Local pH elevation mediated by the intrabacterial urease of Helicobacter pylori cocultured with gastric cells

Athmann, C.; Zeng, Ningxin; Tai, Kang; Marcus, Elizabeth A.; Scott, David; Rektorschek, Marina; Buhmann, Anita; Melchers, Klaus; Sachs, George

doi:10.1172/jci9351

Cited by 53 publications

(47 citation statements)

References 38 publications

(47 reference statements)

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“…It was also noted in these images that the bacteria themselves became brighter as time went on suggesting that some of the BCECF passes through the cell wall into the interior of the bacterium. This observation may be consistent with the periplasmic pH elevation reported by Athmann et al in confocal microscopy studies (53).…”

Section: Discussionsupporting

confidence: 93%

Helicobacter pylori moves through mucus by reducing mucin viscoelasticity

Celli

Turner

Afdhal

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

378

326

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The ulcer-causing gastric pathogen Helicobacter pylori is the only bacterium known to colonize the harsh acidic environment of the human stomach. H. pylori survives in acidic conditions by producing urease, which catalyzes hydrolysis of urea to yield ammonia thus elevating the pH of its environment. However, the manner in which H. pylori is able to swim through the viscoelastic mucus gel that coats the stomach wall remains poorly understood. Previous rheology studies on gastric mucin, the key viscoelastic component of gastric mucus, indicate that the rheology of this material is pH dependent, transitioning from a viscous solution at neutral pH to a gel in acidic conditions. Bulk rheology measurements on porcine gastric mucin (PGM) show that pH elevation by H. pylori induces a dramatic decrease in viscoelastic moduli. Microscopy studies of the motility of H. pylori in gastric mucin at acidic and neutral pH in the absence of urea show that the bacteria swim freely at high pH, and are strongly constrained at low pH. By using two-photon fluorescence microscopy to image the bacterial motility in an initially low pH mucin gel with urea present we show that the gain of translational motility by bacteria is directly correlated with a rise in pH indicated by 2 ,7 -Bis-(2-Carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF), a pH sensitive fluorescent dye. This study indicates that the helicoidal-shaped H. pylori does not bore its way through the mucus gel like a screw through a cork as has previously been suggested, but instead achieves motility by altering the rheological properties of its environment.H. pylori ͉ bacterial motility ͉ rheology ͉ pH ͉ gelation

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Section: Discussionsupporting

confidence: 93%

Helicobacter pylori moves through mucus by reducing mucin viscoelasticity

Celli

Turner

Afdhal

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

378

326

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“…The measurement of cytoplasmic pH and inner membrane potential as well as visualization of the increase of pH in the periplasmic space showed that increased urease activity was able to elevate periplasmic pH (4,36,44,45).…”

Section: Discussionmentioning

confidence: 99%

“…The width of the periplasm is Յ0.1 m, and it is conceivable that the rate of NH 3 diffusion (ϳ2.0 ϫ 10 Ϫ5 cm 2 /s) across the narrow space of the periplasm and out through the outer membrane is so fast that adequate generation of NH 4 ϩ does not occur in the periplasm. Further, NH 3 efflux would be able to elevate the periplasmic pH only if acid entry did not exceed NH 3 production.…”

Section: Discussionmentioning

confidence: 99%

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The Periplasmic α-Carbonic Anhydrase Activity of Helicobacter pylori Is Essential for Acid Acclimation

et al. 2005

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The role of the periplasmic ␣-carbonic anhydrase (␣-CA) (HP1186) in acid acclimation of Helicobacter pylori was investigated. Urease and urea influx through UreI have been shown to be essential for gastric colonization and for acid survival in vitro. Intrabacterial urease generation of NH 3 has a major role in regulation of periplasmic pH and inner membrane potential under acidic conditions, allowing adequate bioenergetics for survival and growth. Since ␣-CA catalyzes the conversion of CO 2 to HCO 3 ؊ , the role of CO 2 in periplasmic buffering was studied using an ␣-CA deletion mutant and the CA inhibitor acetazolamide. Western analysis confirmed that ␣-CA was bound to the inner membrane. Immunoblots and PCR confirmed the absence of the enzyme and the gene in the ␣-CA knockout. In the mutant or in the presence of acetazolamide, there was an ϳ3 log 10 decrease in acid survival. In acid, absence of ␣-CA activity decreased membrane integrity, as observed using membrane-permeant and -impermeant fluorescent DNA dyes. The increase in membrane potential and cytoplasmic buffering following urea addition to wild-type organisms in acid was absent in the ␣-CA knockout mutant and in the presence of acetazolamide, although UreI and urease remained fully functional. At low pH, the elevation of cytoplasmic and periplasmic pH with urea was abolished in the absence of ␣-CA activity. Hence, buffering of the periplasm to a pH consistent with viability depends not only on NH 3 efflux from the cytoplasm but also on the conversion of CO 2 , produced by urease, to HCO 3 ؊ by the periplasmic ␣-CA.

