Journal: mBio

Article Title: Bifidobacterium bifidum Extracellular Sialidase Enhances Adhesion to the Mucosal Surface and Supports Carbohydrate Assimilation

doi: 10.1128/mBio.00928-17

Figure Lengend Snippet: Binding properties of recombinant sialidase peptides. (A-1) Schematic representation of the primary structure of SiaBb2 from B. bifidum ATCC 15696. Amino acid numbering starts at the presumed initiation codon. Sialidase and alpha-galactosidase NEW3 (A-galactosidase_NEW3) domains are depicted as light and dark gray boxes, respectively. The white box at the N terminus and the gridded box at the C terminus indicate a signal peptide and membrane anchor, respectively. (A-2) Schematic representation of SiaBb2-truncated peptides expressed as fusions to 6×His tag at the N terminus (green boxes). (B) Purified SiaBb2-truncated peptides (10 μg per lane) from B. bifidum ATCC 15696 were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and visualized by staining with Coomassie brilliant blue. The molecular mass standard is shown on the left. (C) Sialidase activity of SiaBb2-truncated proteins. Recombinant proteins (50 μM) were incubated with 4-MU-Neu5Ac, and fluorescence was recorded for 30 min. Data are presented as means ± standard deviations ( n = 5). (D) Binding of SiaBb2-truncated peptides to PCM, as determined by enzyme-linked immunosorbent assay. PCM-coated wells were incubated with 0.025 to 50 μM recombinant peptide. Binding was detected with an anti-His antibody. Data are presented as means ± standard deviations ( n = 3). (E) His-SiaBb2 binding to murine colonic tissue sections. Total glycoconjugates were detected by alcian blue staining. Biotinylated His-SiaBb2 specifically reacted with the mucosal surface at the border between the mucus layer and fecal material (black arrowhead) and with goblet cells (gray arrowhead). F, fecal material; M, mucus layer; G, goblet cells. Bars, 200 μm.

Article Snippet: Thus, SiaBb2 is the main determinant of the sialidase-associated phenotype of B. bifidum ATCC 15696 (i.e., carbohydrate assimilation and bacterial adhesion).

Techniques: Binding Assay, Recombinant, Membrane, Purification, Polyacrylamide Gel Electrophoresis, Staining, Activity Assay, Incubation, Fluorescence, Enzyme-linked Immunosorbent Assay

Journal: mBio

Article Title: Bifidobacterium bifidum Extracellular Sialidase Enhances Adhesion to the Mucosal Surface and Supports Carbohydrate Assimilation

doi: 10.1128/mBio.00928-17

Figure Lengend Snippet: SiaBb2 promotes B. bifidum ATCC 15696 degradation of sialyloligosaccharides. (A) Whole-cell lysates (10 μg protein) from the wild-type strain at different culture times (A-1) or from the siabb2 mutant ( Δsiabb2 ) or siabb2 complementation ( Δsiabb2 +p siabb2 ) strain cultured for 12 h (A-2) were analyzed by Western blotting. Reactivity with anti-SiaBb2 antibody is shown. (B) Surface expression of SiaBb2 in B. bifidum ATCC 15696 strains was detected by flow cytometry using an anti-SiaBb2 antibody after 12 h of culture. Preimmune (PI) serum was used for background control. MFI, mean fluorescence intensity. (C) Quantitative analysis of sialidase activity using 2′-(4-methylumbelliferyl)-α- d - N -acetylneuraminic acid (4-MU-Neu5Ac). B. bifidum (12-h culture) was cultured in the presence of 4-MU-Neu5Ac, and fluorescence was recorded for 30 min. Data represent means ± standard deviations ( n = 5). (D) Growth profiles of B. bifidum in mMRSCS medium supplemented with 1% (wt/vol) lactose, HMO, PMO, 6'SL, or acidic PMO as the sole carbon source. MRSCS medium was used as a positive control. Data represent means ± standard deviations ( n = 3). *, P < 0.05 (one-way analysis of variance followed by Tukey’s honestly significant difference post hoc test in pairwise comparisons of wild-type and Δ siabb2 strains at each time point).

