SusE facilitates starch uptake independent of starch binding in B. thetaiotaomicron

The Bacteroides thetaiotaomicron starch utilization system (Sus) is a model system for nutrient acquisition by gut Bacteroidetes, a dominant phylum of gut bacteria. The Sus includes SusCDEFG, which assemble on the cell surface to capture, degrade and import starch. While SusD is an essential starch‐binding protein, the precise role(s) of the partially homologous starch‐binding proteins SusE and SusF has remained elusive. We previously reported that a non‐binding version of SusD (SusD*) supports growth on starch when other members of the multi‐protein complex are present. Here we demonstrate that SusE supports SusD* growth on maltooligosaccharides, and determine the domains of SusE essential for this function. Furthermore, we demonstrate that SusE does not need to bind starch to support growth in the presence of SusD*, suggesting that the assembly of SusCDE is most important for maltooligosaccharide uptake in this context. However, starch binding by proteins SusDEF directs the uptake of maltooligosaccharides of specific lengths, suggesting that these proteins equip the cell to scavenge a range of starch fragments. These data demonstrate that the assembly of core Sus proteins SusCDE is secondary to their glycan binding roles, but glycan binding by Sus proteins may fine tune the selection of glycans from the environment.     

Oligosaccharide binding proteins from Bifidobacterium longum subsp. infantis reveal a preference for host glycans

Bifidobacterium longum subsp. infantis (B. infantis) is a common member of the infant intestinal microbiota, and it has been characterized by its foraging capacity for human milk oligosaccharides (HMO). Its genome sequence revealed an overabundance of the Family 1 of solute binding proteins (F1SBPs), part of ABC transporters and associated with the import of oligosaccharides. In this study we have used the Mammalian Glycan Array to determine the specific affinities of these proteins. This was correlated with binding protein expression induced by different prebiotics including HMO. Half of the F1SBPs in B. infantis were determined to bind mammalian oligosaccharides. Their affinities included different blood group structures and mucin oligosaccharides. Related to HMO, other proteins were specific for oligomers of lacto-N-biose (LNB) and polylactosamines with different degrees of fucosylation. Growth on HMO induced the expression of specific binding proteins that import HMO isomers, but also bind blood group and mucin oligosaccharides, suggesting coregulated transport mechanisms. The prebiotic inulin induced other family 1 binding proteins with affinity for intestinal glycans. Most of the host glycan F1SBPs in B. infantis do not have homologs in other bifidobacteria. Finally, some of these proteins were found to be adherent to intestinal epithelial cells in vitro. In conclusion, this study represents further evidence for the particular adaptations of B. infantis to the infant gut environment, and helps to understand the molecular mechanisms involved in this process.     

Single-molecule dynamics of surface lipoproteins in bacteroides indicate similarities and cooperativity

The gut microbiota comprises hundreds of species with a composition shaped by the available glycans. The well-studied starch utilization system (Sus) is a prototype for glycan uptake in the human gut bacterium Bacteroides thetaiotaomicron (Bt). Each Sus-like system includes outer-membrane proteins, which translocate glycan into the periplasm, and one or more cell-surface glycoside hydrolases, which break down a specific (cognate) polymer substrate. Although the molecular mechanisms of the Sus system are known, how the Sus and Sus-like proteins cooperate remains elusive. Previously, we used single-molecule and super-resolution fluorescence microscopy to show that SusG is mobile on the outer membrane and slows down in the presence of starch. Here, we compare the dynamics of three glycoside hydrolases: SusG, Bt4668, and Bt1760, which target starch, galactan, and levan, respectively. We characterized the diffusion of each surface hydrolase in the presence of its cognate glycan and found that all three enzymes are mostly immobile in the presence of the polysaccharide, consistent with carbohydrate binding. Moreover, experiments in glucose versus oligosaccharides suggest that the enzyme dynamics depend on their expression level. Furthermore, we characterized enzyme diffusion in a mixture of glycans and found that noncognate polysaccharides modify the dynamics of SusG and Bt1760 but not Bt4668. We investigated these systems with polysaccharide mixtures and genetic knockouts and found that noncognate polysaccharides modify hydrolase dynamics through some combination of nonspecific protein interactions and downregulation of the hydrolase. Overall, these experiments extend our understanding of how Sus-like lipoprotein dynamics can be modified by changing carbohydrate conditions and the expression level of the enzyme.     

