Journal: Scientific reports

Article Title: Probiotic Bifidobacterium bifidum strains desialylate MUC13 and increase intestinal epithelial barrier function.

doi: 10.1038/s41598-025-92125-2

Figure Lengend Snippet: Fig. 1. Probiotic Bifidobacterium bifidum strains uniquely modify the MUC13 extracellular domain. (A) Immunoblot analysis of MUC13 modification/degradation and claudin-3 expression in HRT18 monolayers after incubation with different probiotic strains at MOI 10 for 20 h. The full-length 130 kDa MUC13- band that is used for quantification in (B) is marked by an arrow. (B,C) Quantification of protein expression of the 130 kDa MUC13 and 22 kDa claudin-3 bands (relative to uninfected control) in three biological replicates as depicted in A. All immunoblots used for quantification are available in Figure S1 and Figure S2. Statistical analysis was performed on non-normalized data using a one-way ANOVA with Dunnett’s post hoc test to compare each bacterial sample to the uninfected control. * p < 0.05. (D) Fluorescence confocal microscopy images of HRT18 cells stained in green with MUC13-ED (above) and MUC13-CT (below) antibodies after 20 h incubation with B. bifidum strains at MOI 10 and StcE. The maximal intensity projection is depicted. White scale bars represent 20 µM. (E) Quantification of GFP signal in cells stained with MUC13-ED and MUC13-CT antibodies as in (C). Statistical analysis was performed on non-normalized data using a one-way ANOVA with Tukey’s post hoc test. * p < 0.05; ** p < 0.01. (F) Immunoblot analysis of degradation of high-MW MUC13 (130 kDa) after 2 and 20 h incubation with B. bifidum W23, W28, and 20456 strains at MOI 10 and 50 in HRT18 cells. The full-length 130 kDa MUC13- band that is used for quantification in (G) is marked by an arrow. (G) Quantification of protein expression of MUC13 (relative to uninfected control) in three biological replicates as depicted in F. All immunoblots used for analysis are available in Figure S3. Statistical analysis was performed on non-normalized data using a one-way ANOVA or Krustal-Wallis test (when the data was not normally distributed) with Dunnett’s post hoc test to compare each bacterial sample to the uninfected control. * p < 0.05. Bacteria are listed in Table 1. All graphs depict the SEM of three independent experiments.

Article Snippet: The cells were then transferred to the anaerobic chamber, washed twice with anaerobic DPBS, and infected with bacteria in DMEM medium without FCS at a MOI of 10 and 50 for 20 h at 37 °C in anaerobic Species Former name Strain Medium Reference Lactiplantibacillus plantarum Lactobacillus plantarum W1/ WCFS1 MRS Winclove B.V Lactoccocus lactis subspecies lactis W19 MRS Winclove B.V Lacticaseibacillus paracasei Lactobacillus paracasei W20 MRS Winclove B.V Bifidobacterium bifidum W23 DSMZ #58 Winclove B.V Bifidobacterium bifidum W28 DSMZ #58 Winclove B.V Lactobacillus acidophilus W37 MRS Winclove B.V Bifidobacterium animalis subspecies lactis W51 MRS Winclove B.V Bifidobacterium animalis subspecies lactis W53 MRS Winclove B.V Enterococcus faecium W54 MRS Winclove B.V Lactococcus lactis subspecies lactis W58 MRS Winclove B.V Lacticaseibacillus rhamnosus Lactobacillus rhamnosus W71 MRS Winclove B.V Lactobacillus helveticus W74 MRS Winclove B.V Limosilactobacillus reuteri Lactobacillus reuteri W192 MRS Winclove B.V Lactoccocus lactis subspecies cremoris W224 MRS Winclove B.V Bifidobacterium bifidum DSM 20,456 DSMZ #58 DMSZ Table 1.

Techniques: Western Blot, Modification, Expressing, Incubation, Control, Fluorescence, Confocal Microscopy, Staining, Bacteria

Journal: Scientific reports

Article Title: Probiotic Bifidobacterium bifidum strains desialylate MUC13 and increase intestinal epithelial barrier function.

doi: 10.1038/s41598-025-92125-2

Figure Lengend Snippet: 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.

