Carbohydrate polymers drive microbial diversity in the human gut microbiota. It is unclear, however, whether bacterial consortia or single organisms are required to depolymerize highly complex glycans. Here we show that the gut bacterium Bacteroides thetaiotaomicron utilizes the most structurally complex glycan known; the plant pectic polysaccharide rhamnogalacturonan-II, cleaving all but one of its 21 distinct glycosidic linkages. We show that rhamnogalacturonan-II side-chain and backbone deconstruction are coordinated, to overcome steric constraints, and that degradation reveals previously undiscovered enzyme families and novel catalytic activities. The degradome informs revision of the current structural model of RG-II and highlights how individual gut bacteria orchestrate manifold enzymes to metabolize the most challenging glycans in the human diet.

The major nutrients available to human colonic Bacteroides species are glycans exemplified by pectins, a network of covalently linked plant cell wall polysaccharides containing galacturonic acid (GalA). Metabolism of complex carbohydrates by the Bacteroides genus is orchestrated by polysaccharide utilisation loci or PULs. In Bacteroides thetaiotaomicron, a human colonic bacterium, the PULs activated by the different pectin domains have been identified, however, the mechanism by which these loci contribute to the degradation of these GalA-containing polysaccharides is poorly understood. Here we show that each PUL orchestrates the metabolism of specific pectin molecules, recruiting enzymes from two previously unknown glycoside hydrolase (GH) families. The apparatus that depolymerizes the backbone of rhamnogalacturonan-I (RGI) is particularly complex. This system contains several GHs that trim the remnants of other pectin domains attached to RGI, while nine enzymes contribute to the degradation of the backbone comprising a rhamnose-GalA repeating unit. The catalytic properties of the pectin degrading enzymes are optimized to protect the glycan cues that activate the specific PULs ensuring a continuous supply of inducing molecules throughout growth. The contribution of Bacteroides spp. to the metabolism of the pectic network is illustrated by cross-feeding between organisms.

The human microbiota, which plays an important role in health and disease, uses complex carbohydrates as a major source of nutrients. Utilization hierarchy indicates that the host glycosaminoglycans heparin (Hep) and heparan sulfate (HS) are high-priority carbohydrates for , a prominent member of the human microbiota. The sulfation patterns of these glycosaminoglycans are highly variable, which presents a significant enzymatic challenge to the polysaccharide lyases and sulfatases that mediate degradation. It is possible that the bacterium recruits lyases with highly plastic specificities and expresses a repertoire of enzymes that target substructures of the glycosaminoglycans with variable sulfation or that the glycans are desulfated before cleavage by the lyases. To distinguish between these mechanisms, the components of the Hep/HS degrading apparatus were analyzed. The data showed that the bacterium expressed a single-surface endo-acting lyase that cleaved HS, reflecting its higher molecular weight compared with Hep. Both Hep and HS oligosaccharides imported into the periplasm were degraded by a repertoire of lyases, with each enzyme displaying specificity for substructures within these glycosaminoglycans that display a different degree of sulfation. Furthermore, the crystal structures of a key surface glycan binding protein, which is able to bind both Hep and HS, and periplasmic sulfatases reveal the major specificity determinants for these proteins. The locus described here is highly conserved within the human gut , indicating that the model developed is of generic relevance to this important microbial community.

The human gut microbiota (HGM) makes an important contribution to health and disease. It is a complex microbial community of trillions of microbes with a majority of its members represented within two phyla, the Bacteroidetes and Firmicutes, although it also contains species of Actinobacteria and Proteobacteria. Reflecting its importance, the HGM is sometimes referred to as an 'organ' as it performs functions analogous to systemic tissues within the human host. The major nutrients available to the HGM are host and dietary complex carbohydrates. To utilise these nutrient sources, the HGM has developed elaborate, variable and sophisticated systems for the sensing, capture and utilisation of these glycans. Understanding nutrient acquisition by the HGM can thus provide mechanistic insights into the dynamics of this ecosystem, and how it impacts human health. Dietary nutrient sources include a wide variety of simple and complex plant and animal-derived glycans most of which are not degraded by enzymes in the digestive tract of the host. Here we review how various adaptive mechanisms that operate across the major phyla of the HGM contribute to glycan utilisation, focusing on the most complex carbohydrates presented to this ecosystem.