Article Snippet: Thus, SiaBb2 is the main determinant of the sialidase-associated phenotype of B. bifidum ATCC 15696 (i.e., carbohydrate assimilation and bacterial adhesion).

Techniques: Mutagenesis, Cell Culture, Western Blot, Expressing, Flow Cytometry, Control, Fluorescence, Activity Assay, Positive Control

Journal: mBio

Article Title: Bifidobacterium bifidum Extracellular Sialidase Enhances Adhesion to the Mucosal Surface and Supports Carbohydrate Assimilation

doi: 10.1128/mBio.00928-17

Figure Lengend Snippet: SiaBb2 promotes bifidobacterial adhesion to the mucosal surface. (A) Adhesion of B. bifidum ATCC 15696 strains (wild type, Δsiabb2 , and Δsiabb2 +p siabb2 ) to PCM (A-1) or HT29-MTX-E12 epithelial cells (A-2). Bacterial strains (12-h culture) were grown in MRSCS broth. (A-1) Adhesion of B. bifidum to PCM as assayed using diagnostic glass slides. Bound bacteria were stained with methylene blue, and randomly selected fields were imaged with a microscope. (A-2) Cells were added to monolayers of epithelial cells. Adherent bacteria were plated on MRSCS agar and counted. (B) Effect of anti-SiaBb2 antibody on the adhesion of wild-type B. bifidum ATCC 15696 cells to PCM (B-1) or HT29-MTX-E12 epithelial cells (B-2). Bacteria were incubated with anti-SiaBb2 antibody or rabbit preimmune serum (1:100 dilution). (C) Analysis of sialidase activity. B. longum subsp. longum 105-A strains (105-A wild type, pKKT427-Cm r - siabb2 -transformed strain [105-A p siabb2 ], and empty vector control [105-A pEmpty]) were incubated with 4-MU-Neu5Ac, and fluorescence was recorded for 30 min. Data are presented as means ± standard deviations ( n = 4). (D) Cell surface expression of SiaBb2 of B. longum subsp. longum 105-A strains was confirmed by flow cytometry using an anti-SiaBb2 antibody. (E) Adhesion of B. longum subsp. longum 105-A strains to PCM. (F) Effect of anti-SiaBb2 antibody or rabbit preimmune serum on adhesion of B. longum subsp. longum 105-A strains to PCM. (A, B, E, and F) Data are presented as means ± standard deviations ( n = 5). P < 0.05 (one-way analysis of variance followed by Tukey’s honestly significant difference post hoc test in pairwise comparisons among strains). n.s., nonsignificant.

Article Snippet: Thus, SiaBb2 is the main determinant of the sialidase-associated phenotype of B. bifidum ATCC 15696 (i.e., carbohydrate assimilation and bacterial adhesion).

Techniques: Diagnostic Assay, Bacteria, Staining, Microscopy, Incubation, Activity Assay, Transformation Assay, Plasmid Preparation, Control, Fluorescence, Expressing, Flow Cytometry

Journal: mBio

Article Title: Bifidobacterium bifidum Extracellular Sialidase Enhances Adhesion to the Mucosal Surface and Supports Carbohydrate Assimilation

doi: 10.1128/mBio.00928-17

Figure Lengend Snippet: Novel functions of extracellular sialidase SiaBb2 in B. bifidum ATCC 15696. SiaBb2 enhances adhesion to the mucosal surface and supports assimilation of sialylated HMOs and intestinal mucin oligosaccharides by bifidobacteria.

Article Snippet: Thus, SiaBb2 is the main determinant of the sialidase-associated phenotype of B. bifidum ATCC 15696 (i.e., carbohydrate assimilation and bacterial adhesion).

Techniques: