A bacterial sulfoglycosidase highlights mucin O-glycan breakdown in the gut ecosystem.

Mucinolytic bacteria modulate host-microbiota symbiosis and dysbiosis through their ability to degrade mucin O-glycans. However, how and to what extent bacterial enzymes are involved in the breakdown process remains poorly understood. Here we focus on a glycoside hydrolase family 20 sulfoglycosidase (BbhII) from Bifidobacterium bifidum, which releases N-acetylglucosamine-6-sulfate from sulfated mucins. Glycomic analysis showed that, in addition to sulfatases, sulfoglycosidases are involved in mucin O-glycan breakdown in vivo and that the released N-acetylglucosamine-6-sulfate potentially affects gut microbial metabolism, both of which were also supported by a metagenomic data mining analysis. Enzymatic and structural analysis of BbhII reveals the architecture underlying its specificity and the presence of a GlcNAc-6S-specific carbohydrate-binding module (CBM) 32 with a distinct sugar recognition mode that B. bifidum takes advantage of to degrade mucin O-glycans. Comparative analysis of the genomes of prominent mucinolytic bacteria also highlights a CBM-dependent O-glycan breakdown strategy used by B. bifidum.

Comprehensive Analysis of Lipid Composition in Human Foremilk and Hindmilk.

Precision nutrition, also referred to as personalized nutrition, focuses on the individual to determine the individual's most effective eating plan to prevent or treat disease. A precision nutrition for infants requires the determination of the profile of human milk. We compared the lipid profiles of the foremilk (i.e., the initial milk of a breastfeed) and hindmilk (the last milk) of six Japanese subjects and evaluated whether a human milk lipid profile is useful for precision nutrition even though the fat concentration fluctuates during lactation. We detected and quantified 527 species with a lipidome analysis by liquid chromatography-tandem mass spectrometry. The fat concentration in hindmilk (120.6 ± 66.7 µmol/mL) was significantly higher than that in foremilk (68.6 ± 33.3 µmol/mL). While the total carbon number of fatty acids in triglyceride (TG) was highest in C52 for all subjects, the second or third number differed among the subjects. Both the distribution of total carbon number of fatty acids included in TG and the distribution of fatty acids in TG classified by the number of double bonds were almost the same in the foremilk and hindmilk in each subject. The lipids levels containing docosahexaenoic acid and arachidonic acid in total lipids of the foremilk and the hindmilk were almost the same in each subject. Among the sphingolipids and glycerophospholipids, the level of sphingomyelin was the highest in four subjects' milk, and phosphatidylcholine was the highest in the other two subjects' milk. The order of their major species was the same in each foremilk and hindmilk. A clustering heatmap revealed the differences between foremilk and hindmilk in the same subject were smaller than the differences among individuals. Our analyses indicate that a human-milk lipid profile reflects individual characteristics and is a worthwhile focus for precision nutrition.

Priority effects shape the structure of infant-type Bifidobacterium communities on human milk oligosaccharides.

Bifidobacteria are among the first colonizers of the infant gut, and human milk oligosaccharides (HMOs) in breastmilk are instrumental for the formation of a bifidobacteria-rich microbiota. However, little is known about the assembly of bifidobacterial communities. Here, by applying assembly theory to a community of four representative infant-gut associated Bifidobacterium species that employ varied strategies for HMO consumption, we show that arrival order and sugar consumption phenotypes significantly affected community formation. Bifidobacterium bifidum and Bifidobacterium longum subsp. infantis, two avid HMO consumers, dominate through inhibitory priority effects. On the other hand, Bifidobacterium breve, a species with limited HMO-utilization ability, can benefit from facilitative priority effects and dominates by utilizing fucose, an HMO degradant not utilized by the other bifidobacterial species. Analysis of publicly available breastfed infant faecal metagenome data showed that the observed trends for B. breve were consistent with our in vitro data, suggesting that priority effects may have contributed to its dominance. Our study highlights the importance and history dependency of initial community assembly and its implications for the maturation trajectory of the infant gut microbiota.

Changes in intracellular activation-related gene expression and induction of Akt contribute to acquired resistance toward nelarabine in CCRF-CEM cell line.

Drug resistance is a major problem in treatment with nelarabine, and its resolution requires elucidation of the underlying mechanisms. We established two nelarabine-resistant subclones of the human T-cell lymphoblastic leukemia cell line CCRF-CEM. The resistant subclones showed changes in the expression of several genes related to nelarabine intracellular activation and inhibition of apoptosis. Activation of the Akt protein upon nelarabine treatment was observed in both subclones. The combination treatment with nelarabine and PI3K/Akt inhibitors was shown to inhibit cell growth. Cross-resistance was observed with ara-C and not with vincristine, daunorubicin, or etoposide treatment. Thus, changes in the expression of cellular activation-related genes, inhibition of apoptosis, and induction of Akt may be involved in the development of nelarabine resistance in the CCRF-CEM cell model. The use of different classes of chemotherapeutic agents and combination therapy with PI3K/Akt pathway inhibitors may be used to overcome resistance to nelarabine.

Diversification of a Fucosyllactose Transporter within the Genus Bifidobacterium.

Human milk oligosaccharides (HMOs), which are natural bifidogenic prebiotics, were recently commercialized to fortify formula milk. However, HMO assimilation phenotypes of bifidobacteria vary by species and strain, which has not been fully linked to strain genotype. We have recently shown that specialized uptake systems, particularly for the internalization of major HMOs (fucosyllactose [FL]), are associated with the formation of a Bifidobacterium-rich gut microbial community. Phylogenetic analysis revealed that FL transporters have diversified into two clades harboring four clusters within the Bifidobacterium genus, but the underpinning functional diversity associated with this divergence remains underexplored. In this study, we examined the HMO consumption phenotypes of two bifidobacterial species, Bifidobacterium catenulatum subsp. kashiwanohense and Bifidobacterium pseudocatenulatum, both of which possess FL-binding proteins that belong to phylogenetic clusters with unknown specificities. Growth assays, heterologous gene expression experiments, and HMO consumption analyses showed that the FL transporter type from B. catenulatum subsp. kashiwanohense JCM 15439T conferred a novel HMO uptake pattern that includes complex fucosylated HMOs (lacto-N-fucopentaose II and lacto-N-difucohexaose I/II). Further genomic landscape analyses of FL transporter-positive bifidobacterial strains revealed that the H-antigen- or Lewis antigen-specific fucosidase gene(s) and FL transporter specificities were largely aligned. These results suggest that bifidobacteria have acquired FL transporters along with the corresponding gene sets necessary to utilize the imported HMOs. Our results provide insight into the species- and strain-dependent adaptation strategies of bifidobacteria in HMO-rich environments. IMPORTANCE The gut of breastfed infants is generally dominated by health-promoting bifidobacteria. Human milk oligosaccharides (HMOs) from breast milk selectively promote the growth of specific taxa such as bifidobacteria, thus forming an HMO-mediated host-microbe symbiosis. While the coevolution of humans and bifidobacteria has been proposed, the underpinning adaptive strategies employed by bifidobacteria require further research. Here, we analyzed the divergence of the critical fucosyllactose (FL) HMO transporter within Bifidobacterium. We have shown that the diversification of the solute-binding proteins of the FL transporter led to uptake specificities of fucosylated sugars ranging from simple trisaccharides to complex hexasaccharides. This transporter and the congruent acquisition of the necessary intracellular enzymes allow bifidobacteria to consume different types of HMOs in a predictable and strain-dependent manner. These findings explain the adaptation and proliferation of bifidobacteria in the competitive and HMO-rich infant gut environment and enable accurate specificity annotation of transporters from metagenomic data.

Evolutionary adaptation in fucosyllactose uptake systems supports bifidobacteria-infant symbiosis.

