Gut-associated Bifidobacteria Research Articles

Highly conserved bifidobacteria in the human gut: Bifidobacterium longum subsp. longum as a potential modulator of elderly innate immunity.

Aging is a physiological and immunological process involving the deterioration of human health, characterised by the progressive alteration of organs and their functions. The speed and extent of such decline are dependent on lifestyle, environment, and genetic factors. Moreover, with advancing age, humans become progressively more fragile and prone to acute and chronic diseases. Although the intestinal microbiota is predisposed to perturbations that accompany aging and frailty, it is generally accepted that the gut microbiota engages in multiple interactions that affect host health throughout the host life span. In the current study, an exhaustive in silico investigation of gut-associated bifidobacteria in healthy individuals from birth to old age revealed that Bifidobacterium longum subsp. longum is the most prevalent member, especially during infancy and in centenarians. Moreover, B. longum subsp. longum genome reconstruction and strain tracing among human gut microbiomes allowed the identification of prototypes of this taxon in the human gut microbiota of healthy elderly individuals. Such analyses guided culturomics attempts to isolate B. longum subsp. longum strains that matched the genomic content of B. longum subsp. longum prototypes from healthy elderly individuals. The molecular effects of selected B. longum subsp. longum strains on the human host were further investigated using in vitro microbe-host interactions, revealing differences in the host immune system transcriptome, with a reduction in gene expression of inflammation-related cytokines. These intriguing findings support the potential anti-aging effects of elderly associated prototypes of B. longum subsp. longum.

Transmission and Persistence of Infant Gut-Associated Bifidobacteria

Bifidobacterium infantis are the primary colonizers of the infant gut, yet scientific research addressing the transmission of the genus Bifidobacterium to infants remains incomplete. This review examines microbial reservoirs of infant-type Bifidobacterium that potentially contribute to infant gut colonization. Accordingly, strain inheritance from mother to infant via the fecal-oral route is likely contingent on the bifidobacterial strain and phenotype, whereas transmission via the vaginal microbiota may be restricted to Bifidobacterium breve. Additional reservoirs include breastmilk, horizontal transfer from the environment, and potentially in utero transfer. Given that diet is a strong predictor of Bifidobacterium colonization in early life and the absence of Bifidobacterium is observed regardless of breastfeeding, it is likely that additional factors are responsible for bifidobacterial colonization early in life.

Efficient Biosynthesis of Lacto-N-Biose I, a Building Block of Type I Human Milk Oligosaccharides, by a Metabolically Engineered Escherichia coli.

Lacto-N-biose I (LNB), termed a Type 1 disaccharide, is an important building block of human milk oligosaccharides. It shows promising prebiotic activity by stimulating the proliferation of many gut-associated bifidobacteria and thus displays good potential in infant foods or supplements. Enzymatic and microbial approaches to LNB synthesis have been studied, almost all of which involve glycosylation of LNB phosphorylase as the final step. Herein, we report a new and easier microbial LNB synthesis strategy through the route "lactose → lacto-N-triose II (LNTri II) → lacto-N-tetraose (LNT) → LNB". A previously constructed LNT-producing Escherichia coli BL21(DE3) strain was engineered for LNB biosynthesis by introducing Bifidobacterium bifidum LnbB. LNB was efficiently produced, accompanied by lactose regeneration. Genomic integration of key pathway genes related to LNTri II and LNT synthesis was performed to enhance LNB titers. The final engineered strain produced 3.54 and 26.88 g/L LNB by shake-flask and fed-batch cultivation, respectively.

Enzymatic Adaptation of Bifidobacterium bifidum to Host Glycans, Viewed from Glycoside Hydrolyases and Carbohydrate-Binding Modules.