Biochemical Analysis of Interactions between Outer Membrane Proteins That Contribute to Starch Utilization by Bacteroides thetaiotaomicron

An early step in the utilization of starch by Bacteroides thetaiotaomicron is the binding of starch to the bacterial surface. Four starch-associated outer membrane proteins of B. thetaiotaomicron that have no starch-degrading activity have been identified. Two of these, SusC and SusD, have been shown by genetic analysis to be required for starch binding. In this study, we provide the first biochemical evidence that these two proteins interact physically with each other. Both formaldehyde cross-linking and nondenaturing gel electrophoresis experiments showed that SusC and SusD interact to form a complex. Two other proteins encoded by genes in the same operon, SusE and SusF, proved not to be essential for starch utilization and actually decreased starch binding when they were present along with SusC and SusD. Consistent with this, nondenaturing gel analysis revealed that in a strain producing SusC, SusD, and SusE, the SusCD complex was partially destabilized. The strain producing SusC, SusD, and SusE also grew more slowly on starch than a strain producing SusC, SusD, SusE, and SusF (mu(max), 0.29 and 0.37/h, respectively). Thus, SusE appears to interact with the SusCD complex. SusE also interacts with SusF, because SusE was less susceptible to proteinase K digestion when SusF was present, and nondenaturing gel analysis detected a complex formed by these two proteins. Our results indicate that SusC, SusD, SusE, and SusF form a protein complex in the outer membrane but that SusE and SusF are dispensable members of this complex.     

How glycan metabolism shapes the human gut microbiota

Symbiotic microorganisms that reside in the human intestine are adept at foraging glycans and polysaccharides, including those in dietary plants (starch, hemicellulose and pectin), animal-derived cartilage and tissue (glycosaminoglycans and N-linked glycans), and host mucus (O-linked glycans). Fluctuations in the abundance of dietary and endogenous glycans, combined with the immense chemical variation among these molecules, create a dynamic and heterogeneous environment in which gut microorganisms proliferate. In this Review, we describe how glycans shape the composition of the gut microbiota over various periods of time, the mechanisms by which individual microorganisms degrade these glycans, and potential opportunities to intentionally influence this ecosystem for better health and nutrition.     

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.     

Bacteroides in the infant gut consume milk oligosaccharides via mucus-utilization pathways

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

Characterization of four outer membrane proteins involved in binding starch to the cell surface of Bacteroides thetaiotaomicron

Bacteroides thetaiotaomicron, a gram-negative obligate anaerobe, utilizes polysaccharides by binding them to its cell surface and allowing cell-associated enzymes to hydrolyze them into digestible fragments. We use the starch utilization system as a model to analyze the initial steps involved in polysaccharide binding and breakdown. In a recent paper, we reported that one of the outer membrane proteins involved, SusG, had starch-degrading activity but was not sufficient for growth on starch. Moreover, SusG alone did not have detectable starch binding activity. Previous studies have shown that starch binding is essential for starch utilization. In this paper, we report that four other outer membrane proteins, SusC through SusF, are responsible for starch binding. Results of (14)C-starch binding assays show that SusC and SusD both contribute a significant amount of starch binding. SusE also appears to contribute substantially to starch binding. Using affinity chromatography, we show in vitro that these Sus proteins interact to bind starch. Moreover, protease accessibility of either SusC or SusD greatly increased when one was expressed without the other. This finding supports the hypothesis that SusC and SusD interact in the outer membrane. Evidence from additional protease accessibility studies suggests that SusC, SusE, and SusF are exposed on the cell surface. Our results demonstrate that SusC and SusD act as the major starch binding proteins on the cell surface, with SusE enhancing binding. SusF's role in starch utilization has yet to be determined, although the fact that starch protected it from proteolytic attack suggests that it does bind starch.     

