities and behavioral responses during stressor exposure, potentially through effects on the gut microbiota–brain axis.
219 citations
Cites background from "Utilization of Major Fucosylated an..."
...For example, 3′SL and 6′SL promoted growth of Bifidobacterium longum in vitro (Yu et al., 2013)....
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TL;DR: It is proposed that these species possess machinery devoted to selectively pack acidic proteins into the OMV, which could help in securing nutrients for the benefit of the whole bacterial community present in the microbiota, uncovering a novel function for bacterial OMV.
Abstract: Outer membrane vesicles (OMV) are spherical membranous structures released from the outer membrane (OM) of Gram-negative bacteria. OMV have been proposed to play several different roles during both pathogenesis and symbiosis. Despite the fact that OMV were described several decades ago, their biogenesis is a poorly characterized process. Whether OMV are produced by an active mechanism or by passive disintegration of the OM is a still matter of controversy. Bacteroides fragilis and Bacteroides thetaiotaomicron are important members of the human microbiota. In this work, we determined and compared the protein compositions of OM and OMV from B. fragilis and B. thetaiotaomicron. SDS-PAGE analysis of both fractions revealed dramatically different protein profiles. Proteomic analysis of OM and OMV in B. fragilis identified more than 40 proteins found exclusively in OMV and more than 30 proteins detectable only in the OM. The OMV-specific proteome showed a high prevalence of glycosidases and proteases, some of which were shown to be active in vitro . Similar results were obtained for B. thetaiotaomicron. Most of the OMV-exclusive proteins were acidic. Based on these results, we propose that these species possess machinery devoted to selectively pack acidic proteins into the OMV. These OMV equipped with hydrolytic enzymes could help in securing nutrients for the benefit of the whole bacterial community present in the microbiota, uncovering a novel function for bacterial OMV. IMPORTANCE The members of genus Bacteroides are key players in the symbiosis between the human host and the gut microbiota. It is known for its ability to degrade a wide variety of glycans that are not substrates for human glycosidases. The cleaved glycans can be utilized by Bacteroides and other microbiota members, resulting in the production of short-chain fatty acids that are beneficial for the host. Although members of the genus Bacteroides are known to secrete different hydrolases, their secretion pathways remain uncharacterized. In this article, we show that B. fragilis and B. thetaiotaomicron preferentially pack a large number of hydrolases in outer membrane vesicles (OMV). Most of these hydrolases are acidic and were detected exclusively in OMV. This suggests the presence of a molecular mechanism in Bacteroides responsible for the selection of OMV proteins based on their charge. We propose that OMV contribute to the establishment and balance of the gut microbiota.
209 citations
Cites background from "Utilization of Major Fucosylated an..."
...It has been previously established that -L-fucosidase activity in Bacteroides can be induced in the presence of fucosylated human milk oligosaccharides (44)....
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References
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TL;DR: The future use of prebiotics may allow species-level changes in the microbiota, an extrapolation into genera other than the bifidobacteria and lactobacilli, and allow preferential use in disease-prone areas of the body.
Abstract: Prebiotics are non-digestible (by the host) food ingredients that have a beneficial effect through their selective metabolism in the intestinal tract. Key to this is the specificity of microbial changes. The present paper reviews the concept in terms of three criteria: (a) resistance to gastric acidity, hydrolysis by mammalian enzymes and gastrointestinal absorption; (b) fermentation by intestinal microflora; (c) selective stimulation of the growth and/or activity of intestinal bacteria associated with health and wellbeing. The conclusion is that prebiotics that currently fulfil these three criteria are fructo-oligosaccharides, galacto-oligosaccharides and lactulose, although promise does exist with several other dietary carbohydrates. Given the range of food vehicles that may be fortified by prebiotics, their ability to confer positive microflora changes and the health aspects that may accrue, it is important that robust technologies to assay functionality are used. This would include a molecular-based approach to determine flora changes. The future use of prebiotics may allow species-level changes in the microbiota, an extrapolation into genera other than the bifidobacteria and lactobacilli, and allow preferential use in disease-prone areas of the body.
2,312 citations
"Utilization of Major Fucosylated an..." refers background in this paper
...The defining features of prebiotics include their carbohydrate nature, oral ingestion, resistance to digestion by mucosal enzymes, minimal absorption in the proximal gastrointestinal tract, and selective fermentation by beneficial bacteria of the distal gut (Gibson et al. 2004)....
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...The defining features of prebiotics include their carbohydrate nature, oral ingestion, resistance to digestion by mucosal enzymes, minimal absorption in the proximal gastrointestinal tract, and selective fermentation by beneficial bacteria of the distal gut (Gibson et al. 2004)....
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TL;DR: Research on HMOs is certainly needed to increase the knowledge of infant nutrition as it is affected by complex oligosaccharides, but more is needed to know about the metabolism of oligosACcharides in the gastrointestinal tract.