The human gut microbiota established during infancy has persistent effects on health. In vitro studies have suggested that human milk oligosaccharides (HMOs) in breast milk promote the formation of a bifidobacteria-rich microbiota in infant guts; however, the underlying molecular mechanism remains elusive. Here, we characterized two functionally distinct but overlapping fucosyllactose transporters (FL transporter-1 and -2) from Bifidobacterium longum subspecies infantis. Fecal DNA and HMO consumption analyses, combined with deposited metagenome data mining, revealed that FL transporter-2 is primarily associated with the bifidobacteria-rich microbiota formation in breast-fed infant guts. Structural analyses of the solute-binding protein (SBP) of FL transporter-2 complexed with 2'-fucosyllactose and 3-fucosyllactose, together with phylogenetic analysis of SBP homologs of both FL transporters, highlight a unique adaptation strategy of Bifidobacterium to HMOs, in which the gain-of-function mutations enable FL transporter-2 to efficiently capture major fucosylated HMOs. Our results provide a molecular insight into HMO-mediated symbiosis and coevolution between bifidobacteria and humans.

Sharing of human milk oligosaccharides degradants within bifidobacterial communities in faecal cultures supplemented with Bifidobacterium bifidum.

Gut microbiota of breast-fed infants are generally rich in bifidobacteria. Recent studies show that infant gut-associated bifidobacteria can assimilate human milk oligosaccharides (HMOs) specifically among the gut microbes. Nonetheless, little is known about how bifidobacterial-rich communities are shaped in the gut. Interestingly, HMOs assimilation ability is not related to the dominance of each species. Bifidobacterium longum susbp. longum and Bifidobacterium breve are commonly found as the dominant species in infant stools; however, they show limited HMOs assimilation ability in vitro. In contrast, avid in vitro HMOs consumers, Bifidobacterium bifidum and Bifidobacterium longum subsp. infantis, are less abundant in infant stools. In this study, we observed altruistic behaviour by B. bifidum when incubated in HMOs-containing faecal cultures. Four B. bifidum strains, all of which contained complete sets of HMO-degrading genes, commonly left HMOs degradants unconsumed during in vitro growth. These strains stimulated the growth of other Bifidobacterium species when added to faecal cultures supplemented with HMOs, thereby increasing the prevalence of bifidobacteria in faecal communities. Enhanced HMOs consumption by B. bifidum-supplemented cultures was also observed. We also determined the complete genome sequences of B. bifidum strains JCM7004 and TMC3115. Our results suggest B. bifidum-mediated cross-feeding of HMOs degradants within bifidobacterial communities.

Molecular Insight into Evolution of Symbiosis between Breast-Fed Infants and a Member of the Human Gut Microbiome Bifidobacterium longum.

Breast-fed infants generally have a bifidobacteria-rich microbiota with recent studies indicating that human milk oligosaccharides (HMOs) selectively promote bifidobacterial growth. Bifidobacterium bifidum possesses a glycoside hydrolase family 20 lacto-N-biosidase for liberating lacto-N-biose I from lacto-N-tetraose, an abundant HMO unique to human milk, while Bifidobacterium longum subsp. longum has a non-classified enzyme (LnbX). Here, we determined the crystal structure of the catalytic domain of LnbX and provide evidence for creation of a novel glycoside hydrolase family, GH136. The structure, in combination with inhibition and mutation studies, provides insight into the molecular mechanism and broader substrate specificity of this enzyme. Moreover, through genetic studies, we show that lnbX is indispensable for B. longum growth on lacto-N-tetraose and is a key genetic factor for persistence in the gut of breast-fed infants. Overall, this study reveals possible evolutionary routes for the emergence of symbiosis between humans and bifidobacterial species in the infant gut.

Qualitative and Quantitative Analyses of Glycogen in Human Milk.

Identification as well as a detailed analysis of glycogen in human milk has not been shown yet. The present study confirmed that glycogen is contained in human milk by qualitative and quantitative analyses. High-performance anion exchange chromatography (HPAEC) and high-performance size exclusion chromatography with a multiangle laser light scattering detector (HPSEC-MALLS) were used for qualitative analysis of glycogen in human milk. Quantitative analysis was carried out by using samples obtained from the individual milks. The result revealed that the concentration of human milk glycogen varied depending on the mother's condition-such as the period postpartum and inflammation. The amounts of glycogen in human milk collected at 0 and 1-2 months postpartum were higher than in milk collected at 3-14 months postpartum. In the milk from mothers with severe mastitis, the concentration of glycogen was about 40 times higher than that in normal milk.

