Fig. 2

Bifidobacterium bifidum strains W23 and W28 have high sialidase activity that results in desialylation of surface proteins including MUC13. (A) Fucosidase and (B) sialidase activities in bacterial pellets of the B. bifidum strains. Graphs depict experimental results of three independent biological replicates. The commercial α1,2,3,4,6-L-fucosidase (Megazyme, E-FUCHS) was used as a positive control for fucosidase activity. All graphs depict the SEM of three independent experiments. Statistical test: two-tailed independent t-test. **** p < 0.0001. (C) Number of identified O-glycan-targeting CAZymes in the genome of B. bifidum strains 20456. (D) Western blot analysis of sialic acid-containing proteins in the HRT18-∆MUC13 + pMUC13 cell line after 2 h incubation with UV-inactivated B. bifidum strains at MOI 50 probes with lectins SNA (α-2,6 sialic acids) and MAL-II (α-2,3 sialic acids) lectins. Red arrows mark evidently reduced bands after incubation with W23 and W28 compared to the other lanes. Similar results were obtained in two independent experiments. (E) Anti-GFP immunoprecipitation and immunoblot analysis of MUC13-GFP (~ 160 kDa) from HRT18-∆MUC13 + pMUC13 cells line after incubation with UV-inactivated B. bifidum at MOI 50 for 2 h. (F) Anti-GFP immunoprecipitation and immunoblot analysis of MUC13-GFP with MAL-II lectin after incubation with UV-inactivated B. bifidum strains at MOI 200 for 5 h. All immunoblots generated for experiments depicted in (D–F) analysis are available in Figure S4. (G) Immunoblot analysis of MUC13-GFP and actin from HRT18-∆MUC13 + pMUC13 cells incubated with UV-inactivated W28 bacteria at MOI 50 for 4 h. Conditions also included addition of 1.5 mM sialidase inhibitor (DANA) or 1 × Halt protease and phosphatase inhibitor cocktail (p.i.). Cells were also treated with a combination of 200 U/mL of α2,3,6,8,9 neuraminidase A and 0.6 U of α1,2,3,4,6-L-fucosidase. (H) Immunoblot analysis of MUC13-GFP and actin from HRT18-∆MUC13 + pMUC13 cells incubated with UV-inactivated W28 bacteria at MOI 120 for 5 h in the absence or presence of 5.5 mM DANA. All immunoblots generated for experiments depicted in (G,H) are available in Figure S5.