Abstract: Research on human milk oligosaccharides (HMOs) has received much attention in recent years. However, it started about a century ago with the observation that oligosaccharides might be growth factors for a so-called bifidus flora in breast-fed infants and extends to the recent finding of cell adhesion molecules in human milk. The latter are involved in inflammatory events recognizing carbohydrate sequences that also can be found in human milk. The similarities between epithelial cell surface carbohydrates and oligosaccharides in human milk strengthen the idea that specific interactions of those oligosaccharides with pathogenic microorganisms do occur preventing the attachment of microbes to epithelial cells. HMOs may act as soluble receptors for different pathogens, thus increasing the resistance of breast-fed infants. However, we need to know more about the metabolism of oligosaccharides in the gastrointestinal tract. How far are oligosaccharides degraded by intestinal enzymes and does oligosaccharide processing (e.g. degradation, synthesis, and elongation of core structures) occur in intestinal epithelial cells? Further research on HMOs is certainly needed to increase our knowledge of infant nutrition as it is affected by complex oligosaccharides.
979 citations
"Utilization of Major Fucosylated an..." refers background in this paper
...HMOS is the third most abundant fraction of human milk (Newburg et al. 1986; Kunz et al. 2000), accounting for 10% of maternal energy input into milk....
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Book•
01 Jan 1995
TL;DR: The structure of milk, implications for sampling and storage determinants of milk volume and composition carbohydrates in milks, analysis, quantities and significance nitrogenous components of milk milk lipids minerals, ions, and trace elements in milk defence agents in milk comparative analysis of non-human milks contaminants in milk.
Abstract: The structure of milk - implications for sampling and storage determinants of milk volume and composition carbohydrates in milks - analysis, quantities and significance nitrogenous components of milk milk lipids minerals, ions, and trace elements in milk defence agents in milk comparative analysis of non-human milks contaminants in milk.
921 citations
TL;DR: The argument that specific glycans, especially the oligosaccharides, are the major constituent of an innate immune system of human milk whereby the mother protects her infant from enteric and other pathogens through breastfeeding is presented.
Abstract: Breastfed infants have lower morbidity and mortality due to diarrhea than those fed artificially. This had been attributed primarily to the secretory antibodies and prebiotic factors in human milk. Oligosaccharides are the third largest component of human milk. They were initially considered to be functionless by-products of glycoprotein and glycolipid synthesis during milk production. However, in the past few decades it has become apparent that the human milk oligosaccharides are composed of thousands of components, at least some of which protect against pathogens. Oligosaccharide protection against infectious agents may result in part from their prebiotic characteristics, but is thought to be primarily due to their inhibition of pathogen binding to host cell ligands. Most human milk oligosaccharides are fucosylated, and their production depends on enzymes encoded by the genes associated with expression of the Lewis blood group system. The expression of specific fucosylated oligosaccharides in milk thus varies in relation to maternal Lewis blood group type, and is significantly associated with the risk of infectious disease in breastfed infants. Specific fucosylated moieties of oligosaccharides and related glycoconjugates (glycans) are able to inhibit binding and disease by specific pathogens. This review presents the argument that specific glycans, especially the oligosaccharides, are the major constituent of an innate immune system of human milk whereby the mother protects her infant from enteric and other pathogens through breastfeeding. The large input of energy expended by the mother in the synthesis of milk oligosaccharides is consistent with the human reproductive strategy of large parental input into rearing relatively few offspring through a prolonged period of maturation. These protective glycans may prove useful as a basis for the development of novel prophylactic and therapeutic agents that inhibit diseases caused by mucosal pathogens.
634 citations
"Utilization of Major Fucosylated an..." refers background in this paper
...Different patterns of expression, and individual HMOS structures, are associated with diverse functions (Newburg et al. 2005b)....
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...These activities can now be considered in the context of the ability of HMOS to inhibit binding of enteropathogens to their receptors in the intestinal mucosa, with both fucosylated and sialylated HMOS playing an important role in protection from pathogens (Newburg et al. 2005a)....
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TL;DR: It is concluded that campylobacter binding to intestinal H-2 antigen is essential for infection and milk fucosyloligosaccharides and specific fucOSyl α1,2-linked molecules inhibit this binding and may represent a novel class of antimicrobial agents.
569 citations
"Utilization of Major Fucosylated an..." refers background in this paper
...Several enteric pathogens such as stable toxin of Escherichia coli, Campylobacter, and noroviruses, are inhibited by fucosylated glycans (Ruiz-Palacios et al. 2003; Newburg, Ruiz-Palacios, Altaye, Chaturvedi, Guerrero, et al. 2004; Newburg, Ruiz-Palacios, Altaye, Chaturvedi, Meinzen-Derr, et al.…...
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...Several enteric pathogens such as stable toxin of Escherichia coli, Campylobacter, and noroviruses, are inhibited by fucosylated glycans (Ruiz-Palacios et al. 2003; Newburg, Ruiz-Palacios, Altaye, Chaturvedi, Guerrero, et al. 2004; Newburg, Ruiz-Palacios, Altaye, Chaturvedi, Meinzen-Derr, et al. 2004)....
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