Structure determination by MALDI-IRMPD mass spectrometry and exoglycosidase digestions of O-linked oligosaccharides from Xenopus borealis egg jelly

Differences in the fertilization behavior of Xenopus borealis from X. laevis and X. tropicalis suggest differences in the glycosylation of the egg jellies. To test this assumption, O-linked glycans were chemically released from the egg jelly coat glycoproteins of X. borealis. Over 50 major neutral glycans were observed, and no anionic glycans were detected from the released O-glycan pool. Preliminary structures of ～30 neutral oligosaccharides were determined using matrix-assisted laser desorption/ionization (MALDI) infrared multiphoton dissociation tandem mass spectrometry (MS). The mass fingerprint of a group of peaks for the core-2 structure of O-glycans was conserved in the tandem mass spectra and was instrumental in rapid and efficient structure determination. Among the 29 O-glycans, 22 glycans contain the typical core-2 structure, 3 glycans have the core-1 structure and 2 glycans contained a previously unobserved core structure with hexose at the reducing end. There were seven pairs of structural isomers observed in the major O-linked oligosaccharides. To further elucidate the structures of a dozen O-linked glycans, specific and targeted exoglycosidase digestions were carried out and the products were monitored with MALDI-MS. Reported here are the elucidated structures of O-linked oligosaccharides from glycoproteins of X. borealis egg jelly coats. The structural differences in O-glycans from jelly coats of X. borealis and its close relatives may provide a better understanding of the structure-function relationships and the role of glycans in the fertilization process within Xenopodinae.     

Molecular details of a starch utilization pathway in the human gut symbiont Eubacterium rectale

Eubacterium rectale is a prominent human gut symbiont yet little is known about the molecular strategies this bacterium has developed to acquire nutrients within the competitive gut ecosystem. Starch is one of the most abundant glycans in the human diet, and E. rectale increases in vivo when the host consumes a diet rich in resistant starch, although it is not a primary degrader of this glycan. Here we present the results of a quantitative proteomics study in which we identify two glycoside hydrolase 13 family enzymes, and three ABC transporter solute‐binding proteins that are abundant during growth on starch and, we hypothesize, work together at the cell surface to degrade starch and capture the released maltooligosaccharides. EUR_21100 is a multidomain cell wall anchored amylase that preferentially targets starch polysaccharides, liberating maltotetraose, whereas the membrane‐associated maltogenic amylase EUR_01860 breaks down maltooligosaccharides longer than maltotriose. The three solute‐binding proteins display a range of glycan‐binding specificities that ensure the capture of glucose through maltoheptaose and some α1,6‐branched glycans. Taken together, we describe a pathway for starch utilization by E. rectale DSM 17629 that may be conserved among other starch‐degrading Clostridium cluster XIVa organisms in the human gut.     

Structure of the carbohydrate units of human amniotic fluid fibronectin

Human amniotic fluid fibronectin was found to contain three types of carbohydrates: complex-type N-glycosidic glycans, lactosaminoglycans, and O-glycosidic glycans. The structures of the complex-type glycans were established by carbohydrate and methylation analysis, Smith degradation, sequential exoglycosidase treatments, lectin chromatography, and DEAE-Sephadex chromatography. Lactosaminoglycans were analyzed by fast atom bombardment mass spectrometry, and the O-glycosidically-linked oligosaccharides by gas-liquid chromatography-mass spectrometry and high-pressure liquid chromatography. The results show that amniotic fluid fibronectin contains 2 mol of biantennary and 2-3 mol of triantennary, complex-type N-glycosidic glycans. Unlike the N-glycosidic glycans of human adult plasma fibronectin, which contain only traces of fucose and are completely sialylated, the glycans from amniotic fluid fibronectin are fucosylated and only partially sialylated. The complex-type N-glycosidic glycans present in amniotic fluid fibronectin also include a fractional amount (0.1 mol) of glycans with a polylactosaminyl structure. In addition, 4 mol of O-glycosidic oligosaccharides, which have not previously been described in fibronectins, were found in amniotic fluid fibronectin. The major oligosaccharides in this fraction have the structures Gal beta 1----3GalNAcol, NeuNAc alpha 2----3Gal beta 1----3GalNAcol and NeuNAc alpha 2----3Gal beta 1----3(NeuNAc alpha 2----6)GalNAcol. O-glycosidically linked oligosaccharides were also detected in human adult plasma fibronectin but in smaller amounts than in amniotic fluid fibronectin. These results show that amniotic fluid fibronectin differs from plasma fibronectin with regard to the number of glycans attached to the polypeptide and that the glycans present in these two fibronectins differ in structure.     

