Bifidobacterium longum subsp. infantis uses two different β-galactosidases for selectively degrading type-1 and type-2 human milk oligosaccharides

ABSTRACTHuman 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 asBifidobacterium longumsubsp.infantisandBifidobacterium bifidumcan utilize human milk oligosaccharides (HMO)in vitroas the sole carbon source, while the relatively fewB. longumsubsp.longumandBifidobacterium breveisolates tested appear less adapted to these substrates. Considering the high frequency at whichB. breveis isolated from breast-fed infant feces, we postulated that someB. brevestrains can more vigorously consume HMO and thus are enriched in the breast-fed infant gastrointestinal tract. To examine this, a number ofB. breveisolates 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. AllB. brevestrains 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 allB. brevestrains 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 inB. breveis 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.

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Bifidobacterium bifidum Lacto-N-Biosidase, a Critical Enzyme for the Degradation of Human Milk Oligosaccharides with a Type 1 Structure

Applied and Environmental Microbiology ◽

10.1128/aem.00149-08 ◽

2008 ◽

Vol 74 (13) ◽

pp. 3996-4004 ◽

Cited By ~ 147

Author(s):

Jun Wada ◽

Takuro Ando ◽

Masashi Kiyohara ◽

Hisashi Ashida ◽

Motomitsu Kitaoka ◽

...

Keyword(s):

Human Milk ◽

Enteric Bacteria ◽

Bifidobacterium Bifidum ◽

Glycoside Hydrolase Family ◽

Carbohydrate Binding ◽

Amino Acid Residues ◽

Human Milk Oligosaccharides ◽

Milk Oligosaccharides ◽

Hydrolase Family

ABSTRACT Breast-fed infants often have intestinal microbiota dominated by bifidobacteria in contrast to formula-fed infants. We found that several bifidobacterial strains produce a lacto-N-biosidase that liberates lacto-N-biose I (Galβ1,3GlcNAc; type 1 chain) from lacto-N-tetraose (Galβ1,3GlcNAcβ1,3Galβ1,4Glc), which is a major component of human milk oligosaccharides, and subsequently isolated the gene from Bifidobacterium bifidum JCM1254. The gene, designated lnbB, was predicted to encode a protein of 1,112 amino acid residues containing a signal peptide and a membrane anchor at the N and C termini, respectively, and to possess the domain of glycoside hydrolase family 20, carbohydrate binding module 32, and bacterial immunoglobulin-like domain 2, in that order, from the N terminus. The recombinant enzyme showed substrate preference for the unmodified β-linked lacto-N-biose I structure. Lacto-N-biosidase activity was found in several bifidobacterial strains, but not in the other enteric bacteria, such as clostridia, bacteroides, and lactobacilli, under the tested conditions. These results, together with our recent finding of a novel metabolic pathway specific for lacto-N-biose I in bifidobacterial cells, suggest that some of the bifidobacterial strains are highly adapted for utilizing human milk oligosaccharides with a type 1 chain.

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Assessment of the synbiotic properites of human milk oligosaccharides and Bifidobacterium longum subsp. infantis in vitro and in humanised mice

Beneficial Microbes ◽

10.3920/bm2016.0138 ◽

2017 ◽

Vol 8 (2) ◽

pp. 281-289 ◽

Cited By ~ 4

Author(s):

S. Musilova ◽

N. Modrackova ◽

P. Hermanova ◽

T. Hudcovic ◽

R. Svejstil ◽

...

Keyword(s):

Caesarean Section ◽

Human Milk ◽

Pathogenic Bacteria ◽

Bifidobacterium Longum ◽

Mode Of Delivery ◽

Human Milk Oligosaccharides ◽

Milk Oligosaccharides ◽

High Concentrations

The mode of delivery plays a crucial role in infant gastrointestinal tract colonisation, which in the case of caesarean section is characterised by the presence of clostridia and low bifidobacterial counts. Gut colonisation can be modified by probiotics, prebiotics or synbiotics. Human milk oligosaccharides (HMOs) are infant prebiotics that show a bifidogenic effect. Moreover, genome sequencing of Bifidobacterium longum subsp. infantis within the infant microbiome revealed adaptations for milk utilisation. This study aimed to evaluate the synbiotic effect of B. longum subsp. infantis, HMOs and human milk (HM) both in vitro and in vivo (in a humanised mouse model) in the presence of faecal microbiota from infants born by caesarean section. The combination of B. longum and HMOs or HM reduced the clostridia and G-bacteria counts both in vitro and in vivo. The bifidobacterial population in vitro significantly increased and produce high concentrations of acetate and lactate. In vitro competition assays confirmed that the tested bifidobacterial strain is a potential probiotic for infants and, together with HMOs or HM, acts as a synbiotic. It is also able to inhibit potentially pathogenic bacteria. The synbiotic effects identified in vitro were not observed in vivo. However, there was a significant reduction in clostridia counts in both experimental animal groups (HMOs + B. longum and HM + B. longum), and a specific immune response via increased interleukin (IL)-10 and IL-6 production. Animal models do not perfectly mimic human conditions; however, they are essential for testing the safety of functional foods.