Certain species of the genus Bifidobacterium represent human symbionts. Many studies have shown that the establishment of symbiosis with such bifidobacterial species confers various beneficial effects on human health. Among the more than ten (sub)species of human gut-associated Bifidobacterium that have significantly varied genetic characteristics at the species level, Bifidobacterium bifidum is unique in that it is found in the intestines of a wide age group, ranging from infants to adults. This species is likely to have adapted to efficiently degrade host-derived carbohydrate chains, such as human milk oligosaccharides (HMOs) and mucin O-glycans, which enabled the longitudinal colonization of intestines. The ability of this species to assimilate various host glycans can be attributed to the possession of an adequate set of extracellular glycoside hydrolases (GHs). Importantly, the polypeptides of those glycosidases frequently contain carbohydrate-binding modules (CBMs) with deduced affinities to the target glycans, which is also a distinct characteristic of this species among members of human gut-associated bifidobacteria. This review firstly describes the prevalence and distribution of B. bifidum in the human gut and then explains the enzymatic machinery that B. bifidum has developed for host glycan degradation by referring to the functions of GHs and CBMs. Finally, we show the data of co-culture experiments using host-derived glycans as carbon sources, which underpin the interesting altruistic behavior of this species as a cross-feeder.

Varied Pathways of Infant Gut-Associated Bifidobacterium to Assimilate Human Milk Oligosaccharides: Prevalence of the Gene Set and Its Correlation with Bifidobacteria-Rich Microbiota Formation.

The infant’s gut microbiome is generally rich in the Bifidobacterium genus. The mother’s milk contains natural prebiotics, called human milk oligosaccharides (HMOs), as the third most abundant solid component after lactose and lipids, and of the different gut microbes, infant gut-associated bifidobacteria are the most efficient in assimilating HMOs. Indeed, the fecal concentration of HMOs was found to be negatively correlated with the fecal abundance of Bifidobacterium in infants. Given these results, two HMO molecules, 2′-fucosyllactose and lacto-N-neotetraose, have recently been industrialized to fortify formula milk. As of now, however, our knowledge about the HMO consumption pathways in infant gut-associated bifidobacteria is still incomplete. The recent studies indicate that HMO assimilation abilities significantly vary among different Bifidobacterium species and strains. Therefore, to truly maximize the effects of prebiotic and probiotic supplementation in commercialized formula, we need to understand HMO consumption behaviors of bifidobacteria in more detail. In this review, we summarized how different Bifidobacterium species/strains are equipped with varied gene sets required for HMO assimilation. We then examined the correlation between the abundance of the HMO-related genes and bifidobacteria-rich microbiota formation in the infant gut through data mining analysis of a deposited fecal microbiome shotgun sequencing dataset. Finally, we shortly described future perspectives on HMO-related studies.

Structural basis for broad substrate specificity of UDP-glucose 4-epimerase in the human milk oligosaccharide catabolic pathway of Bifidobacterium longum

Infant gut-associated bifidobacteria has a metabolic pathway that specifically utilizes lacto-N-biose I (Gal-β1,3-GlcNAc) and galacto-N-biose (Gal-β1,3-GalNAc) from human milk and mucin glycans. UDP-glucose 4-epimerase (GalE) from Bifidobacterium longum (bGalE) catalyzes epimerization reactions of UDP-Gal into UDP-Glc and UDP-GalNAc into UDP-GlcNAc with the same level of activity that is required to send galacto-hexoses into glycolysis. Here, we determined the crystal structures of bGalE in three ternary complex forms: NAD+/UDP, NAD+/UDP-GlcNAc, and NAD+/UDP-Glc. The broad specificity of bGalE was explained by structural features of the binding pocket for the N-acetyl or C2 hydroxy group of the substrate. Asn200 is located in a pocket of the C2 group, and its side chain adopts different conformations in the complex structures with UDP-Glc and UDP-GlcNAc. On the other side, Cys299 forms a large pocket for the C5 sugar ring atom. The flexible C2 pocket and the large C5 pocket of bGalE are suitable for accommodating both the hydroxy and N-acetyl groups of the substrate during sugar ring rotation in the catalytic cycle. The substrate specificity and active site structure of bGalE were distinct from those of Esherichia coli GalE but similar to those of human GalE.

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.

Impact of gut-associated bifidobacteria and their phages on health: two sides of the same coin?

