Mechanisms of acid resistance due to the urease system of Helicobacter pylori  David R. Scott, Elizabeth A. Marcus, David L. Weeks, George Sachs  Gastroenterology  Volume 123, Issue 1, Pages 187-195 (July 2002) DOI: 10.1053/gast.2002.34218 Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 1 Urease immunofluorescent labeling of H. pylori. Localization of urease in H. pylori is shown in A (methanol fixed, membrane permeabilized) and in B (unfixed, membrane intact) by using UreA antibodies. SYTO 9 staining of H. pylori in C (methanol fixed) and D (unfixed) allowed identification of individual organisms. The absence of surface-bound fluorescence in the unfixed bacteria indicates the lack of surface-bound urease. Therefore, because surface-bound urease does not exist, it cannot contribute to the acid resistance of H. pylori. The average intensity per organism of the fixed and unfixed conditions was 1946.9 ± 47.4 (n = 200) and 3.5 ± 0.5 (n = 200), respectively. Gastroenterology 2002 123, 187-195DOI: (10.1053/gast.2002.34218) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 2 Urease activity of H. pylori preincubated at pH 5.5 and 7.3. H. pylori was incubated at either pH 5.5 (●) or pH 7.3 (○) for the times indicated. Urease activity was determined at pH 7.0 in the presence of the nonionic detergent C12E8 to allow urea access to intrabacterial urease. Acid-incubated H. pylori total urease activity increased 3–4-fold, whereas the urease activity of H. pylori incubated under neutral pH conditions remained unchanged. Urease activity is expressed as the percentage of activity at time 0. Gastroenterology 2002 123, 187-195DOI: (10.1053/gast.2002.34218) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 3 Activity of H. pylori urease incubated at pH 5.5 and 7.3. H. pylori was incubated at either pH 5.5 (■) or pH 7.3 (□) for 180 minutes. Urease activity was determined at pH 7.0 in the presence of the nonionic detergent C12E8 to allow urea access to intrabacterial urease. Chloramphenicol (CP) was used to inhibit protein synthesis; dimethyl glyoxime was used to remove nickel from the medium and the bacteria. These data show that the increase in urease activity seen in H. pylori incubated at pH 5.5 does not require de novo synthesis of enzyme or urea hydrolysis but is nickel and UreI dependent, suggesting that there is an increase in the conversion of apoenzyme to active enzyme at acidic medium pH. Urease activity is expressed as the percentage of activity at time 0. WT, wild-type. Gastroenterology 2002 123, 187-195DOI: (10.1053/gast.2002.34218) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 4 Western blot analysis of UreA, UreB, UreE, and UreI of H. pylori. H. pylori were incubated at pH 5.5 or pH 7.3 for 180 minutes. H. pylori total protein was size-fractionated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis on 10%–20% tricine gradient gels and transferred onto nitrocellulose. Western blot analysis was performed with 4 different antibodies: anti-UreA (1:100,000 dilution), anti-UreB (1:10,000), anti-UreI (1:2,000), and anti-UreE (1:500). No difference in the amount of protein was detected between bacteria incubated at pH 5.5 and 7.3. These data show that the increase in urease activity of acid-preincubated H. pylori is not due to an increase in the protein levels of the structural subunits or 2 of the accessory genes. Gastroenterology 2002 123, 187-195DOI: (10.1053/gast.2002.34218) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 5 Acidic medium pH activation of urease of intact H. pylori preincubated at pH 5.5 or pH 8.0. H. pylori was incubated in HPB at either pH 5.5 (■) or pH 8.0 (□) for 180 minutes. Urease activity of the intact organism was determined at medium pH 5.0 and pH 7.0. UreI-dependent activation of urease in acid-preincubated organisms resulted in a 2.5-fold higher activation as compared with organisms preincubated at neutral pH, consistent with the higher levels of intrabacterial urease found in these organisms. Gastroenterology 2002 123, 187-195DOI: (10.1053/gast.2002.34218) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 6 Survival of H. pylori at pH 2.5 for 30 minutes. H. pylori were incubated at either pH 5.0 or pH 7.3 for 90 minutes, followed by incubation at pH 2.5 for 30 minutes in the presence of 5 mmol/L of urea. These data correlate with the levels of urease activity seen at pH 5.5 and 7.3, showing the functional role of the increase in active urease at pH 5.5 for acid resistance. Survival was determined by colony counting, and the results are expressed as the percentage of survival before the pH 2.5 challenge for each preincubation pH. Gastroenterology 2002 123, 187-195DOI: (10.1053/gast.2002.34218) Copyright © 2002 American Gastroenterological Association Terms and Conditions