Method for Evaluation of the Requirements of B-group Vitamins Using Tryptophan Metabolites in Human Urine.

Tryptophan metabolism is directly involved with B-group vitamins such as vitamin B2, niacin, and vitamin B6, and indirectly with vitamin B1 and pantothenic acid. We evaluated the validity of requirements of B-group vitamins set by the Dietary Reference Intakes for the Japanese (DRI-J). We investigated the fate of dietary tryptophan in 10 Japanese adult men who ate the same diet based on DRI-J during a 4-week study. Vitamin mixtures were administered based on the amounts in the basal diet during weeks 2, 3, and 4. Daily urine samples were collected eight times (days 1 and 5 in each week). Administration of vitamin mixtures had no effect on tryptophan metabolites such as anthranilic acid, kynurenic acid, xanthurenic acid, 3-hydroxyanthranilic acid, and quinolinic acid within individuals. Surplus administration of B-group vitamins against DRI-J requirements did not elicit beneficial effects on tryptophan metabolism. Our findings supported the requirements of B-group vitamins set by the DRI-J.

Relationship Between Urinary Concentrations of Nine Water-soluble Vitamins and their Vitamin Intakes in Japanese Adult Males.

Excess water-soluble vitamins are thought to be eliminated in the urine. We have reported a strong relationship between water-soluble vitamin intake and urinary excretion in females. The relationship, however, is not well understood in males. In the present experiment, 10 Japanese male subjects were given a standard Japanese diet for the first week. The subjects remained on the same diet, and a synthesized water-soluble vitamin mixture containing one time the Dietary Reference Intakes (DRIs) for Japanese was given for the second week, three times the DRIs for the third week, and six times the DRIs for the fourth week. Twenty-four-hour urine samples were collected each week. Urinary excretion levels for seven of the nine water-soluble vitamin levels, excluding vitamin B12 and folate, increased linearly and sharply in a dose-dependent manner. These results suggest that measuring urinary water-soluble vitamins can be good nutritional markers for assessing vitamin intakes in humans.

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

The breast-fed infant intestine is often colonized by particular bifidobacteria, and human milk oligosaccharides (HMOs) are considered to be bifidogenic. Recent studies showed that Bifidobacterium longum subsp. infantis can grow on HMOs as the sole carbon source. This ability has been ascribed to the presence of a gene cluster (HMO cluster-1) contained in its genome. However, the metabolism of HMOs by the organism remains unresolved because no enzymatic studies have been completed. In the present study, we characterized ß-galactosidases of this subspecies to understand how the organism degrades type-1 (Galß1-3GlcNAc) and type-2 (Galß1-4GlcNAc) isomers of HMOs. The results revealed that the locus tag Blon_2016 gene, which is distantly located from the HMO cluster-1, encodes a novel ß-galactosidase (Bga42A) with a significantly higher specificity for lacto-N-tetraose (LNT; Galß1-3GlcNAcß1-3Galß1-4Glc) than for lacto-N-biose I (Galß1-3GlcNAc), lactose (Lac) and type-2 HMOs. The proposed name of Bga42A is LNT ß-1,3-galactosidase. The Blon_2334 gene (Bga2A) located within the HMO cluster-1 encodes a ß-galactosidase specific for Lac and type-2 HMOs. Real-time quantitative reverse transcription-polymerase chain reaction analysis revealed the physiological significance of Bga42A and Bga2A in HMO metabolism. The organism therefore uses two different ß-galactosidases to selectively degrade type-1 and type-2 HMOs. Despite the quite rare occurrence in nature of ß-galactosidases acting on type-1 chains, the close homologs of Bga42A were present in the genomes of infant-gut associated bifidobacteria that are known to consume LNT. The predominance of type-1 chains in HMOs and the conservation of Bga42A homologs suggest the coevolution of these bifidobacteria with humans.