Bifidobacteria are among the first microbial colonisers of the human infant gut post-partum. Their early appearance and dominance in the human infant gut and the reported health-promoting or probiotic status of several bifidobacterial strains has culminated in intensive research efforts that focus on their activities as part of the gut microbiota and the concomitant implications for human health. In this mini-review, we evaluate current knowledge on the genomics of this diverse bacterial genus, and on the genetic and functional adaptations that have underpinned the success of bifidobacteria in colonising the infant gut. The growing interest in functional genomics of bifidobacteria has also created interest in the interactions of bifidobacteria and their (bacterio)phages. While virulent phages of bifidobacteria have yet to be isolated, the incidence of integrated (pro)phages in bifidobacterial genomes are widely reported and this mini-review considers the role of these so-called bifidoprophages in modulating bifidobacterial populations in the human gastrointestinal tract and the implications for existing and future development of probiotic therapies.

Carbohydrate-binding module 74 is a novel starch-binding domain associated with large and multidomain α-amylase enzymes.

Microbacterium aurum B8.A is a bacterium that originates from a potato starch-processing plant and employs a GH13 α-amylase (MaAmyA) enzyme that forms pores in potato starch granules. MaAmyA is a large and multi-modular protein that contains a novel domain at its C terminus (Domain 2). Deletion of Domain 2 from MaAmyA did not affect its ability to degrade starch granules but resulted in a strong reduction in granular pore size. Here, we separately expressed and purified this Domain 2 in Escherichia coli and determined its likely function in starch pore formation. Domain 2 independently binds amylose, amylopectin, and granular starch but does not have any detectable catalytic (hydrolytic or oxidizing) activity on α-glucan substrates. Therefore, we propose that this novel starch-binding domain is a new carbohydrate-binding module (CBM), the first representative of family CBM74 that assists MaAmyA in efficient pore formation in starch granules. Protein sequence-based BLAST searches revealed that CBM74 occurs widespread, but in bacteria only, and is often associated with large and multi-domain α-amylases containing family CBM25 or CBM26 domains. CBM74 may specifically function in binding to granular starches to enhance the capability of α-amylase enzymes to degrade resistant starches (RSs). Interestingly, the majority of family CBM74 representatives are found in α-amylases originating from human gut-associated Bifidobacteria, where they may assist in resistant starch degradation. The CBM74 domain thus may have a strong impact on the efficiency of RS digestion in the mammalian gastrointestinal tract.

Comparative transcriptomics reveals key differences in the response to milk oligosaccharides of infant gut-associated bifidobacteria.

Breast milk enhances the predominance of Bifidobacterium species in the infant gut, probably due to its large concentration of human milk oligosaccharides (HMO). Here we screened infant-gut isolates of Bifidobacterium longum subsp. infantis and Bifidobacterium bifidum using individual HMO, and compared the global transcriptomes of representative isolates on major HMO by RNA-seq. While B. infantis displayed homogeneous HMO-utilization patterns, B. bifidum were more diverse and some strains did not use fucosyllactose (FL) or sialyllactose (SL). Transcriptomes of B. bifidum SC555 and B. infantis ATCC 15697 showed that utilization of pooled HMO is similar to neutral HMO, while transcriptomes for growth on FL were more similar to lactose than HMO in B. bifidum. Genes linked to HMO-utilization were upregulated by neutral HMO and SL, but not by FL in both species. In contrast, FL induced the expression of alternative gene clusters in B. infantis. Results also suggest that B. bifidum SC555 does not utilize fucose or sialic acid from HMO. Surprisingly, expression of orthologous genes differed between both bifidobacteria even when grown on identical substrates. This study highlights two major strategies found in Bifidobacterium species to process HMO, and presents detailed information on the close relationship between HMO and infant-gut bifidobacteria.