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Oligosaccharides Released from Milk Glycoproteins Are Selective Growth Substrates for Infant-Associated Bifidobacteria

Applied and Environmental Microbiology ◽

10.1128/aem.00547-16 ◽

2016 ◽

Vol 82 (12) ◽

pp. 3622-3630 ◽

Cited By ~ 67

Author(s):

Sercan Karav ◽

Annabelle Le Parc ◽

Juliana Maria Leite Nobrega de Moura Bell ◽

Steven A. Frese ◽

Nina Kirmiz ◽

...

Keyword(s):

Mass Spectrometry ◽

Human Milk ◽

Bifidobacterium Longum ◽

Bovine Milk ◽

Selective Growth ◽

Whey Protein Concentrate ◽

Human Milk Oligosaccharides ◽

Milk Oligosaccharides ◽

Content Type ◽

Growth Substrates

ABSTRACTMilk, in addition to nourishing the neonate, provides a range of complex glycans whose construction ensures a specific enrichment of key members of the gut microbiota in the nursing infant, a consortium known as the milk-oriented microbiome. Milk glycoproteins are thought to function similarly, as specific growth substrates for bifidobacteria common to the breast-fed infant gut. Recently, a cell wall-associated endo-β-N-acetylglucosaminidase (EndoBI-1) found in various infant-borne bifidobacteria was shown to remove a range of intactN-linked glycans. We hypothesized that these released oligosaccharide structures can serve as a sole source for the selective growth of bifidobacteria. We demonstrated that EndoBI-1 releasedN-glycans from concentrated bovine colostrum at the pilot scale. EndoBI-1-releasedN-glycans supported the rapid growth ofBifidobacterium longumsubsp.infantis(B. infantis), a species that grows well on human milk oligosaccharides, but did not support growth ofBifidobacterium animalissubsp.lactis(B. lactis), a species which does not. Conversely,B. infantisATCC 15697 did not grow on the deglycosylated milk protein fraction, clearly demonstrating that the glycan portion of milk glycoproteins provided the key substrate for growth. Mass spectrometry-based profiling revealed thatB. infantisconsumed 73% of neutral and 92% of sialylatedN-glycans, whileB. lactisdegraded only 11% of neutral and virtually no (<1%) sialylatedN-glycans. These results provide mechanistic support thatN-linked glycoproteins from milk serve as selective substrates for the enrichment of infant-associated bifidobacteria capable of carrying out the initial deglycosylation. Moreover, releasedN-glycans were better growth substrates than the intact milk glycoproteins, suggesting that EndoBI-1 cleavage is a key initial step in consumption of glycoproteins. Finally, the variety ofN-glycans released from bovine milk glycoproteins suggests that they may serve as novel prebiotic substrates with selective properties similar to those of human milk oligosaccharides.IMPORTANCEIt has been previously shown that glycoproteins serve as growth substrates for bifidobacteria. However, which part of a glycoprotein (glycans or polypeptides) is responsible for this function was not known. In this study, we used a novel enzyme to cleave conjugatedN-glycans from milk glycoproteins and tested their consumption by various bifidobacteria. The results showed that the glycans selectively stimulated the growth ofB. infantis, which is a key infant gut microbe. The selectivity of consumption of individualN-glycans was determined using advanced mass spectrometry (nano-liquid chromatography chip–quadrupole time of flight mass spectrometry [nano-LC-Chip-Q-TOF MS]) to reveal thatB. infantiscan consume the range of glycan structures released from whey protein concentrate.

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Bifidobacterium bifidum Lacto-N-Biosidase, a Critical Enzyme for the Degradation of Human Milk Oligosaccharides with a Type 1 Structure

Applied and Environmental Microbiology ◽

10.1128/aem.01847-09 ◽

2009 ◽

Vol 75 (19) ◽

pp. 6414-6414

Author(s):

Jun Wada ◽

Takuro Ando ◽

Masashi Kiyohara ◽

Hisashi Ashida ◽

Motomitsu Kitaoka ◽

...

Keyword(s):

Human Milk ◽

Bifidobacterium Bifidum ◽

Human Milk Oligosaccharides ◽

Milk Oligosaccharides

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A novel class of human milk oligosaccharides based on 6-galactosyllactose and containing N-acetylglucosamine branches extended by oligogalactoses

10.1101/2021.05.27.445961 ◽

2021 ◽

Author(s):

Franz-Georg Hanisch ◽

Clemens Kunz

Keyword(s):

