Simulated digestions of free oligosaccharides and mucin-type O-glycans reveal a potential role for Clostridium perfringens

Abstract

The development of a stable human gut microbiota occurs within the first year of life. Many open questions remain about how microfloral species are influenced by the composition of milk, in particular its content of human milk oligosaccharides (HMOs). The objective is to investigate the effect of the human HMO glycome on bacterial symbiosis and competition, based on the glycoside hydrolase (GH) enzyme activities known to be present in microbial species. We extracted from UniProt a list of all bacterial species catalysing glycoside hydrolase activities (EC 3.2.1.-), cross-referencing with the BRENDA database, and obtained a set of taxonomic lineages and CAZy family data. A set of 13 documented enzyme activities was selected and modelled within an enzyme simulator according to a method described previously in the context of biosynthesis. A diverse population of experimentally observed HMOs was fed to the simulator, and the enzymes matching specific bacterial species were recorded, based on their appearance of individual enzymes in the UniProt dataset. Pairs of bacterial species were identified that possessed complementary enzyme profiles enabling the digestion of the HMO glycome, from which potential symbioses could be inferred. Conversely, bacterial species having similar GH enzyme profiles were considered likely to be in competition for the same set of dietary HMOs within the gut of the newborn. We generated a set of putative biodegradative networks from the simulator output, which provides a visualisation of the ability of organisms to digest HMO and mucin-type O-glycans. B. bifidum, B. longum and C. perfringens species were predicted to have the most diverse GH activity and therefore to excel in their ability to digest these substrates. The expected cooperative role of Bifidobacteriales contrasts with the surprising capacities of the pathogen. These findings indicate that potential pathogens may associate in human gut based on their shared glycoside hydrolase digestive apparatus, and which, in the event of colonisation, might result in dysbiosis. The methods described can readily be adapted to other enzyme categories and species as well as being easily fine-tuneable if new degrading enzymes are identified and require inclusion in the model.

Figure 1

Numbers of distinct bacterial sulfatase (EC 3.1.6.-) and glycoside hydrolase (EC 3.2.1.-) activities in UniProt, cross-referenced with the BRENDA database, and CAZy. Sulfatases not being exclusively carbohydrate-active, no members of EC 3.1.6 are listed in the CAZy database. The BRENDA values were not sourced directly from the BRENDA database but represent EC numbers that are cross-referenced in UniProtKB.

Figure 2

Mapping the enzymes used in carbohydrate (HMO) digestion to the full complement of simulated glycoside hydrolase enzymes available to an organism. (A) Krona diagram showing the GH enzyme profiles on the inner ring, which reflect simulated digestion of a heterogeneous population of HMOs, map to one or more profiles of available enzymes in the outer ring, which corresponds with the bacterial species. (B) Unique enzyme profile values belonging to the six named spaces in (A), Bifidobacterium bifidum, Bifidobacterium longum, Clostridium butyricum, Clostridium perfringens, Oribacterium sinus and Treponema succinifaciens. The enzyme profile value is illustrated by a vector of cells (1 to 13, left to right, representing the enzymes of Table 1) filled according to a colour representing the substituent removed, or bond cleaved: red (l-fucose); yellow (d-galactose); orange (GalNAc); blue (GlcNAc/HexNAc) magenta (sialic acid); grey (lacto-N-biose); white (no activity). A complete set of enzyme profile values is shown in Fig. 9, while interactive Krona diagrams are provided in Supplementary Information (Krona), in which each segment can be expanded to reveal the species associated with each profile.

Figure 3

Simulated networks of HMO degradation by glycoside hydrolases expressed in Bacteria when each of 226 unique human milk oligosaccharides were submitted to the simulator. (A) Network obtained with all enzymes of the model available. (B) Networks corresponding to the enzyme profiles p4360 and p4888, representing Bifidobacterium spp. bifidum and longum, respectively. (C) Network p6912 (Clostridium perfringens). (D) Network p7984 (E. coli and other species). Between panels (A–D), increased fragmentation of the networks occurs, as the number of available GH enzymes active towards HMO substrates decreases (B–D). Nodes are coloured according to type of core structure based on the reducing end of the HMO: red (lacto-N-tetraose), cyan (lacto-N-neotetraose), blue (lacto-N-hexaose), orange (lacto-N-neohexaose), grey (other).