Article Snippet: The cells were then transferred to the anaerobic chamber, washed twice with anaerobic DPBS, and infected with bacteria in DMEM medium without FCS at a MOI of 10 and 50 for 20 h at 37 °C in anaerobic Species Former name Strain Medium Reference Lactiplantibacillus plantarum Lactobacillus plantarum W1/ WCFS1 MRS Winclove B.V Lactoccocus lactis subspecies lactis W19 MRS Winclove B.V Lacticaseibacillus paracasei Lactobacillus paracasei W20 MRS Winclove B.V Bifidobacterium bifidum W23 DSMZ #58 Winclove B.V Bifidobacterium bifidum W28 DSMZ #58 Winclove B.V Lactobacillus acidophilus W37 MRS Winclove B.V Bifidobacterium animalis subspecies lactis W51 MRS Winclove B.V Bifidobacterium animalis subspecies lactis W53 MRS Winclove B.V Enterococcus faecium W54 MRS Winclove B.V Lactococcus lactis subspecies lactis W58 MRS Winclove B.V Lacticaseibacillus rhamnosus Lactobacillus rhamnosus W71 MRS Winclove B.V Lactobacillus helveticus W74 MRS Winclove B.V Limosilactobacillus reuteri Lactobacillus reuteri W192 MRS Winclove B.V Lactoccocus lactis subspecies cremoris W224 MRS Winclove B.V Bifidobacterium bifidum DSM 20,456 DSMZ #58 DMSZ Table 1.

Techniques: Activity Assay, Positive Control, Two Tailed Test, Glycoproteomics, Western Blot, Incubation, Immunoprecipitation, Generated, Bacteria

Journal: Scientific reports

Article Title: Probiotic Bifidobacterium bifidum strains desialylate MUC13 and increase intestinal epithelial barrier function.

doi: 10.1038/s41598-025-92125-2

Figure Lengend Snippet: Fig. 3. Adherence of Bifidobacterium bifidum strains to HRT18 monolayers and penetration of the secreted mucus layer in Caco-2 ALI/VIP cultures. (A) FISH staining in combination with confocal microscopy of UV-killed B. bifidum adhesion to HRT18 monolayers at MOI 10 and 50 for 20 h using a 16S PNA probe (green). White scale bars represent 20 µM. (B) Adhesion of B. bifidum W23, W28, and 20456 strains to HRT18 monolayers assessed by quantification of colony-forming units (CFUs). The graph represents the average and SEM of three independent experiments. Statistical test: one-way ANOVA with Tukey’s correction. (C) Schematic representation of Caco-2 cells cultured in Transwells under liquid–liquid interface (LLI) and air– liquid interface (ALI) with the basolateral addition of vasointestinal peptide (VIP). Caco-2 ALI/VIP cultures produce a secreted mucus layer on the apical surface41 (D) Fluorescence confocal microscopy images of Caco-2 cells grown under ALI/VIP conditions to induce mucus formation incubated with pre-stained W23, W28, 20456, and W1 bacteria (green) at MOI 50 for 4 h. The secreted mucus layer was stained with the lectin Jacalin (orange) and DAPI was used for the nuclei (blue). The middle of the image shows the maximal intensity projection of the mucus and bacterial stainings, while the orthogonal view depicts single planes for clarity. White scale bars represent 20 µM. (E) 3D rendering of images is shown in (B). (F) Immunoblot analysis of MUC13 modification in the Caco-2 ALI/VIP cultures after incubation with B. bifidum strains at MOI 50 for 4 h. All immunoblots used for quantification are available in Figure S6. (G) Quantification of protein abundance of the high and low MW MUC13 (relative to actin) as shown in (D). Graph depicts experimental results of three independent biological replicates. Statistical test: one-way ANOVA with Dunnett’s post hoc test. * p < 0.05.

Article Snippet: The cells were then transferred to the anaerobic chamber, washed twice with anaerobic DPBS, and infected with bacteria in DMEM medium without FCS at a MOI of 10 and 50 for 20 h at 37 °C in anaerobic Species Former name Strain Medium Reference Lactiplantibacillus plantarum Lactobacillus plantarum W1/ WCFS1 MRS Winclove B.V Lactoccocus lactis subspecies lactis W19 MRS Winclove B.V Lacticaseibacillus paracasei Lactobacillus paracasei W20 MRS Winclove B.V Bifidobacterium bifidum W23 DSMZ #58 Winclove B.V Bifidobacterium bifidum W28 DSMZ #58 Winclove B.V Lactobacillus acidophilus W37 MRS Winclove B.V Bifidobacterium animalis subspecies lactis W51 MRS Winclove B.V Bifidobacterium animalis subspecies lactis W53 MRS Winclove B.V Enterococcus faecium W54 MRS Winclove B.V Lactococcus lactis subspecies lactis W58 MRS Winclove B.V Lacticaseibacillus rhamnosus Lactobacillus rhamnosus W71 MRS Winclove B.V Lactobacillus helveticus W74 MRS Winclove B.V Limosilactobacillus reuteri Lactobacillus reuteri W192 MRS Winclove B.V Lactoccocus lactis subspecies cremoris W224 MRS Winclove B.V Bifidobacterium bifidum DSM 20,456 DSMZ #58 DMSZ Table 1.