Contribution of N-linked glycans to the conformation and function of intercellular adhesion molecules (ICAMs)

The crystal structures of the glycosylated N-terminal two domains of ICAM-1 and ICAM-2 provided a framework for understanding the role of glycosylation in the structure and function of intercellular adhesion molecules (ICAMs). The most conserved glycans were less flexible in the structures, interacting with protein residues and contributing to receptor folding and expression. The first N-linked glycan in ICAM-2 contacts an exposed tryptophan residue, defining a conserved glycan-W motif critical for the conformation of the integrin binding domain. The absence of this motif in human ICAM-1 exposes regions used in receptor dimerization and rhinovirus recognition. Experiments with soluble molecules having the N-terminal two domains of human ICAMs identified glycans of the high mannose type N-linked to the second domain of the dendritic cell-specific ICAM-grabbing nonintegrin lectin-ligands ICAM-2 and ICAM-3. About 40% of those receptor molecules bear endoglycosidase H sensitive glycans responsible of the lectin binding activity. High mannose glycans were absent in ICAM-1, which did not bind to the lectin, but they appeared in ICAM-1 mutants with additional N-linked glycosylation and lectin binding activity. N-Linked glycosylation regulate both conformation and immune related functions of ICAM receptors.     

Structural Characterization by Multistage Mass Spectrometry (MSn) of Human Milk Glycans Recognized by Human Rotaviruses

We have shown that recombinant forms of VP8* domains of the human rotavirus outer capsid spike protein VP4 from human neonatal strains (N155(G10P[11]) and RV3(G3P[6]) and a bovine strain (B223) recognize unique glycans within the repertoire of human milk glycans. The accompanying study by Yu et al.², describes a human milk glycan shotgun glycan microarray that led to the identification of 32 specific glycans in the human milk tagged glycan library that were recognized by these human rotaviruses. These microarray analyses also provided a variety of metadata about the recognized glycan structures compiled from anti-glycan antibody and lectin binding before and after specific glycosidase digestions, along with compositional information from mass analysis by matrix-assisted laser desorption ionization-mass spectrometry. To deduce glycan sequence and utilize information predicted by analyses of metadata from each glycan, 28 of the glycan targets were retrieved from the tagged glycan library for detailed sequencing using sequential disassembly of glycans by ion-trap mass spectrometry. Our aim is to obtain a deeper structural understanding of these key glycans using an orthogonal approach for structural confirmation in a single ion trap mass spectrometer. This sequential ion disassembly strategy details the complexities of linkage and branching in multiple compositions, several of which contained isomeric mixtures including several novel structures. The application of this approach exploits both library matching with standard materials and de novo approaches. This combination together with the metadata generated from lectin and antibody-binding data before and after glycosidase digestions provide a heretofore-unavailable level of analytical detail to glycan structure analysis. The results of these studies showed that, among the 28 glycan targets analyzed, 27 unique structures were identified, and 23 of the human milk glycans recognized by human rotaviruses represent novel structures not previously described as glycans in human milk. The functional glycomics analysis of human milk glycans provides significant insight into the repertoire of glycans comprising the human milk metaglycome.     

Carbohydrate-binding specificity of Tetracarpidium conophorum lectin.

The carbohydrate-binding specificity of a novel plant lectin isolated from the seeds of Tetracarpidium conophorum (Nigerian walnut) has been studied by quantitative hapten inhibition assays and by determining the behavior of a number of oligosaccharides and glycopeptides on lectin-Sepharose affinity columns. The Tetracarpidium lectin shows preference for simple, unbranched oligosaccharides containing a terminal Gal beta 1----4GlNAc sequence over a Gal beta 1----3GlcNAc sequence and substitution by sialic acid or fucose of the terminal galactose residue, the subterminal N-acetylglucosamine or more distally located sugar residues of oligosaccharides reduce binding activity. Branched complex-type glycans containing either Gal beta 1----4GlcNAc or Gal beta 1----3GlcNAc termini bind with higher affinity than simpler oligosaccharides. The lectin shows highest affinity for a tri-antennary glycan carrying Gal beta 1----4GlcNAc substituents on C-2 and C-4 of Man alpha 1----3 and C-2 of Man alpha 1----6 core residues. Bi- and tri-glycans lacking this branching pattern bind more weakly. Tetra-antennary glycans and mono- and di-branched hybrid-type glycans also bind weakly to the immobilized lectin. Therefore, Tetracarpidium lectin complements the binding specificities of well-known lectins such as Datura stramonium agglutinin, Phaseolus vulgaris agglutinin, and lentil lectin and will be a useful additional tool for the identification and separation of complex-type glycans.     

