Nicole M. Koropatkin

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.

Complex Glycan Catabolism by the Human Gut Microbiota: The Bacteroidetes Sus-like Paradigm

Trillions of microbes inhabit the distal gut of adult humans. They have evolved to compete efficiently for nutrients, including a wide array of chemically diverse, complex glycans present in our diets, secreted by our intestinal mucosa, and displayed on the surfaces of other gut microbes. Here, we review how members of the Bacteroidetes, one of two dominant gut-associated bacterial phyla, process complex glycans using a series of similarly patterned, cell envelope-associated multiprotein systems. These systems provide insights into how gut, as well as terrestrial and aquatic, Bacteroidetes survive in highly competitive ecosystems.

A Dietary Fiber-Deprived Gut Microbiota Degrades the Colonic Mucus Barrier and Enhances Pathogen Susceptibility

Despite the accepted health benefits of consuming dietary fiber, little is known about the mechanisms by which fiber deprivation impacts the gut microbiota and alters disease risk. Using a gnotobiotic mouse model, in which animals were colonized with a synthetic human gut microbiota composed of fully sequenced commensal bacteria, we elucidated the functional interactions between dietary fiber, the gut microbiota, and the colonic mucus barrier, which serves as a primary defense against enteric pathogens. We show that during chronic or intermittent dietary fiber deficiency, the gut microbiota resorts to host-secreted mucus glycoproteins as a nutrient source, leading to erosion of the colonic mucus barrier. Dietary fiber deprivation, together with a fiber-deprived, mucus-eroding microbiota, promotes greater epithelial access and lethal colitis by the mucosal pathogen, Citrobacter rodentium. Our work reveals intricate pathways linking diet, the gut microbiome, and intestinal barrier dysfunction, which could be exploited to improve health using dietary therapeutics.

A discrete genetic locus confers xyloglucan metabolism in select human gut Bacteroidetes

A well-balanced human diet includes a significant intake of non-starch polysaccharides, collectively termed ‘dietary fibre’, from the cell walls of diverse fruits and vegetables. Owing to the paucity of alimentary enzymes encoded by the human genome, our ability to derive energy from dietary fibre depends on the saccharification and fermentation of complex carbohydrates by the massive microbial community residing in our distal gut. The xyloglucans (XyGs) are a ubiquitous family of highly branched plant cell wall polysaccharides whose mechanism(s) of degradation in the human gut and consequent importance in nutrition have been unclear. Here we demonstrate that a single, complex gene locus in Bacteroides ovatus confers XyG catabolism in this common colonic symbiont. Through targeted gene disruption, biochemical analysis of all predicted glycoside hydrolases and carbohydrate-binding proteins, and three-dimensional structural determination of the vanguard endo-xyloglucanase, we reveal the molecular mechanisms through which XyGs are hydrolysed to component monosaccharides for further metabolism. We also observe that orthologous XyG utilization loci (XyGULs) serve as genetic markers of XyG catabolism in Bacteroidetes, that XyGULs are restricted to a limited number of phylogenetically diverse strains, and that XyGULs are ubiquitous in surveyed human metagenomes. Our findings reveal that the metabolism of even highly abundant components of dietary fibre may be mediated by niche species, which has immediate fundamental and practical implications for gut symbiont population ecology in the context of human diet, nutrition and health.

Starch Catabolism by a Prominent Human Gut Symbiont Is Directed by the Recognition of Amylose Helices

The human gut microbiota performs functions that are not encoded in our Homo sapiens genome, including the processing of otherwise undigestible dietary polysaccharides. Defining the structures of proteins involved in the import and degradation of specific glycans by saccharolytic bacteria complements genomic analysis of the nutrient-processing capabilities of gut communities. Here, we describe the atomic structure of one such protein, SusD, required for starch binding and utilization by Bacteroides thetaiotaomicron, a prominent adaptive forager of glycans in the distal human gut microbiota. The binding pocket of this unique alpha-helical protein contains an arc of aromatic residues that complements the natural helical structure of starch and imposes this conformation on bound maltoheptaose. Furthermore, SusD binds cyclic oligosaccharides with higher affinity than linear forms. The structures of several SusD/oligosaccharide complexes reveal an inherent ligand recognition plasticity dominated by the three-dimensional conformation of the oligosaccharides rather than specific interactions with the composite sugars.

SusG: A Unique Cell-Membrane-Associated α-Amylase from a Prominent Human Gut Symbiont Targets Complex Starch Molecules

SusG is an alpha-amylase and part of a large protein complex on the outer surface of the bacterial cell and plays a major role in carbohydrate acquisition by the animal gut microbiota. Presented here, the atomic structure of SusG has an unusual extended, bilobed structure composed of amylase at one end and an unprecedented internal carbohydrate-binding motif at the other. Structural studies further demonstrate that the carbohydrate-binding motif binds maltooligosaccharide distal to, and on the opposite side of, the amylase catalytic site. SusG has an additional starch-binding site on the amylase domain immediately adjacent to the active cleft. Mutagenesis analysis demonstrates that these two additional starch-binding sites appear to play a role in catabolism of insoluble starch. However, elimination of these sites has only a limited effect, suggesting that they may have a more important role in product exchange with other Sus components.