Open–close structural change upon ligand binding and two magnesium ions required for the catalysis of N-acetylhexosamine 1-kinase

Infant gut-associated bifidobacteria possess a metabolic pathway to utilize lacto-N-biose (Gal-β1,3-GlcNAc) and galacto-N-biose (Gal-β1,3-GalNAc) from human milk and glycoconjugates specifically. In this pathway, N-acetylhexosamine 1-kinase (NahK) catalyzes the phosphorylation of GlcNAc or GalNAc at the anomeric C1 position with ATP. Crystal structures of NahK have only been determined in the closed state. In this study, we determined open state structures of NahK in three different forms (apo, ADP complex, and ATP complex). A comparison of the open and closed state structures revealed an induced fit structural change defined by two rigid domains. ATP binds to the small N-terminal domain, and binding of the N-acetylhexosamine substrate to the large C-terminal domain induces a closing conformational change with a rotation angle of 16°. In the nucleotide binding site, two magnesium ions bridging the α-γ and β-γ phosphates were identified. A mutational analysis indicated that a residue coordinating both of the two magnesium ions (Asp228) is essential for catalysis. The involvement of two magnesium ions in the catalytic machinery is structurally similar to the catalytic structures of protein kinases and aminoglycoside phosphotransferases, but distinct from the structures of other anomeric kinases or sugar 6-kinases. These findings help to elucidate the possible evolutionary adaptation of substrate specificities and induced fit mechanism.

Hydrolysis of milk gangliosides by infant-gut associated bifidobacteria determined by microfluidic chips and high-resolution mass spectrometry.

Gangliosides are receiving considerable attention because they participate in diverse biological processes. Milk gangliosides appear to block pathogen adhesion and modify the intestinal ecology of newborns. However, the interaction of milk gangliosides with gut bifidobacteria has been little investigated. The digestion products of a mixture of gangliosides isolated from milk following incubation with six strains of bifidobacteria were studied using nanoHPLC Chip Q-TOF MS. To understand ganglioside catabolism in vitro, the two major milk gangliosides--GM3 and GD3--remaining in the media after incubation with bifidobacteria were quantified. Individual gangliosides were identified through postprocessing precursor ion scans, and quantitated with the "find by molecular feature" algorithm of MassHunter Qualitative Analysis software. Bifidobacterium infantis and B. bifidum substantially degraded the GM3 and GD3, whereas B. longum subsp. longum and B. animalis subsp. lactis only showed moderate degradation. MALDI FTICR MS analysis enabled a deeper investigation of the degradation and identified ganglioside degradation specifically at the outer portions of the glycan molecules. These results indicate that certain infant gut-associated bifidobacteria have the ability to degrade milk gangliosides releasing sialic acid, and that these glycolipids could play a prebiotic role in the infant gut.

Lacto-N-biosidase Encoded by a Novel Gene of Bifidobacterium longum Subspecies longum Shows Unique Substrate Specificity and Requires a Designated Chaperone for Its Active Expression

Infant gut-associated bifidobacteria possess species-specific enzymatic sets to assimilate human milk oligosaccharides, and lacto-N-biosidase (LNBase) is a key enzyme that degrades lacto-N-tetraose (Galβ1-3GlcNAcβ1-3Galβ1-4Glc), the main component of human milk oligosaccharides, to lacto-N-biose I (Galβ1-3GlcNAc) and lactose. We have previously identified LNBase activity in Bifidobacterium bifidum and some strains of Bifidobacterium longum subsp. longum (B. longum). Subsequently, we isolated a glycoside hydrolase family 20 (GH20) LNBase from B. bifidum; however, the genome of the LNBase(+) strain of B. longum contains no GH20 LNBase homolog. Here, we reveal that locus tags BLLJ_1505 and BLLJ_1506 constitute LNBase from B. longum JCM1217. The gene products, designated LnbX and LnbY, respectively, showed no sequence similarity to previously characterized proteins. The purified enzyme, which consisted of LnbX only, hydrolyzed via a retaining mechanism the GlcNAcβ1-3Gal linkage in lacto-N-tetraose, lacto-N-fucopentaose I (Fucα1-2Galβ1-3GlcNAcβ1-3Galβ1-4Glc), and sialyllacto-N-tetraose a (Neu5Acα2-3Galβ1-3GlcNAcβ1-3Galβ1-4Gal); the latter two are not hydrolyzed by GH20 LNBase. Among the chromogenic substrates examined, the enzyme acted on p-nitrophenyl (pNP)-β-lacto-N-bioside I (Galβ1-3GlcNAcβ-pNP) and GalNAcβ1-3GlcNAcβ-pNP. GalNAcβ1-3GlcNAcβ linkage has been found in O-mannosyl glycans of α-dystroglycan. Therefore, the enzyme may serve as a new tool for examining glycan structures. In vitro refolding experiments revealed that LnbY and metal ions (Ca(2+) and Mg(2+)) are required for proper folding of LnbX. The LnbX and LnbY homologs have been found only in B. bifidum, B. longum, and a few gut microbes, suggesting that the proteins have evolved in specialized niches.