Techniques: Staining, Confocal Microscopy, Cell Culture, Fluorescence, Incubation, Bacteria, Western Blot, Modification, Quantitative Proteomics

Journal: Scientific reports

Article Title: Probiotic Bifidobacterium bifidum strains desialylate MUC13 and increase intestinal epithelial barrier function.

doi: 10.1038/s41598-025-92125-2

Figure Lengend Snippet: Fig. 4. Bifidobacterium bifidum strains W23 and W28 enhance intestinal barrier properties. (A) Immunofluorescence confocal images of HRT18 cells grown in glass slides until full confluency and infected with B. bifidum strains W23, W28, and 20456 at MOI 10 for 20 h. Cells were stained for occludin (green), ZO-1 (red), and DAPI (white). The maximum intensity projection is depicted. White scale bars represent 20 µM. (B) Immunofluorescence images of HRT18 cultures grown in Transwells incubated with W23, W28, 20456, W1, and W37 at MOI 100 for 28 h. Cultures were stained for occludin (green), ZO-1 (red), and DAPI (white). Z-stacks were captured from the apical surface until the appearance of nuclei and maximum intensity projections are depicted. White scale bars represent 20 µM. (C) Immunoblot analysis of MUC13 modification and expression of claudins-1, -3, and -4 in HRT18 Transwell cultures after incubation with bacterial strains W23, W28, 20456, W1, and W37 at MOI 100 for 28 h and during a challenge with 100 ng/mL IL-1β and 100 ng/mL TNF-α. The control sample only received the cytokine challenge without bacteria. All immunoblots used for quantification are available in Figure S7. (D) Quantification of protein expression of MUC13 and claudins-1, -3, and -4 (relative to actin) as shown in (C). All graphs depict the SEM of three independent experiments. Statistical analysis was performed on non-normalized data using a one-way ANOVA with Dunnett’s post hoc test to compare each bacterial sample to the uninfected control. * p < 0.05. (E) TEER measurements of HRT18 Transwell cultures over time during incubation with W23, W28, 20456, W1, and W37 strains at MOI 100 and cytokine challenge at baseline as described for C. (F) TEER values at the 28 h timepoint depicted in E. Graphs depict SEM of three independent biological replicates. Statistical test: two-way ANOVA with Dunnett’s post hoc test. **** p < 0.0001.

Article Snippet: The cells were then transferred to the anaerobic chamber, washed twice with anaerobic DPBS, and infected with bacteria in DMEM medium without FCS at a MOI of 10 and 50 for 20 h at 37 °C in anaerobic Species Former name Strain Medium Reference Lactiplantibacillus plantarum Lactobacillus plantarum W1/ WCFS1 MRS Winclove B.V Lactoccocus lactis subspecies lactis W19 MRS Winclove B.V Lacticaseibacillus paracasei Lactobacillus paracasei W20 MRS Winclove B.V Bifidobacterium bifidum W23 DSMZ #58 Winclove B.V Bifidobacterium bifidum W28 DSMZ #58 Winclove B.V Lactobacillus acidophilus W37 MRS Winclove B.V Bifidobacterium animalis subspecies lactis W51 MRS Winclove B.V Bifidobacterium animalis subspecies lactis W53 MRS Winclove B.V Enterococcus faecium W54 MRS Winclove B.V Lactococcus lactis subspecies lactis W58 MRS Winclove B.V Lacticaseibacillus rhamnosus Lactobacillus rhamnosus W71 MRS Winclove B.V Lactobacillus helveticus W74 MRS Winclove B.V Limosilactobacillus reuteri Lactobacillus reuteri W192 MRS Winclove B.V Lactoccocus lactis subspecies cremoris W224 MRS Winclove B.V Bifidobacterium bifidum DSM 20,456 DSMZ #58 DMSZ Table 1.

Techniques: Immunofluorescence, Infection, Staining, Incubation, Western Blot, Modification, Expressing, Control, Bacteria