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Local pH elevation mediated by the intrabacterial urease of Helicobacter pylo
ri cocultured with gastric cells
J. Clin. Invest. 106:339–347 (2000).
IntroductionIntroduction 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.
The action of urease in The action of urease in H.pH.p
© (2001) American Society for Microbiology.
IntroductionIntroduction Helicobacter pylori will generate large quantities of N
H3 that converts to NH4+ 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 C12E8 permeabilizes the inner membrane of the organism with resultant full urease activity , as urea penetration is no longer rate limiting.
IntroductionIntroduction The work reported here used a coculture system wit
h 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.
IntroductionIntroduction
Ure-I, and all medium pH changes are completely inhibited by 10 mM flurofamide, a urease suicide inhibitor.
The generation of NH3 at pH 5.5 had no effect on the intracellular pH of AGS cells to which the bacteria adhered until medium pH increased.
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 OD6
00 of 0.4 ( about 1×108cfu/ml ) and cocultured for 1 hour in microaerophilic condition.
Bacteria:cell concentration=100:1
BCECF: pH ↑, fluorescence ↑
Add 5mM urea after BCECF added
Wt
Using pH electrode to estimate buffer pH change when 5mM urea adding
Mt
1 mM flurofamide
MtAdd 0.01% C12E8 to Mt
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.
This cellular pH shift wasaccompanied by Ca2+ release from intracellular stores(Figure 5).
Detection [Ca+2] ,[Ca+2] , ↑
BCECF-AM and Fura-2 AM were loaded into AGS cells and fluorescence measuredwith video microscopy
Detection pH
DiscussionDiscussion
1. The neutral pH optimum urease is limited in its activity, at a 1. The neutral pH optimum urease is limited in its activity, at a neutral medium pH, by the slow pentration of urea into the orneutral medium pH, by the slow pentration of urea into the organism, the major barrier being the inner membrane.ganism, the major barrier being the inner membrane.
2. The 2. The periplasmperiplasm is the first site at which an elevation of pH is e is the first site at which an elevation of pH is e
xpected with the addition of urea to wild-type organisms in acxpected with the addition of urea to wild-type organisms in acidic media.idic 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.
DiscussionDiscussion
4.4.In the wild-type organisms, but not in the mutants, the continuIn the wild-type organisms, but not in the mutants, the continuing efflux of NHing efflux of NH33 from the cytoplasm to the periplasm and th from the cytoplasm to the periplasm and th
en across the outer membrane should secondarily alkalinize then across the outer membrane should secondarily alkalinize the medium.e medium.
5.In the 5.In the Ure1-deletion mutantsUre1-deletion mutants, it is predicted that a slow alkali, it is predicted that a slow alkalization of the medium is expected. If the membrane barrier to zation of the medium is expected. If the membrane barrier to urea is disrupted in the mutants,, the addition of urea should rurea is disrupted in the mutants,, the addition of urea should result in an even more rapid alkalization than even in wild-typesult in an even more rapid alkalization than even in wild-type organisms at acidic pH and should be seen first in the e organisms at acidic pH and should be seen first in the cytcytoplasm.oplasm.