Acid resistance of Helicobacter pylori depends on the UreI membrane protein and an inner membrane proton barrier

ureI encodes an inner membrane protein of Helicobacter pylori. The role of the bacterial inner membrane and UreI in acid protection and regulation of cytoplasmic urease activity in the gastric microorganism was studied. The irreversible inhibition of urease when the organism was exposed to a protonophore (3,3',4', 5-tetrachlorsalicylanide; TCS) at acidic pH showed that the inner membrane protected urease from acid. Isogenic ureI knockout mutants of several H. pylori strains were constructed by replacing the ureI gene of the urease gene cluster with a promoterless kanamycin resistance marker gene (kanR). Mutants carrying the modified ureAB-kanR-EFGH operon all showed wild-type levels of urease activity at neutral pH in vitro. The mutants resisted media of pH > 4.0 but not of pH < 4.0. Whereas wild-type bacteria showed high levels of urease activity below pH 4.0, this ability was not retained in the ureI mutants, resulting in inhibition of metabolism and cell death. Gene complementation experiments with plasmid-derived H. pylori ureI restored wild-type properties. The activation of urease activity found in structurally intact but permeabilized bacteria treated with 0.01% detergent (polyoxy-ethylene-8-laurylether; C12E8), suggested a membrane-limited access of urea to internal urease at neutral pH. Measurement of 14C-urea uptake into Xenopus oocytes injected with ureI cRNA showed acid activation of uptake only in injected oocytes. Acceleration of urea uptake by UreI therefore mediates the increase of intracellular urease activity seen under acidic conditions. This increase of urea permeability is essential for H. pylori survival in environments below pH 4.0. ureI-independent urease activity may be sufficient for maintenance of bacterial viability above pH 4.0.     

Acid survival of Helicobacter pylori: how does urease activity trigger cytoplasmic pH homeostasis?

Helicobacter pylori can survive for several hours at pH 1 in the presence of urea. Under these conditions, the organism maintains its cytoplasmic pH at a value close to neutral. The role of the cytoplasmically located urease enzyme in this process is a matter of debate. We propose that cytoplasmic ammonia generated by the action of urease is protonated by H(+) ions leaking in from the acidic medium and that the NH(4)(+) formed is extruded from the cytoplasm via an as-yet-unidentified transport system. This mechanism is compared with the general mechanism of cytoplasmic pH homeostasis in microorganisms.     

Presumptive mechanisms of peptic ulceration by Helicobacter pylori VacA involving mucoprotease and CagA.

Helicobacter pylori vacuolating toxin (VacA) appears to be unusually stable, not only against extreme pH conditions or high temperatures, but also against common organic solvents or detergents. Under acidic conditions, its activity was markedly increased in the manner of temperature-independent, suggesting a spontaneous activation. A similar finding was also observed under alkaline conditions, however, it should have an appropriate temperature. From these observations, the mechanisms of VacA activation were suggested to be so redundant that either the case of acidic or basic amino acid residues could be involved in the VacA activation. Separately, we also found that the VacA production by H. pylori was pH-dependent: Its production was increased at a low pH region with a broad range (1.0-5.0), and at a high pH region with a narrow range (8.0-9.0). Astonishingly, a highly immunogenic CagA did not appear to be expressed under the acidic conditions. Its expression, however, was shown to be enhanced when the surrounding pH of this bacterium was raised. In contrast, mucoproteolytic activity in the H. pylori membrane was found to be increased at acidic conditions. Considering these observations, together with the stomach and duodenal pH of humans, two presumptive mechanisms of H. pylori VacA-associated ulceration may be deduced; namely, an acid- and an alkali-dependent type, involving mucoprotease and CagA, respectively.     

The changes of proteomes components of Helicobacter pylori in response to acid stress without urea

Acid stress is the most obvious challenge Helicobacter pylori encounters in human stomach. The urease system is the basic process used to maintain periplasmic and cytoplasmic pH near neutrality when H. pylori is exposed to acidic condition. However, since the urea concentration in gastric juice is approximately 1 mM, considered possibly insufficient to ensure the survival of H. pylori, it is postulated that additional mechanisms of pH homeostasis may contribute to the acid adaptation in H. pylori. In order to identify the acid-related proteins other than the urease system we have compared the proteome profiles of H. pylori strain 26695 exposed to different levels of external pH (7.4, 6.0, 5.0, 4.0, 3.0, and 2.0) for 30 min in the absence of urea using 2-DE. Differentially expressed proteins were identified by MALDI-TOF-TOF-MS analysis, which turned out to be 36 different proteins. The functions of these proteins included ammonia production, molecular chaperones, energy metabolism, cell envelope, response regulator and some proteins with unknown function. SOM analysis indicated that H. pylori responds to acid stress through multi-mechanisms involving many proteins, which depend on the levels of acidity the cells encounter.     

