Acid-dependent adherence of Helicobacter pylori urease to diverse polysaccharides  Faustino C. Icatlo, Hideo Goshima, Nobutake Kimura, Yoshikatsu Kodama  Gastroenterology  Volume 119, Issue 2, Pages 358-367 (August 2000) DOI: 10.1053/gast.2000.9372 Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 1 Acid-functional adherence of urease to eukaryotic cell membrane glycolipids. MKN45 cells were cultivated for 7 days using RPMI 1640 in Falcon tissue culture microplates. Biotinylated urease (2.5 μg/mL) or whole H. pylori cells (24-hour broth culture, optical density of 0.07 at 550 nm) was diluted or suspended in 20 mmol/L phosphate saline/0.05% Tween 20 at different pH levels and incubated in MKN45 cell for 1 hour at 37°C. After 5 washes with pH-matched buffers, cells were fixed at 65°C for 10 minutes, probed with streptavidin–horseradish peroxidase, and developed with o-phenylenediamine. Urease (○) bound to MKN45 gastric epithelial cells at pH 2.0–5.0 but failed to adhere at pH 5.5–7.0. Whole H. pylori cells (●) bound at all pH levels. Gastroenterology 2000 119, 358-367DOI: (10.1053/gast.2000.9372) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 2 pH-controlled adherence of urease to H. pylori surface structures. Immulon 2 plates were coated with purified OMP (5 μg/mL; ▴), LPS (10 μg/mL; ▵), or whole H. pylori cells (optical density of 0.07 at 550 nm) prewashed with either pH 4.0 (○) or pH 7.0 (●) phosphate buffer (0.1 mol/L). Biotinylated urease was diluted with 20 mmol/L phosphate saline/0.05% Tween 20 at different pH levels and incubated for 1 hour at 37°C on bacteria/OMP/LPS-coated wells. After 5 washes with pH-matched buffers, cells were fixed at 65°C for 10 minutes, probed with streptavidin–horseradish peroxidase, and developed with o-phenylenediamine. The general similarity in peak adherence by urease to all tested substrates suggests a common binding site on the bacterial outer membrane, particularly the exposed sugar-containing O-antigen of native LPS. The possible retention of other acid-dependent adherent structures on the surface of bacterial cells via washing with pH 4.0 buffer was not a critical factor in urease adherence because cells washed with pH 7.0 buffer gave a similar adherence curve. Gastroenterology 2000 119, 358-367DOI: (10.1053/gast.2000.9372) Copyright © 2000 American Gastroenterological Association Terms and Conditions