Volume 135, Issue 6, Pages 2043-2054.e2 (December 2008) Helicobacter pylori Lipopolysaccharide Interacts With TFF1 in a pH-Dependent Manner  Emer P. Reeves, Tehmeena Ali, Paul Leonard, Stephen Hearty, Richard O'Kennedy, Felicity E.B. May, Bruce R. Westley, Christine Josenhans, Melanie Rust, Sebastian Suerbaum, Angeline Smith, Brendan Drumm, Marguerite Clyne  Gastroenterology  Volume 135, Issue 6, Pages 2043-2054.e2 (December 2008) DOI: 10.1053/j.gastro.2008.08.049 Copyright © 2008 AGA Institute Terms and Conditions

Figure 1 The identification of H pylori adhesins that bind TFF1. (A) Inhibition of H pylori binding to TFF1 dimer immobilized on a chip surface in the presence of TFF1 dimer, glycosylated TFF2, and TFF3 dimer. Binding was measured by surface plasmon resonance. The binding response at each protein concentration was divided by the cell binding response determined in the absence of TFF (R0) to give a normalized binding response (R/R0). (B) Bacterial whole cell lysates of H pylori strains PU4 and N6 were electrophoresed on a polyacrylamide gel and stained with Coomassie blue or transferred to PVDF and probed with recombinant biotinylated TFF1 dimer and glycosylated TFF2 or TFF3 dimer. (C) Subcellular fractions of H pylori strain N6 (bacterial cytosol, inner membranes, outer membrane proteins, and flagella) were electrophoresed on a polyacrylamide gel and stained with Coomassie blue. (D) The subcellular distribution of the bacterial adhesin was determined after transfer to PVDF membrane and incubation with recombinant biotinylated TFF1 dimer. Gastroenterology 2008 135, 2043-2054.e2DOI: (10.1053/j.gastro.2008.08.049) Copyright © 2008 AGA Institute Terms and Conditions

Figure 2 Characterization of the H pylori adhesin. Outer membrane proteins (OMP) derived from strain N6 were treated with sodium metaperiodate (SM) or proteinase K (PK), separated by polyacrylamide gel electrophoresis, and stained with Alcian blue or transferred to PVDF membrane and incubated with TFF1 dimer. Gastroenterology 2008 135, 2043-2054.e2DOI: (10.1053/j.gastro.2008.08.049) Copyright © 2008 AGA Institute Terms and Conditions

Figure 3 TFF1 dimer interact with H pylori LPS. (A) H pylori LPS electrophoresed on a polyacrylamide gel and stained with (1) alcian blue. LPS was transferred to PVDF membrane and incubated with (2) TFF1 dimer or antibodies to (3) Lex or (4) Ley. (B) Surface plasmon resonance analysis of purified LPS binding to immobilized TFF1 dimer. (C) Direct interaction between TFF1 and purified LPS. TFF1 dimer (0.25 μg) was incubated with different amounts of purified LPS (0.00 μg, 0.15 μg, and 1.5 μg), electrophoresed on a nondenaturing gel, and stained with Coomassie blue or transferred to PVDF membrane and incubated with a monoclonal antibody against TFF1. The position of the TFF1 dimer and the TFF1/LPS complex are indicated by an arrow and arrowheads, respectively. Gastroenterology 2008 135, 2043-2054.e2DOI: (10.1053/j.gastro.2008.08.049) Copyright © 2008 AGA Institute Terms and Conditions

Figure 4 Characterization of the interaction between H pylori LPS and TFF1 dimer. (A) Overlayed representative raw SPR binding sensograms comparing TFF-binding propensity of wild-type bacteria (N6) with that of H pylori mutant strains. The mean (n = 3) reference-subtracted binding responses are indicated in brackets. (B and C) LPS from H pylori strain N6 or from isogenic mutants was electrophoresed on a polyacrylamide gel and stained with (B) Alcian blue or (C) transferred to PVDF membrane and probed with TFF1 dimer. Representative results from one of 3 separate experiments are shown. Gastroenterology 2008 135, 2043-2054.e2DOI: (10.1053/j.gastro.2008.08.049) Copyright © 2008 AGA Institute Terms and Conditions

Figure 5 The effect of glycosidases and specific monosaccharides on the interaction of H pylori LPS with TFF1 dimer. (A) LPS was treated with fucosidase, mannosidase, glucosidase, or galactosidase at various concentrations, transferred to PVDF membrane, and incubated with TFF1 dimer. (B) The binding of TFF1 to oligosaccharides was tested using neutral oligosaccharides conjugated to HSA. D-Fucose, D-mannose-, D-glucose-β-PAP-HSA and HSA immobilized on PVDF membrane and incubated with recombinant TFF1 dimer. (C) Inhibition of TFF1 binding to LPS by monosaccharides was tested by pre-incubating various monosaccharides (1 mol/L) with recombinant TFF1 dimer before its incubation with immobilized LPS. Antibodies specific for TFF1 were used to identify levels of bound protein. Immunoreactive bands were quantified by densitometry. Gastroenterology 2008 135, 2043-2054.e2DOI: (10.1053/j.gastro.2008.08.049) Copyright © 2008 AGA Institute Terms and Conditions

Figure 6 Binding of TFF1 dimer and TFF3 dimer to H pylori RF-LPS is pH sensitive. (A) Binding of TFF1 dimer or (B) binding of TFF3 dimer to PVDF-immobilized LPS was assessed over a pH range of 3.0–7.0. Antibodies specific for TFF1 or TFF3 were used to identify levels of bound protein (insets). Immunoreactive bands were quantitated by densitometry. (C and D) Binding of bovine serum albumin (BSA)- or RF-LPS–coated latex beads to (C) TFF1 dimer or (D) TFF3 dimer over the pH range 4–7. The percentage of beads that were positive for bound TFF1 or TFF3 is shown in each plot. Representative results from one of 3 separate experiments are shown. Gastroenterology 2008 135, 2043-2054.e2DOI: (10.1053/j.gastro.2008.08.049) Copyright © 2008 AGA Institute Terms and Conditions

Figure 7 Binding of H pylori to TFF1 dimer in the presence of mucus gel. Bacteria were incubated in a mucus gel solution containing TFF1 dimer and immunopurified with anti-H pylori antibody conjugated to magnetic beads. Fluorescently labeled bacteria (red; A) stained with TFF1 antibody (green; B) are clearly visible adherent to the magnetic beads. (Original magnification 600×.) Arrows indicate coincidental objects of note. Gastroenterology 2008 135, 2043-2054.e2DOI: (10.1053/j.gastro.2008.08.049) Copyright © 2008 AGA Institute Terms and Conditions

Figure 8 LPS interacts with gastric tissue in a TFF1-dependent manner. (A) LPS-coated fluorescein isothiocyanate latex beads incubated with gastric biopsy tissue localized to the surface mucous cells and the gastric pits. (B) A reduction in adherence of LPS-coated fluorescein isothiocyanate latex beads to the surface mucous cells was observed following preincubation of beads with TFF1 dimer. (Original magnification 400×.) Gastroenterology 2008 135, 2043-2054.e2DOI: (10.1053/j.gastro.2008.08.049) Copyright © 2008 AGA Institute Terms and Conditions