Physiology of consumption of human milk oligosaccharides by infant gut-associated bifidobacteria.

The bifidogenic effect of human milk oligosaccharides (HMOs) has long been known, yet the precise mechanism underlying it remains unresolved. Recent studies show that some species/subspecies of Bifidobacterium are equipped with genetic and enzymatic sets dedicated to the utilization of HMOs, and consequently they can grow on HMOs; however, the ability to metabolize HMOs has not been directly linked to the actual metabolic behavior of the bacteria. In this report, we clarify the fate of each HMO during cultivation of infant gut-associated bifidobacteria. Bifidobacterium bifidum JCM1254, Bifidobacterium longum subsp. infantis JCM1222, Bifidobacterium longum subsp. longum JCM1217, and Bifidobacterium breve JCM1192 were selected for this purpose and were grown on HMO media containing a main neutral oligosaccharide fraction. The mono- and oligosaccharides in the spent media were labeled with 2-anthranilic acid, and their concentrations were determined at various incubation times using normal phase high performance liquid chromatography. The results reflect the metabolic abilities of the respective bifidobacteria. B. bifidum used secretory glycosidases to degrade HMOs, whereas B. longum subsp. infantis assimilated all HMOs by incorporating them in their intact forms. B. longum subsp. longum and B. breve consumed lacto-N-tetraose only. Interestingly, B. bifidum left degraded HMO metabolites outside of the cell even when the cells initiate vegetative growth, which indicates that the different species/subspecies can share the produced sugars. The predominance of type 1 chains in HMOs and the preferential use of type 1 HMO by infant gut-associated bifidobacteria suggest the coevolution of the bacteria with humans.

Urinary excretion of vitamin B12 depends on urine volume in Japanese female university students and elderly.

Recent studies have shown that urinary excretion of water-soluble vitamins reflects their intake in humans. However, some have reported that physical characteristics and urine volume may affect the amount of vitamin compounds found in urine. We hypothesized that physical characteristics and urine volume could affect urinary excretion of B-group vitamins. Twenty-four-hour urine samples were collected from 186 free-living Japanese women aged 19 to 21 years and 104 free-living Japanese subjects aged 70 to 84 years. Correlations between urinary output of each B-group vitamin and body height, body weight, body mass index, body surface area, urine volume, and urinary creatinine were determined. Only urinary vitamin B(12) was strongly correlated to urine volume in young (r = 0.683, P < .001) and elderly (r = 0.523, P < .001) subjects. To confirm this finding, 20 Japanese adults were orally administered 1.5 mg of cyanocobalamin (500-fold higher daily intake); and correlations between urinary vitamin B(12) and urine volume were determined. The load of cyanocobalamin increased vitamin B(12) content in the urine by only 1.3-fold. Urinary vitamin B(12) was strongly correlated with urine volume on the day before taking, the day of taking, and the day after taking cyanocobalamin (r = 0.745, P < .001; r = 0.897, P < .0001; and r = 0.855, P < .0001, respectively). We conclude that urinary excretion of vitamin B(12) is dependent upon urine volume, but not on intake of vitamin B(12). Physical characteristics and urine volume are less important for B-group vitamins except for vitamin B(12) as biomarker.

Molybdenum and chromium concentrations in breast milk from Japanese women.

Molybdenum (Mo) and chromium (Cr) in 79 Japanese breast milk samples were measured by inductively coupled plasma-mass spectrometry. For Mo, 51 samples (64.6%) showed less than 5 ng/ml and only 12 samples (15.2%) showed more than 10 ng/ml. The range and median were <0.1 to 25.91 and 3.18 ng/ml respectively. For Cr, 38 samples (48.1%) showed less than 1 ng/ml, 20 samples (25.3%) showed 1 to 2 ng/ml, and only six samples (7.6%) showed more than 5 ng/ml. The range and median were <0.1 to 18.67 and 1.00 ng/ml respectively.

Ovalbumin-related gene Y protein bears carbohydrate chains of the ovomucoid type.