Glycans are major nutrients for the human gut microbiota (HGM). Arabinogalactan proteins (AGPs) comprise a heterogenous group of plant glycans in which a β1,3-galactan backbone and β1,6-galactan side chains are conserved. Diversity is provided by the variable nature of the sugars that decorate the galactans. The mechanisms by which nutritionally relevant AGPs are degraded in the HGM are poorly understood. Here we explore how the HGM organism Bacteroides thetaiotaomicron metabolises AGPs. We propose a sequential degradative model in which exo-acting glycoside hydrolase (GH) family 43 β1,3-galactanases release the side chains. These oligosaccharide side chains are depolymerized by the synergistic action of exo-acting enzymes in which catalytic interactions are dependent on whether degradation is initiated by a lyase or GH. We identified two GHs that establish two previously undiscovered GH families. The crystal structures of the exo-β1,3-galactanases identified a key specificity determinant and departure from the canonical catalytic apparatus of GH43 enzymes. Growth studies of Bacteroidetes spp. on complex AGP revealed three keystone organisms that facilitated utilisation of the glycan by 17 recipient bacteria, which included B. thetaiotaomicron . A surface endo-β1,3-galactanase, when engineered into B. thetaiotaomicron , enabled the bacterium to utilise complex AGPs and act as a keystone organism.

The human gut microbiota (HGM), which is critical to human health, utilises complex glycans as its major carbon source. Glycosaminoglycans represent an important, high priority, nutrient source for the HGM. Pathways for the metabolism of various glycosaminoglycan substrates remain ill-defined. Here we perform a biochemical, genetic and structural dissection of the genetic loci that orchestrates glycosaminoglycan metabolism in the organism Bacteroides thetaiotaomicron. Here, we report: the discovery of two previously unknown surface glycan binding proteins which facilitate glycosaminoglycan import into the periplasm; distinct kinetic and genetic specificities of various periplasmic lyases which dictate glycosaminoglycan metabolic pathways; understanding of endo sulfatase activity questioning the paradigm of how the 'sulfation problem' is handled by the HGM; and 3D crystal structures of the polysaccharide utilisation loci encoded sulfatases. Together with comparative genomic studies, our study fills major gaps in our knowledge of glycosaminoglycan metabolism by the HGM.

Glycans are the major carbon sources available to the human colonic microbiota. Numerous N-glycosylated proteins are found in the human gut, from both dietary and host sources, including immunoglobulins such as IgA which are secreted into the intestine at high levels. Here we show that many mutualistic gut Bacteroides spp. have the capacity to utilise complex N-glycans (CNGs) as nutrients, including those from immunoglobulins. Detailed mechanistic studies using transcriptomic, biochemical, structural and genetic techniques reveal the pathway employed by B. thetaiotaomicron (Bt) for CNG degradation. The breakdown process involves an extensive enzymatic apparatus encoded by multiple non-adjacent loci and comprises 19 different carbohydrate-active enzymes (CAZymes) from different families, including a CNG specific endo-glycosidase activity. Furthermore, CNG degradation involves the activity of CAZymes that have previously been implicated in the degradation of other 2 classes of glycan. This complex and diverse apparatus provides Bt with the capacity to access the myriad different structural variants of CNGs likely to be found in the intestinal niche.

The mucosal microbiota is recognised as an important factor for our health, with many disease states linked to imbalances in the normal community structure. Hence, there is considerable interest in identifying the molecular basis of human-microbe interactions. In this work we investigated the capacity of microbes to thrive on mucosal surfaces, either as mutualists, commensals or pathogens, using comparative genomics to identify co-occurring molecular traits. We identified a novel domain we named M60-like/PF13402 (new Pfam entry PF13402), which was detected mainly among proteins from animal host mucosa-associated prokaryotic and eukaryotic microbes ranging from mutualists to pathogens. Lateral gene transfers between distantly related microbes explained their shared M60-like/PF13402 domain. The novel domain is characterised by a zinc-metallopeptidase-like motif and is distantly related to known viral enhancin zinc-metallopeptidases. Signal peptides and/or cell surface anchoring features were detected in most microbial M60-like/PF13402 domain-containing proteins, indicating that these proteins target an extracellular substrate. A significant subset of these putative peptidases was further characterised by the presence of associated domains belonging to carbohydrate-binding module family 5/12, 32 and 51 and other glycan-binding domains, suggesting that these novel proteases are targeted to complex glycoproteins such as mucins. An in vitro mucinase assay demonstrated degradation of mammalian mucins by a recombinant form of an M60-like/PF13402-containing protein from the gut mutualist Bacteroides thetaiotaomicron. This study reveals that M60-like domains are peptidases targeting host glycoproteins. These peptidases likely play an important role in successful colonisation of both vertebrate mucosal surfaces and the invertebrate digestive tract by both mutualistic and pathogenic microbes. Moreover, 141 entries across various peptidase families described in the MEROPS database were also identified with carbohydrate-binding modules defining a new functional context for these glycan-binding domains and providing opportunities to engineer proteases targeting specific glycoproteins for both biomedical and industrial applications.