Human Milk ◽

Infant Nutrition ◽

Maldi Mass Spectrometry ◽

The Novel ◽

Human Milk Oligosaccharides ◽

Milk Oligosaccharides ◽

New Class ◽

Terminal Galactose ◽

Branched Chains

Human milk oligosaccharides (HMOs) have attracted much attention in recent years not only as a prebiotic factor, but in particular as an essential component in infant nutrition related to their impact in innate immunity. The backbone structures of complex HMOs generally contain single or repetitive lacto-N-biose (type 1) or lactosamine (type 2) units in either linear or branched chains extending from a lactose core. While all known branched structures originate from 3,6-substitution of the lactosyl core galactose, we here describe a new class of HMOs that tentatively branch at terminal galactose of 6-galactosyllactose. Another novel feature of this class of HMOs was found in linear oligo-galactosyl chains linked to one of the N-acetylglucosamine (GlcNAc) branches. The novel structures exhibit general formulas with hexose vs. hexosamine contents of 5/2 to 8/2 and can be designated as high-galactose (HG)-HMOs. In addition, up to three fucosyl residues are linked to the octa- to dodecasaccharides, which were detected in two human milk samples from Lewis blood group defined donors. Structural analyses of methylated glycans and their alditols comprised MALDI mass spectrometry, ESI-(CID)MS and linkage analyses by GC-MS of the derived partially methylated alditol acetates. Enzymatic degradation by application of β1-3,4-specific galactosidase supported the presence of terminal galactose linked [beta]1-6 to one of the two GlcNAc branches.

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Reconstruction of the Bifidobacterial Pan-Secretome Reveals the Network of Extracellular Interactions between Bifidobacteria and the Infant Gut

Applied and Environmental Microbiology ◽

10.1128/aem.00796-18 ◽

2018 ◽

Vol 84 (16) ◽

Cited By ~ 5

Author(s):

Gabriele Andrea Lugli ◽

Walter Mancino ◽

Christian Milani ◽

Sabrina Duranti ◽

Francesca Turroni ◽

...

Keyword(s):

Human Milk ◽

Protein Translocation ◽

Bifidobacterium Longum ◽

Extracellular Proteins ◽

Glycoside Hydrolases ◽

Secreted Proteins ◽

Human Milk Oligosaccharides ◽

Secretion Systems ◽

Milk Oligosaccharides ◽

Content Type

ABSTRACT The repertoire of secreted proteins decoded by a microorganism represents proteins released from or associated with the cell surface. In gut commensals, such as bifidobacteria, these proteins are perceived to be functionally relevant, as they regulate the interaction with the gut environment. In the current study, we screened the predicted proteome of over 300 bifidobacterial strains among the currently recognized bifidobacterial species to generate a comprehensive database encompassing bifidobacterial extracellular proteins. A glycobiome analysis of this predicted bifidobacterial secretome revealed that a correlation exists between particular bifidobacterial species and their capability to hydrolyze human milk oligosaccharides (HMOs) and intestinal glycoconjugates, such as mucin. Furthermore, an exploration of metatranscriptomic data sets of the infant gut microbiota allowed the evaluation of the expression of bifidobacterial genes encoding extracellular proteins, represented by ABC transporter substrate-binding proteins and glycoside hydrolases enzymes involved in the degradation of human milk oligosaccharides and mucin. Overall, this study provides insights into how bifidobacteria interact with their natural yet highly complex environment, the infant gut.IMPORTANCE The ecological success of bifidobacteria relies on the activity of extracellular proteins that are involved in the metabolism of nutrients and the interaction with the environment. To date, information on secreted proteins encoded by bifidobacteria is incomplete and just related to few species. In this study, we reconstructed the bifidobacterial pan-secretome, revealing extracellular proteins that modulate the interaction of bifidobacteria with their natural environment. Furthermore, a survey of the secretion systems between bifidobacterial genomes allowed the identification of a conserved Sec-dependent secretion machinery in all the analyzed genomes and the Tat protein translocation system in the chromosomes of 23 strains belonging to Bifidobacterium longum subsp. longum and Bifidobacterium aesculapii.

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Bifidobacterium longum subsp. infantis ATCC 15697 α-Fucosidases Are Active on Fucosylated Human Milk Oligosaccharides

Applied and Environmental Microbiology ◽

10.1128/aem.06762-11 ◽

2011 ◽

Vol 78 (3) ◽

pp. 795-803 ◽

Cited By ~ 119

Author(s):

David A. Sela ◽

Daniel Garrido ◽

Larry Lerno ◽

Shuai Wu ◽

Kemin Tan ◽

...

Keyword(s):

Human Milk ◽

Turnover Rate ◽

Bifidobacterium Longum ◽

Small Mass ◽

Human Milk Oligosaccharides ◽

High Turnover ◽

Milk Oligosaccharides ◽

Content Type ◽

Biochemical Kinetics ◽

Time Dependent Effect

ABSTRACTBifidobacterium longumsubsp.infantisATCC 15697 utilizes several small-mass neutral human milk oligosaccharides (HMOs), several of which are fucosylated. Whereas previous studies focused on endpoint consumption, a temporal glycan consumption profile revealed a time-dependent effect. Specifically, among preferred HMOs, tetraose was favored early in fermentation, with other oligosaccharides consumed slightly later. In order to utilize fucosylated oligosaccharides, ATCC 15697 possesses several fucosidases, implicating GH29 and GH95 α-l-fucosidases in a gene cluster dedicated to HMO metabolism. Evaluation of the biochemical kinetics demonstrated that ATCC 15697 expresses three fucosidases with a high turnover rate. Moreover, several ATCC 15697 fucosidases are active on the linkages inherent to the HMO molecule. Finally, the HMO cluster GH29 α-l-fucosidase possesses a crystal structure that is similar to previously characterized fucosidases.

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