Crystal Structures of a Glycoside Hydrolase Family 20 Lacto-N-biosidase from Bifidobacterium bifidum

Human milk oligosaccharides contain a large variety of oligosaccharides, of which lacto-N-biose I (Gal-β1,3-GlcNAc; LNB) predominates as a major core structure. A unique metabolic pathway specific for LNB has recently been identified in the human commensal bifidobacteria. Several strains of infant gut-associated bifidobacteria possess lacto-N-biosidase, a membrane-anchored extracellular enzyme, that liberates LNB from the nonreducing end of human milk oligosaccharides and plays a key role in the metabolic pathway of these compounds. Lacto-N-biosidase belongs to the glycoside hydrolase family 20, and its reaction proceeds via a substrate-assisted catalytic mechanism. Several crystal structures of GH20 β-N-acetylhexosaminidases, which release monosaccharide GlcNAc from its substrate, have been determined, but to date, a structure of lacto-N-biosidase is unknown. Here, we have determined the first three-dimensional structures of lacto-N-biosidase from Bifidobacterium bifidum JCM1254 in complex with LNB and LNB-thiazoline (Gal-β1,3-GlcNAc-thiazoline) at 1.8-Å resolution. Lacto-N-biosidase consists of three domains, and the C-terminal domain has a unique β-trefoil-like fold. Compared with other β-N-acetylhexosaminidases, lacto-N-biosidase has a wide substrate-binding pocket with a -2 subsite specific for β-1,3-linked Gal, and the residues responsible for Gal recognition were identified. The bound ligands are recognized by extensive hydrogen bonds at all of their hydroxyls consistent with the enzyme's strict substrate specificity for the LNB moiety. The GlcNAc sugar ring of LNB is in a distorted conformation near (4)E, whereas that of LNB-thiazoline is in a (4)C1 conformation. A possible conformational pathway for the lacto-N-biosidase reaction is discussed.

Endo-β-N-acetylglucosaminidases from Infant Gut-associated Bifidobacteria Release Complex N-glycans from Human Milk Glycoproteins

Breastfeeding is one of the main factors guiding the composition of the infant gut microbiota in the first months of life. This process is shaped in part by the high amounts of human milk oligosaccharides that serve as a carbon source for saccharolytic bacteria such as Bifidobacterium species. Infant-borne bifidobacteria have developed various molecular strategies for utilizing these oligosaccharides as a carbon source. We hypothesized that these species also interact with N-glycans found in host glycoproteins that are structurally similar to free oligosaccharides in human milk. Endo-β-N-acetylglucosaminidases were identified in certain isolates of Bifidobacterium longum subsp. longum, B. longum subsp. infantis, and Bifidobacterium breve, and their presence correlated with the ability of these strains to deglycosylate glycoproteins. An endoglycosidase from B. infantis ATCC 15697, EndoBI-1, was active toward all major types of N-linked glycans found in glycosylated proteins. Its activity was not affected by core fucosylation or extensive fucosylation, antenna number, or sialylation, releasing several N-glycans from human lactoferrin and immunoglobulins A and G. Extensive N-deglycosylation of whole breast milk was also observed after coincubation with this enzyme. Mutation of the active site of EndoBI-1 did not abolish binding to N-glycosylated proteins, and this mutant specifically recognized Man(3)GlcNAc(2)(α1-6Fuc), the core structure of human N-glycans. EndoBI-1 is constitutively expressed in B. infantis, and incubation of the bacterium with human or bovine lactoferrin led to the induction of genes associated to import and consumption of human milk oligosaccharides, suggesting linked regulatory mechanisms among these glycans. This work reveals an unprecedented interaction of bifidobacteria with host N-glycans and describes a novel endoglycosidase with broad specificity on diverse N-glycan types, potentially a useful tool for glycoproteomics studies.

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.