Figure 4

Symbols of common monosaccharides and cores found in human milk oligosaccharides and mucin-type O-glycans.

Figure 5

Simulated pathways of biosynthesis and glycoside hydrolase-catalysed degradation of the human milk oligosaccharide TFS-LNO. In the biosynthetic network, only products leading to TFS-LNO are included. Selected edges are labelled according to the enzymes assumed to be active at those steps of the pathway. Highlighted nodes represent experimentally characterised structures: grey node (LNTri II), blue nodes representing structures with a lacto-N-hexaose core (Fig. 4). Abbreviations used: LNTri II (lacto-N-triose II); TFS-LNO (a trifucosyl,monosialyllacto-N-octaose).

Figure 6

Predicted potential energy (PE) scores assigned to Bacteria fed a population of human milk oligosaccharides and mucin-type O-glycans. For each unique enzyme profile, with a subset of the simulated enzymes, a simulated glycoside hydrolase degradation network was generated, and the number of monosaccharides released was counted (the action of lacto-N-biosidase was counted as 0.5 instead of 1). Each profile was then scored based on the ratio of this value to the maximum possible value for the HMO dataset, with all enzymes active. Colours are assigned from grey through blue for the PE scores (0–1). (A) Non-gut species, grouped by taxonomic Class. (B) Gut bacterial species, grouped by taxonomic Order. The position of C. perfringens is indicated by an arrow. C Numbers of simulated enzymes available to gut and non-gut species. D Frequency distribution of potential energy scores of HMO-fed bacteria. Highly scoring gut species, fed in silico on HMOs (A, right) were B. bifidum (p4360), B. longum (p4888) and C. perfringens (p6912); the highest scoring on mucin-type O-glycans (B, right) were B. bifidum (p4360) and C. perfringens (p6912). Data are sourced from UniProtKB (https://uniprot.org). For comparison with the CAZy dataset, see Extended Data Fig. E2.

Figure 7

Predicted competition and symbiosis between gut bacteria, based on available enzyme profiles. For each bit in profiles p₁ and p₂, a syntrophic score increased by 1 when the bitwise comparison p₁ XOR p₁ = 1 (Boolean true), decreased by 0.5 when p₁ AND p₂ = 1 (Boolean true), and left unchanged when p1 AND p₂ = 0 (Boolean false). A syntrophic pair (green) and competitive pair of species (red) are indicated. See text for details.

Figure 8

O-glycan degradation networks by glycoside hydrolases. 434 mucin-type GalNAc-linked glycans were submitted to the Glycologue glycoside hydrolase simulator for digestion by the enzymes of Table 1. Larger nodes are experimentally observed O-glycans, coloured according to the core type (cf. Figure 4): core 1 (yellow); core 2 (green); core 3 (cyan); core 4 (blue); core 5 (orange); smaller grey nodes are inferred intermediates.

Figure 9

Comparison of UniProt- and CAZy-derived enzyme profiles of bacteria and simulated potential energy scores based on HMOs. A list of the top ten species in decreasing order of the number of simulated enzymes shown in Table 1, along with an enzyme profile value and a vector of cells (1 to 13, left to right) filled according to a colour representing the substituent removed, or bond cleaved. Colour key: red (l-fucose); yellow (d-galactose); orange (GalNAc); blue (GlcNAc/HexNAc) magenta (sialic acid); grey (lacto-N-biose); white denotes a complete absence of activity. The enzyme profile value is calculated from the source database for each organism. The relative potential energy (P.E.) score matching the enzyme profile of a species fed on a mixture of HMOs. The unique GH enzyme profile values obtained from UniProt/BRENDA (left panels) and CAZy (right panels) are shown beneath, divided into gut and non-gut categories. Each heatmap possesses, as a reference point p0, which is the default value with all enzymes available.