1. Local pH elevation mediated by the intrabacterial urease of Helicobacter pylori cocultured with gastric cells J. Clin. Invest. 106:339–347 (2000).

2. Introduction   The neutralophile Helicobacter pylori is a pathogen that resists gastric acidity and colonizes the human stomach. Helicobacter pylori is an etiologic agent of gastritis, gastric ulcer, and gastric cancer.   However, with increasing acidity, urease activity increases between 10- and 20-fold as the pH falls from 6.0 to 5.0, and thereafter remains steady down to pH 2.5   UreI, that encodes a H + gated urea channel, allowing an increase of urea permeability of the bacterial membrane by at least 300-fold as medium pH becomes acidic.

3. The action of urease in H.p © (2001) American Society for Microbiology.

4. Introduction   Helicobacter pylori will generate large quantities of NH 3 that converts to NH 4 + in gastric acid.   Alkalinization of the cytoplasm of many eukaryotic cells results in elevation of intracellular calcium and release of cytokines such as IL-1b   0.01% concentration of C 12 E 8 permeabilizes the inner membrane of the organism with resultant full urease activity, as urea penetration is no longer rate limiting.

5. Introduction   The work reported here used a coculture system with gastric-derived AGS cells and H. pylori.   Both confocal and video microscopy were used to define the site(s) at which intrabacterial urease has a buffering action.

6. Introduction   Ure-I, and all medium pH changes are completely inhibited by 10 mM flurofamide, a urease suicide inhibitor.   The generation of NH 3 at pH 5.5 had no effect on the intracellular pH of AGS cells to which the bacteria adhered until medium pH increased.

7. Coculture of AGS cells and bacteria Condition: AGS cells were washed with the growth media lacking antibiotics. Cover slips were transferred into a new six-well plate with each well containing 2 mL of the same medium. H. pylori was added at an OD 600 of 0.4 ( about 1×10 8 cfu/ml ) and cocultured for 1 hour in microaerophilic condition. Bacteria:cell concentration=100:1

9. BCECF: pH ↑, fluorescence ↑ Add 5mM urea after BCECF added

10. Wt Using pH electrode to estimate buffer pH change when 5mM urea adding Mt 1 mM flurofamide Mt Add 0.01% C 12 E 8 to Mt

11. BCECF-acid present to monitor peribacterial and medium pH changes. SNARF-AM at 10 mM was used to load the AGS cells at 37°C for 30 minutes and monitor intracellular pH changes as a function of urea addition at pH 5.5.

12. This cellular pH shift was accompanied by Ca 2+ release from intracellular stores (Figure 5). [Ca+2], Detection [Ca+2], ↑ BCECF-AM and Fura-2 AM were loaded into AGS cells and fluorescence measured with video microscopy Detection pH

13. Discussion 1. The neutral pH optimum urease is limited in its activity, at a neutral medium pH, by the slow pentration of urea into the organism, the major barrier being the inner membrane. 1. The neutral pH optimum urease is limited in its activity, at a neutral medium pH, by the slow pentration of urea into the organism, the major barrier being the inner membrane. 2. The periplasm is the first site at which an elevation of pH is expected with the addition of urea to wild-type organisms in acidic media. 2. The periplasm is the first site at which an elevation of pH is expected with the addition of urea to wild-type organisms in acidic media. 3. From these data, we had deduced that the elevation of membrane potential at acidic pH with urea addition was due to a rise in periplasmic pH. Here, this is directly demonstrated in the confocal images.

14. Discussion 4. In the wild-type organisms, but not in the mutants, the continuing efflux of NH 3 from the cytoplasm to the periplasm and then across the outer membrane should secondarily alkalinize the medium. 4. In the wild-type organisms, but not in the mutants, the continuing efflux of NH 3 from the cytoplasm to the periplasm and then across the outer membrane should secondarily alkalinize the medium. 5.In the Ure1-deletion mutants, it is predicted that a slow alkalization of the medium is expected. If the membrane barrier to urea is disrupted in the mutants,, the addition of urea should result in an even more rapid alkalization than even in wild-type organisms at acidic pH and should be seen first in the cytoplasm. 5.In the Ure1-deletion mutants, it is predicted that a slow alkalization of the medium is expected. If the membrane barrier to urea is disrupted in the mutants,, the addition of urea should result in an even more rapid alkalization than even in wild-type organisms at acidic pH and should be seen first in the cytoplasm.