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, 106 (3), 339-47

Local pH Elevation Mediated by the Intrabacterial Urease of Helicobacter Pylori Cocultured With Gastric Cells

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Local pH Elevation Mediated by the Intrabacterial Urease of Helicobacter Pylori Cocultured With Gastric Cells

C Athmann et al. J Clin Invest.

Abstract

Helicobacter pylori resists gastric acidity by modulating the proton-gated urea channel UreI, allowing for pH(out)-dependent regulation of urea access to intrabacterial urease. We employed pH- and Ca(2+)-sensitive fluorescent dyes and confocal microscopy to determine the location, rate, and magnitude of pH changes in an H. pylori-AGS cell coculture model, comparing wild-type bacteria with nonpolar ureI-deletion strains (ureI-ve). Addition of urea at pH 5.5 to the coculture resulted first in elevation of bacterial periplasmic pH, followed by an increase of medium pH and then pH in AGS cells. No change in periplasmic pH occurred in ureI-deletion mutants, which also induced a slower increase in the pH of the medium. Pretreatment of the mutant bacteria with the detergent C(12)E(8) before adding urea resulted in rapid elevation of bacterial cytoplasmic pH and medium pH. UreI-dependent NH(3) generation by intrabacterial urease buffers the bacterial periplasm, enabling acid resistance at the low urea concentrations found in gastric juice. Perfusion of AGS cells with urea-containing medium from coculture at pH 5.5 did not elevate pH(in) or [Ca(2+)](in), unless the conditioned medium was first neutralized to elevate the NH(3)/NH(4)(+) ratio. Therefore, cellular effects of intrabacterial ammonia generation under acidic conditions are indirect and not through a type IV secretory complex. The pH(in) and [Ca(2+)](in) elevation that causes the NH(3)/NH(4)(+) ratio to increase after neutralization of infected gastric juice may contribute to the gastritis seen with H. pylori infection.