We have recently established the monoclonal antibodies (mAbs) specific to the major food allergen, ovomucoid, as mAb 7D, recognizing the carbohydrate moiety of ovomucoid, and mAb 6H, the peptide moiety (Biosci. Biothechnol. Biochem., 68, 2490-2497, (2004)). Using these mAbs, we found commercially available ovalbumin preparations contaminated with a considerable amount of ovomucoid together with other glycoproteins. To examine the contaminants, egg white was subjected to cation-exchange chromatography. An unidentified protein was found in egg white that reacted with mAb 7D but not with mAb 6H, having a molecular size of about 52 kDa and a blocked N-terminus. Two internal amino acid sequences of the fragments obtained after a lysyl endopeptidase and a hydroxylamine treatment revealed the protein to be ovalbumin Y (ovalbumin-related gene Y protein). We conclude that ovalbumin Y is a unique chimeric glycoprotein having an amino acid sequence similar to that of ovalbumin, but having a carbohydrate moiety similar to that of ovomucoid.

Oligoclonal Enzyme-linked Immunosorbent Assay Capable of Determining the Major Food Allergen, Ovomucoid, Irrespective of the Degree of Heat Denaturation.

We have recently established an enzyme-linked immunosorbent assay (ELISA) for total ovomucoid determination, irrespective of the degree of its heat denaturation, by using a monoclonal antibody (mAb) 7D specific to the carbohydrate moiety of ovomucoid (Biosci Biothechnol Biochem, 68, 2490-2497, 2004). Two novel methods have been developed to improve the ELISA. First, its sensitivity was enhanced 100 times by using an oligoclonal cocktail of mAb 7D and two other mAbs with different epitopes as a second antibody. Second, it was shown that usage of denaturing reagents such as SDS and beta-mercaptoethanol for extraction was acceptable for ELISA within a range of stability of a first antibody on a solid phase. Properties of the oligoclonal sandwich ELISA system thus constructed were discussed in connection with allergen labeling.

Recognition of native and/or thermally induced denatured forms of the major food allergen, ovomucoid, by human IgE and mouse monoclonal IgG antibodies.

Human sera obtained from children with egg allergy reacted well with both native and heated ovomucoid (OM). Ovalbumin is present in egg white in a 5 times greater quantity than OM; however, it easily aggregates and becomes difficult to extract by heating. For accurate food allergen labeling of processed food, therefore, OM should be evaluated with the determination of egg white protein in consideration of heat denaturation. Three kinds of monoclonal antibodies and sandwich ELISA tests were established which are able to recognize the native and/or heat-denatured forms of OM. The usefulness of these characteristic mAbs and ELISA tests are discussed in relation to allergen labeling, monitoring food processing, and movement or change of dietary protein in vivo.

Cloning and characterization of a novel mammalian zinc transporter, zinc transporter 5, abundantly expressed in pancreatic beta cells.

Intracellular homeostasis for zinc is achieved through the coordinate regulation of specific transporters engaged in zinc influx, efflux, and intracellular compartmentalization. We have identified a novel mammalian zinc transporter, zinc transporter 5 (ZnT-5), by virtue of its similarity to ZRC1, a zinc transporter of Saccharomyces cerevisiae, a member of the cation diffusion facilitator family. Human ZnT-5 (hZnT-5) cDNA encodes a 765-amino acid protein with 15 predicted membrane-spanning domains. hZnT-5 was ubiquitously expressed in all tested human tissues and abundantly expressed in the pancreas. In the human pancreas, hZnT-5 was expressed abundantly in insulin-containing beta cells that contain zinc at the highest level in the body. The hZnT-5 immunoreactivity was found to be associated with secretory granules by electron microscopy. The hZnT-5-derived zinc transport activity was detected using the Golgi-enriched vesicles prepared from hZnT-5-induced HeLa/hZnT-5 cells in which exogenous hZnT-5 expression is inducible by the Tet-on gene regulation system. This activity was dependent on time, temperature, and concentration and was saturable. Moreover, zinc at a high concentration (10 mm) inhibited the growth of yeast expressing hZnT-5. These results suggest that ZnT-5 plays an important role for transporting zinc into secretory granules in pancreatic beta cells.