The Structure of a Cyanobacterial Bicarbonate Transport Protein, CmpA

Cyanobacteria, blue-green algae, are the most abundant autotrophs in aquatic environments and form the base of the food chain by fixing carbon and nitrogen into cellular biomass. To compensate for the low selectivity of Rubisco for CO2 over O2, cyanobacteria have developed highly efficient CO2-concentrating machinery of which the ABC transport system CmpABCD from Synechocystis PCC 6803 is one component. Here, we have described the structure of the bicarbonate-binding protein CmpA in the absence and presence of bicarbonate and carbonic acid. CmpA is highly homologous to the nitrate transport protein NrtA. CmpA binds carbonic acid at the entrance to the ligand-binding pocket, whereas bicarbonate binds in nearly an identical location compared with nitrate binding to NrtA. Unexpectedly, bicarbonate binding is accompanied by a metal ion, identified as Ca2+ via inductively coupled plasma optical emission spectrometry. The binding of bicarbonate and metal appears to be highly cooperative and suggests that CmpA may co-transport bicarbonate and calcium or that calcium acts a cofactor in bicarbonate transport.

Polysaccharide Degradation by the Intestinal Microbiota and Its Influence on Human Health and Disease.

Carbohydrates comprise a large fraction of the typical diet, yet humans are only able to directly process some types of starch and simple sugars. The remainder transits the large intestine where it becomes food for the commensal bacterial community. This is an environment of not only intense competition but also impressive cooperation for available glycans, as these bacteria work to maximize their energy harvest from these carbohydrates during their limited transit time through the gut. The species within the gut microbiota use a variety of strategies to process and scavenge both dietary and host-produced glycans such as mucins. Some act as generalists that are able to degrade a wide range of polysaccharides, while others are specialists that are only able to target a few select glycans. All are members of a metabolic network where substantial cross-feeding takes place, as by-products of one organism serve as important resources for another. Much of this metabolic activity influences host physiology, as secondary metabolites and fermentation end products are absorbed either by the epithelial layer or by transit via the portal vein to the liver where they can have additional effects. These microbially derived compounds influence cell proliferation and apoptosis, modulate the immune response, and can alter host metabolism. This review summarizes the molecular underpinnings of these polysaccharide degradation processes, their impact on human health, and how we can manipulate them through the use of prebiotics.

Multidomain Carbohydrate-binding Proteins Involved in Bacteroides thetaiotaomicron Starch Metabolism

Background: Bacteroides thetaiotaomicron is a prototype for understanding carbohydrate metabolism by colonic bacteria. Results: Two nonenzymatic membrane proteins involved in starch metabolism are composed of tandem carbohydrate-binding modules that each bind starch differently. Conclusion: B. thetaiotaomicron has evolved multiple starch-binding modules to compete for different forms of starch. Significance: Learning how gut bacteria degrade carbohydrates is crucial for understanding their role in nutrition. Human colonic bacteria are necessary for the digestion of many dietary polysaccharides. The intestinal symbiont Bacteroides thetaiotaomicron uses five outer membrane proteins to bind and degrade starch. Here, we report the x-ray crystallographic structures of SusE and SusF, two outer membrane proteins composed of tandem starch specific carbohydrate-binding modules (CBMs) with no enzymatic activity. Examination of the two CBMs in SusE and three CBMs in SusF reveals subtle differences in the way each binds starch and is reflected in their Kd values for both high molecular weight starch and small maltooligosaccharides. Thus, each site seems to have a unique starch preference that may enable these proteins to interact with different regions of starch or its breakdown products. Proteins similar to SusE and SusF are encoded in many other polysaccharide utilization loci that are possessed by human gut bacteria in the phylum Bacteroidetes. Thus, these proteins are likely to play an important role in carbohydrate metabolism in these abundant symbiotic species. Understanding structural changes that diversify and adapt related proteins in the human gut microbial community will be critical to understanding the detailed mechanistic roles that they perform in the complex digestive ecosystem.

Dynamic responses of Bacteroides thetaiotaomicron during growth on glycan mixtures

Bacteroides thetaiotaomicron (Bt) is a human colonic symbiont that degrades many different complex carbohydrates (glycans), the identities and amounts of which are likely to change frequently and abruptly from meal‐to‐meal. To understand how this organism reacts to dynamic growth conditions, we challenged it with a series of different glycan mixtures and measured responses involved in glycan catabolism. Our results demonstrate that individual Bt cells can simultaneously respond to multiple glycans and that responses to new glycans are extremely rapid. The presence of alternative carbohydrates does not alter response kinetics, but reduces expression of some glycan utilization genes as well as the cells sensitivity to glycans that are present in lower concentration. Growth in a mixture containing 12 different glycans revealed that Bt preferentially uses some before others. This metabolic hierarchy is not changed by prior exposure to lower priority glycans because re‐introducing high priority substrates late in culture re‐initiates repression of genes involved in degrading those with lower priority. At least some carbohydrate prioritization effects occur at the level of monosaccharide recognition. Our results provide insight into how a bacterial glycan generalist modifies its responses in dynamic glycan environments and provide essential knowledge to interpret related metabolic behaviour in vivo.