Figures

Figure 1
Figure 1
Localization of H. pylori in coculture with AGS cells. (a) AGS cells were stained with the dye DiSC3(5) after 1-hour coculture with H. pylori. BCECF-free acid was used to illuminate the bacteria after addition of urea. Both dyes were monitored simultaneously using confocal microscopy. ×63. (b) These images show a perpendicular cut through the same sample as panel a in the X and in the Y plane as shown by the corresponding lines of a. H. pylori are located primarily just above the regions of contact between the AGS cells. (c) A higher magnification view to illustrate that the peribacterial region of individual organisms shows the change in BCECF fluorescence. ×100. The bars correspond to 3 or 10 μm as defined by the confocal software.
Figure 2
Figure 2
Localization of pH elevation with urea addition at pHout 5.5. H. pylori cocultured with AGS cells were perfused with medium containing BCECF-free acid and then 5 mM urea added as detailed in the text. Changes of fluorescence were then observed as a function of time using confocal microscopy at ×100 optical magnification and ×3 or ×10 electronic magnification. (a) The addition of 5 mM urea initially resulted in an increase of BCECF fluorescence clearly visible at the periphery of the wild-type organisms. (b) Higher magnification of a single wild-type organism emphasizing the peripheral increase of BCECF fluorescence obtained with urea addition. This restricted region of pH elevation is the periplasm. (c) Pretreatment with 0.01% C12E8 resulted in bacterial cytoplasmic alkalinization after the addition of urea to wild-type organisms. (d) Addition of 5 mM urea resulted in little or no change of fluorescence (pH) in the periplasm of the H. pylori ureI-ve mutant with the addition of 5 mM urea. (e) Pretreatment of the ureI-ve mutants with 0.01% C12E8 followed by urea addition increased cytoplasmic pH as monitored by the rise in BCECF fluorescence within the organism. The arrow points to a region where the image is electronically magnified to show the internal, compared with surface, fluorescence. Each bar corresponds to 3 μm as defined by the confocal software.
Figure 3
Figure 3
Bacterial and medium pH effects of urea addition to H. pylori-AGS coculture with either wild-type or ureI mutant strains. (a and b) The effect of urea addition on medium and bacterial pH in coculture with H. pylori expressing UreI. H. pylori and AGS cells in coculture were perfused with medium containing BCECF-free acid at the indicated pH and then 5 mM urea was added. A calibrated pH microelectrode was placed in the chamber to monitor medium pH and confocal microscopy was used to monitor the regions of change of pH after the addition of 5 mM urea using wild-type or ureI-deletion mutants as detailed in Methods. The numbering in the confocal images corresponds in time to the images above the pH tracings. (a) At a medium pH of 5.5, there was a rapid rise of medium pH after the addition of 5 mM urea. Within 2 minutes, the pH had risen to 6.0 and reached a steady-state pH of about 7.0 within 5 minutes. In the image sequence above the pH curve from the same experiment, the fluorescence increased markedly first on or at the bacteria, evident in the first two or three images. The increase in fluorescence then spread into the medium as medium pH increased and is close to maximum at about 4 minutes (see Table 1). (b) In contrast, when urea is added at 5 mM at a medium pH of 7.4, there was little change of medium pH or the pH around the organism. After 20 minutes, the pH had only increased by 0.2 U. (c and d) The effect of 5 mM urea addition on medium and bacterial pH in coculture with H. pylori ureI–negative mutants. (c) When urea was added at pH 5.5 to the ureI-ve strain, there was a slow rise of medium pH, reaching about 6.5 after 5 minutes and then rising slowly to 7.2 at the end of the experiments. There was no obvious change of fluorescence with the addition of urea in the vicinity of the organisms, although they have considerable surface urease activity (11). (d) Urea addition in the presence of 0.01% C12E8 to the ureI-deletion mutant resulted in a rapid change in medium pH, faster than that seen with the wild-type organisms, reaching pH 7.1 within 5 minutes and continuing to increase to more than 8.0, in contrast to the untreated wild-type organisms. In the fluorescence images, there was a similar increase in the fluorescence of the medium reflecting the pH changes monitored with the pH electrode. (e) When 5 mM urea was added in the presence of 10 μM flurofamide at pH 5.5, no change in either bacterial or medium pH could be observed, showing the dependence of these changes on urease activity.
Figure 4
Figure 4
Time course of the effect of NH3 generation on peribacterial, medium, and AGS cell pH. SNARF-AM was used to monitor the effect of 5 mM urea addition to the pHin of the AGS cells in bacterial coculture with BCECF-acid present to monitor peribacterial and medium pH changes. Fluorescence changes were analyzed quantitatively on the bacteria, in the medium and in the cells in regions of interest defined by the confocal software (confocal images, rectangles) and displayed as a time course of fluorescence (graphs below). Three time points are illustrated in the confocal images corresponding to the time course shown in the time plot underneath. There was first a large change in periplasmic pH followed by an increase of medium pH. No change was observed in the AGS cells until medium pH had risen. The earliest fluorescence change was seen over the bacteria, and then the increase spread into the medium and then into the AGS cells (see Table 1).
Figure 5
Figure 5
The effect of coculture perfusate on pHin and [Ca2+]in of AGS cells. These experiments were designed to test the effects of ammonia generated by intrabacterial urease on the AGS cells cocultured with H. pylori either at acidic or neutral pH. Bacteria and cells in coculture were perfused with 5 mM urea at pH 7.4 or 5.5 for 30 minutes, as illustrated in the diagram above the graphs, which shows the protocol for pH 5.5 perfusion without or with 10 μM flurofamide (also see Methods). The AGS cells were loaded in the microscopy chamber with BCECF-AM for pHin measurement or Fura 2-AM for [Ca2+]in measurements. The coculture was reperfused with the conditioned perfusate generated at pH 7.4 or 5.5 either before (2 curves on left) or after (2 curves on right) adjusting the pH to 7.4 with NaOH, and changes of pHin and [Ca2+]in were measured. The perfusion using conditioned medium at acidic pH resulted in no change in cell pH or calcium, whereas if the medium from the coculture was first neutralized to pH 7.4 with NaOH, a robust increase in intracellular pH or a transient increase in intracellular calcium was observed. The presence of flurofamide in the initial perfusion prevented any changes from being observed in the reperfusion at pH 7.4 showing that the effect observed at pH 7.4 reperfusion was due to the urease activity present during the pH 5.5 initial perfusion.

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