Fungal β-glucan-facilitated cross-feeding activities between Bacteroides and Bifidobacterium species

Abstract

The human gut microbiota (HGM) is comprised of a very complex network of microorganisms, which interact with the host thereby impacting on host health and well-being. β-glucan has been established as a dietary polysaccharide supporting growth of particular gut-associated bacteria, including members of the genera Bacteroides and Bifidobacterium, the latter considered to represent beneficial or probiotic bacteria. However, the exact mechanism underpinning β-glucan metabolism by gut commensals is not fully understood. We show that mycoprotein represents an excellent source for β-glucan, which is consumed by certain Bacteroides species as primary degraders, such as Bacteroides cellulosilyticus WH2. The latter bacterium employs two extracellular, endo-acting enzymes, belonging to glycoside hydrolase families 30 and 157, to degrade mycoprotein-derived β-glucan, thereby releasing oligosaccharides into the growth medium. These released oligosaccharides can in turn be utilized by other gut microbes, such as Bifidobacterium and Lactiplantibacillus, which thus act as secondary degraders. We used a cross-feeding approach to track how both species are able to grow in co-culture.

Fig. 1. Polysaccharide utilization systems in Bacteroides species for β-glucan degradation.

A Structures of fungal (mycoprotein), yeast, laminarin, barley and pustulan β-glucan. B Growth Bacteroides ovatus, DSM, WH2 and BT on mycoprotein as measured on a plate reader. All growths have been produced in 3 different independent replicates (n = 3). C Number of proteins expressed by Baccell WH2 on the use of β-glucan from Mycoprotein as identified by proteome analysis. The proteomics have been produced in 3 different independent replicates (n = 3). D GUL structure in Baccell WH2 (GUL-1 and GUL-2) and BT acting on mycoprotein, yeast and pustulan.

Fig. 2. HPLC analysis of the enzymatic reactions of glycoside hydrolases in Baccell WH2 and BT on fungal β-glucan.

All HPLC experiments have been produced in 3 different independent replicates (n = 3). A Time course of BcellWH2_01931 (GH157) with mycoprotein β-glucan. B Time course of BcellWH2_01931 (GH157) with linear β-1,3-glucan. C Time course of BcellWH2_02537 (GH30_3) on mycoprotein β-glucan. D Time course of BcellWH2_02537 (GH30_3) with pustulan. E Time course of BcellWH2_01931 (GH157) and BcellWH2_02537 (GH30_3) together on linear β-1,3-glucan. F HPLC of BT3312 (GH30_3) on mycoprotein β-glucan. G HPLC of BT3314 (GH3) on mycoprotein β-glucan.

Fig. 3. Characterization of oligosaccharides released by Bacteroides when using β-1,3-glucan.

A HPLC chromatogram of the growth media of Baccell WH2 on fungal β-glucan. B Same as Panel A with BT. C Purified oligosaccharide from Baccell WH2 after Gel Filtration (GF) column. D Purified oligosaccharide from BT after GF column. E LC/MS of Baccell WH2 supernatant grown on fungal β-glucan. F LC/MS of BT supernatant grown on fungal β-glucan. All HPLC and LC/MS experiments have been performed in 3 different independent replicates (n = 3).

Fig. 4. Consumption of β-glucan oligosaccharides released by Bacteroides into the growth media by Bifidobacterium species.

A Growth of Bifidobacterium with fungal β-glucan supernatant from Baccell WH2. B HPLC analysis of supernatants before and after growth of Bifidobacterium longum subsp. longum on supernantants from Baccell WH2. C Growth of Bifidobacterium with fungal β-glucan supernatant from BT. D HPLC analysis of supernatants before and after growth of Bifidobacterium longum subspecie longum on cell-free supernatant of BT grown on fungal β-glucan. E HPLC analysis of supernatants before and after growth of Bi breve UCC2003 and L. plantarum on cell-free supernatants of BT grown on fungal β-glucan. All growths and HPLC experiments have been produced in 3 different independent replicates (n = 3).

Fig. 5. Cross-feeding experiments between Baccell WH2 and Bifidobacterium and Lactiplantibacillus spp.

A Colony forming units of Baccell WH2 + Bi. longum subsp. longum. B Percentage of Baccell WH2 + Bi. longum subsp. longum. C Colony forming units of Baccell WH2 + Bi. Breve UCC2003. D Percentage of Baccell WH2 + Bi. breve UCC2003. All cross-feeding experiments have been produced in 3 different independent replicates (n = 3).

Fig. 6. Cross-feeding experiments between BT and Bifidobacterium and Lactiplantibacillus spp.

A Colony forming units of BT + Bi. breve UCC2003. B Percentage of BT + Bi. breve UCC2003. C Colony forming units of BT + Bi. longum subsp. longum. D Percentage of BT + Bi. longum subsp. longum. All cross-feeding experiments have been produced in 3 different independent replicates (n = 3).