Recent insight into oligosaccharide uptake and metabolism in probiotic bacteria

Abstract

In recent years, a plethora of studies have demonstrated the paramount physiological importance of the gut microbiota on various aspects of human health and development. Particular focus has been set on probiotic members of this community, the best studied of which are assigned into the Lactobacillus and Bifidobacterium genera. Effects such as pathogen exclusion, alleviation of inflammation and allergies, colon cancer, and other bowel disorders are attributed to the activity of probiotic bacteria, which selectively ferment prebiotics comprising mainly non-digestible oligosaccharides. Thus, glycan metabolism is an important attribute of probiotic action and a factor influencing the composition of the gut microbiota.

In the quest to understand the molecular mechanism of this selectivity for certain glycans, we have explored the routes of uptake and utilization of a variety of oligosaccharides differing in size, composition, and glycosidic linkages. A combination of “omics” technologies bioinformatics, enzymology and protein characterization proved fruitful in elucidating the protein transport and catabolic machinery conferring the utilization of glucosides, galactosides, and xylosides in the two clinically validated probiotic strains Lactobacillus acidophilus NCFM and Bifidobacterium animalis subsp. lactis Bl-04. Importantly, we have been able to identify and in some cases validate the specificity of several transport systems, which are otherwise poorly annotated. Further, we have demonstrated for the first time that non-naturally occurring tri- and tetra-saccharides are internalized and efficiently utilized by probiotic bacteria in some cases better than well-established natural prebiotics.

Selected highlights of these data are presented, emphasising the importance and the diversity of oligosaccharide transport in probiotic bacteria.     

Minority species influences microbiota formation: the role of Bifidobacterium with extracellular glycosidases in bifidus flora formation in breastfed infant guts

The human body houses a variety of microbial ecosystems, such as the microbiotas on the skin, in the oral cavity and in the digestive tract. The gut microbiota is one such ecosystem that contains trillions of bacteria, and it is well established that it can significantly influence host health and diseases. With the advancement in bioinformatics tools, numerous comparative studies based on 16S ribosomal RNA (rRNA) gene sequences, metabolomics, pathological and epidemical analyses have revealed the correlative relationship between the abundance of certain taxa and disease states or amount of certain causative bioactive compounds. However, the 16S rRNA-based taxonomic analyses using next-generation sequencing (NGS) technology essentially detect only the majority species. Although the entire gut microbiome consists of 10¹³ microbial cells, NGS read counts are given in multiples of 10⁶, making it difficult to determine the diversity of the entire microbiota. Some recent studies have reported instances where certain minority species play a critical role in creating locally stable conditions for other species by stabilizing the fundamental microbiota, despite their low abundance. These minority species act as ‘keystone species’, which is a species whose effect on the community is disproportionately large compared to its relative abundance. One of the attributes of keystone species within the gut microbiota is its extensive enzymatic capacity for substrates that are rare or difficult to degrade for other species, such as dietary fibres or host-derived complex glycans, like human milk oligosaccharides (HMOs). In this paper, we propose that more emphasis should be placed on minority taxa and their possible role as keystone species in gut microbiota studies by referring to our recent studies on HMO-mediated microbiota formation in the infant gut.     

Diet drives convergent evolution of gut microbiomes in bamboo-eating species

Gut microbiota plays a critical role in host physiology and health. The coevolution between the host and its gut microbes facilitates animal adaptation to its specific ecological niche. Multiple factors such as host diet and phylogeny modulate the structure and function of gut microbiota. However, the relative contribution of each factor in shaping the structure of gut microbiota remains unclear. The giant(Ailuropoda melanoleuca) and red(Ailurus styani) pandas belong to different families of order Carnivora. They have evolved as obligate bamboo-feeders and can be used as a model system for studying the gut microbiome convergent evolution. Here, we compare the structure and function of gut microbiota of the two pandas with their carnivorous relatives using 16S rRNA and metagenome sequencing. We found that both panda species share more similarities in their gut microbiota structure with each other than each species shares with its carnivorous relatives. This indicates that the specialized herbivorous diet rather than host phylogeny is the dominant driver of gut microbiome convergence within Arctoidea.Metagenomic analysis revealed that the symbiotic gut microbiota of both pandas possesses a high level of starch and sucrose metabolism and vitamin B12 biosynthesis. These findings suggest a diet-driven convergence of gut microbiomes and provide new insight into host-microbiota coevolution of these endangered species.