The claim of primacy of human gut Bacteroides ovatus in dietary cellobiose degradation

Figures & data

Figure 1. Growth curves of HGM grown on cello-oligosaccharides were detected.

Notes: (a, b) The growth curve of BO and BC grown on 2.5 and 5 mg/mL cellobiose as the sole carbon source, respectively, was tested. (c) The growth curve of probiotics grown on 5 mg/mL cellobiose were detected. (d, e) The growth curve of BO and BC grown on 2.5 and 5 mg/mL cellotetrose as the sole carbon source, respectively, were analyzed. (f) Probiotics grew on 5 mg/mL cellotetrose were measured. Data represent technical triplicates (n = 3). Abbreviation: BO (Bacteroides ovatus), BC (Bacteroides cellulosilyticus), LR (Lactobacillus reuteri), LGG (Lactobacillus rhamnosus) and BL (Bifidobacterium longum).

Figure 2. The degradation products of cellobiose were analyzed by TLC.

(a) The supernatants of BO grown on cellobiose were analyzed. (b) BO was cultured either with glucose or cellobiose, and cells were collected till log phase, respectively. Then, cells were incubated with cellobiose, and the end products were analyzed. (c) Cells were treated with or without proteinase K for 6 h and then incubated with cellobiose for analyzing. (d) Collected cells were treated with or without proteinase K for 12 h and then incubated with cellobiose for analyzing. 1 means glucose; 2 means cellobiose.

Figure 3. Polysaccharide utilization loci (PULs) were screened by RNA-seq and confirmed by RT-qPCR.

Notes: (a) Up-regulated genes were selected based on Log2FC ≥ 3 and showed by heatmap. (b, c) The up-regulated genes were arranged into two PULs and confirmed by qPCR. (d) Schematic diagram of PUL1. (e) Schematic diagram of PUL2. Data represent technical triplicates (n = 3).

Figure 4. Two new cellulases were determined.

Notes: (a) Cellobiose activated enzymes and glycan binding proteins were tested by Western blotting. (b, c) Proteins cellar location was measured by immunofluorescence and Western blotting. PK means proteinase K. (d – f) Enzymatic activity of BACOVA_02626^GH5, BACOVA_026-30^GH5 and BACOVA_02745^GH3 were analyzed by HPAEC-PAD.

Figure 5. Structure biology of BACOVA_02626^GH5 and BACOVA_02630^GH5.

Notes: (a) The structure of BACOVA_02626^GH5 was predicted by AlphaFold2. (b) The structure of BACOVA_02630^GH5 was predicted by AlphaFold2. (c, d) The catalytic pocket of BACOVA_02626^GH5 was predicted by point-site. The catalytic residues of BACOVA_02626 was Glu 154 and Glu 241 (the orange arrow showed). (e, f) The catalytic pocket of BACOVA_02630^GH5 was predicted by point-site. The catalytic residues of BACOVA_02630 was Glu 181 and Glu 293 (the blue arrow showed). Binding predication of glucose with cellulase (g) BACOVA_02626^GH5 and (h) BACOVA_02630^GH5 by docking.

Figure 6. Function of cellobiose on the composition of gut microbiota was analyzed by diversity sequencing based on 16S rRNA.

(a) Schematic diagram of animal test. (b) Body weight of mice. (c) sobs index. (d, e) Principal component analysis (PCA) plots of fecal microbiota based on the ANOSIM and Adonis, respectively, were analyzed on genus level (R = 0.2153, p = 0.009). (f) The composition of gut microbiota was analyzed based on the top 50 species at genus level showed by Heatmap. (g) LEfSe analysis on genus level was analyzed (LDA = 2.5). (h) Compared cellobiose group with vehicle group on genus level. Data were presented as the mean ± SD, and n = 10.

Figure 7. Predication of metabolic function of bacteria and inflammatory factors detection.

(a) Functional activity of bacteria was predicted by PICRUSt2 tool. (b) Carbohydrate related enzymes were predicted by Tax4Fun tool. (c) KEGG pathways were predicted by PICRUSt1 tool. (d – f) mRNA expression level of TNF-α, IL-6 and H2R from colonic tissue were measured by qPCR. (g) Protein level of colonic TNF-α and IL-6 were analyzed by IHC. Data represent technical triplicates (n = 10).

Figure 8. Model of degradation of cellobiose by human gut BO. Two new cellulases on the cell surface conferred the degradation of cellobiose into glucose were determined. In vivo test, we observed that cellobiose reshaped the composition of gut microbiota, and the abundance of LR and BO were enriched significantly after eight weeks administration by gavage.