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“…nuclear factor-B THE GASTRIC MUCOSA IS FREQUENTLY infected with the gram-negative bacterium Helicobacter pylori, colonizing the antral part of the gastric epithelium in which the pH is elevated to 3-4, which allows optimal growth (7). Previous studies have revealed that urease activity of H. pylori depends on urea uptake through a specific channel, which is open at pH values between 3 and 6 but closes at levels Ͼ7 (6,19,28). During long-term infection as well as long-term therapy with proton pump inhibitors, the infection may also proceed to the gastric corpus, resulting in atrophy of gastric glands, hypochlorhydria, and possibly the development of gastric adenocarcinoma (14,32,33).…”

mentioning

confidence: 99%

Helicobacter pyloriinduces apoptosis of rat gastric parietal cells

Neu¹,

Randlkofer²,

Neuhofer³

et al. 2002

American Journal of Physiology-Gastrointestinal and Liver Physi

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Gastric Helicobacter pylori infection may lead to multifocal atrophic corpus gastritis associated with loss of epithelial cells as well as glandular structures. The current work investigated H. pylori effects on cell death of isolated, nontransformed rat parietal cells (PC). Highly enriched rat PC (>97%) were isolated from gastric mucosa and cultured in serum-free medium over 24 h. The cells were cocultured over 8 h with cytotoxin-associated immunodominant protein (cagA)+/vacuolating toxin (vacA)+ or with cagA−/vacA− H. pylori laboratory strains and also with H. pylori mutants deleted in several genes of the cag pathogenicity island. Staphylococcus aureus or Campylobacter jejuni were used as controls. Apoptosis was determined by terminal deoxynucleotidyl transferase dUTP nick-end labeling staining and electron microscopy. Interleukin (IL)-8 and cytokine-induced neutrophil chemoattractant (CINC)-1 secretion was measured by ELISA. Activation of nuclear factor-κB (NF-κB) was studied in nuclear extracts of PC by electrophoretic mobility shift assay. Apoptosis of PC was induced in a concentration- and time-dependent manner by cagA+/vacA+ H. pylori strains but not by cagA−/vacA−negative strains or by the cagE knockout mutant. S. aureusand C. jejuni had no effect. PC showed no IL-8 or CINC-1 secretion on exposure to cagA+/vacA+ H. pylori. cagA+/vacA+ strains induced activation of NF-κB complexes in nuclear extracts of PC, which were composed of p65 and p50 subunits. No significant stimulation of NF-κB activation was detected by incubation of PC with the cagE knockout mutant. Preincubation of PC with antisense but not missense oligodeoxynucleotides against the p65 subunit significantly reduced DNA binding to the κB recognition sequence. The p65 oligonucleotides as well as the proteasome inhibitor N-CBZ-isoleucin-glutamin-(o-t-butyl-)-alanin-leucin and the nitric oxide synthase inhibitor N G-monomethyl-l-arginine completely prevented PC apoptosis induced by cagA+/vacA+ strains. In summary, cagE presence appears to be essential for H. pylori-induced apoptosis of gastric parietal cells, and this effect is dependent on the activation of NF-κB and production of nitric oxide.

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Local pH elevation mediated by the intrabacterial urease of Helicobacter pylori cocultured with gastric cells

Cited by 53 publications

References 38 publications

Helicobacter pylori moves through mucus by reducing mucin viscoelasticity

Helicobacter pylori moves through mucus by reducing mucin viscoelasticity

The Periplasmic α-Carbonic Anhydrase Activity of Helicobacter pylori Is Essential for Acid Acclimation

Helicobacter pyloriinduces apoptosis of rat gastric parietal cells

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