Energetics of Helicobacter pylori and its implications for the mechanism of urease-dependent acid tolerance at pH 1

In the presence of urea the neutrophilic human pathogen Helicobacter pylori survives for several hours at pH 1 with concomitant cytoplasmic pH homeostasis. To study this effect in detail, the transmembrane proton motive force and cytoplasmic urease activity of H. pylori were determined at various pH values. In the absence of urea, the organism maintained a close-to-neutral cytoplasm and an internally negative membrane potential at external pH values greater than 4 to 5. In the presence of urea, H. pylori accomplished cytoplasmic pH homeostasis down to an external pH of 1.2. At this external pH, the cytoplasmic pH was 4.9 and the membrane potential was slightly negative inside. The latter finding is in contrast to the situation in acidophiles, which develop inside-positive membrane potentials under similar conditions. Measurements of the time course of the membrane potential confirmed that addition of urea to the cells led to hyperpolarization. Most likely, this effect was due to electrogenic export of ammonium cations from the cytoplasm. The urease activity of intact cells increased nearly exponentially with decreasing external pH. This activation was not due to enhanced gene expression at low external pH values. In cell extracts the pH optimum of urease activity was dependent on the buffer system and was about pH 5 in sodium citrate buffer. Since this is the cytoplasmic pH of the cells at pH 1 to 2, we propose that cytoplasmic pH is a factor in the in vivo activation of the urease at low external pH values. The mechanism by which urease activity leads to cytoplasmic pH homeostasis in H. pylori is discussed.     

Conformational stability of Helicobacter pylori flavodoxin: fit to function at pH 5

Flavodoxin is an essential protein for Helicobacter pylori, a pathogen living in the very acidic environment of the gastric tract and responsible for several diseases. We report the conformational stability of the protein in neutral and acidic pH. The apoprotein remains native between pH 12 and 5 and adopts a monomeric molten globule conformation at more acidic pH values. The equilibrium unfolding in urea appears two-state for either conformation, but the native one coexists with a hidden equilibrium intermediate of very similar properties. The stability of H. pylori apoflavodoxin is higher than that of the Anabaena homologue throughout the entire pH interval, which may be related to better charge compensation. H. pylori apoflavodoxin is strongly stabilized by its FMN cofactor. A global analysis of apo- and holoflavodoxin equilibrium unfolding, with and without excess FMN, indicates that the cofactor only binds to the native state. Some physical-chemical properties of the protein may represent an adaptation to the acidic environment. Unlike the apoflavodoxin from Anabaena, which becomes highly insoluble at pH 5.0, that from H. pylori remains soluble to at least 40 microm. This fact, together with the high stability of the apoprotein at this low pH that can arise in the bacteria cytoplasm, seems useful to allow newly synthesized apoflavodoxin molecules to fold and remain soluble to accomplish cofactor binding, which in turn increases the stability. Also, whenever the cytoplasmic pH drops to 5, preexisting flavodoxin molecules will remain folded and soluble and will retain the FMN cofactor, thus remaining functional.     

Helicobacter pylori utilises urea for amino acid synthesis

Abstract Helicobacter pylori has one of the highest urease activities of all known bacteria. Its enzymatic production of ammonia protects the organism from acid damage by gastric juice. The possibility that the urease activity allows the bacterium to utilise urea as a nitrogen source for the synthesis of amino acids was investigated. H. pylori (NCTC 11638) was incubated with 50 mM urea, enriched to 5 atom% excess ¹⁵N, that is the excess enrichment of ¹⁵N above the normal background, in the presence of either NaCl pH 6.0, or 0.2M citrate pH 6.0. E. coli (NCTC 9001) was used as a urease-negative control. ¹⁵N enrichment was detected by isotope ratio mass spectrometry. H. pylori showed intracellular incorporation of ¹⁵N in the presence of citrate buffer pH 6.0 but there was no significant incorporation of ¹⁵N in unbuffered saline or by E. coli in either pH 6.0 citrate buffer or unbuffered saline. The intracellular fate of the urea-nitrogen was determined by means of gas chromatography/mass spectrometry following incubation with ¹⁵N enriched 5 mM urea in the presence of either 0.2 M citrate buffer pH 6.0 or 0.2 M acetate buffer pH 6.0. After 5 min incubation in either buffer the ¹⁵n label appeared in glutamate, glutamine, phenylalanine, aspartate and alanine. It appears, therefore, that at pH and urea concentrations typical of the gastric mucosal surface, H. pylori utilises exogenous urea as a nitrogen source for amino acid synthesis. The ammonia produced by H. pylori urease activity thus facilitates the organism's nitrogen metabolism at neutral pH as well as protecting it from acid damage at low pH.     

Extracellular pH modulates Helicobacter pylori-induced vacuolation and VacA toxin internalization in human gastric epithelial cells

In this study we investigated whether an acidic extracellular pH may inhibit H. pylori-induced internalization of bacterial virulence factors by gastric epithelium, thus preventing ingestion of potentially dangerous luminal contents and resulting cellular damage. The interaction of H. pylori VacA toxin and ammonia (produced by H. pylori urease) with partly polarized gastric MKN 28 cells in culture was investigated at neutral and moderately acidic pH (6.2, compatible with cell viability) by means of neutral red dye uptake and ultrastructural immunocytochemistry. We found that acidic extracellular pH virtually abolished both VacA-dependent and ammonia-dependent cell vacuolation, as shown by the neutral red test, and caused a 50% decrease in VacA internalization into endosomal vesicles and vacuoles, as assessed by quantitation of immunogold particles. In addition, acidic pH blocked endosomal internalization of H. pylori outer membrane vesicles, a convenient indicator of endocytosis. Our data raise the possibility that suppression of gastric acid may increase H. pylori-induced gastric damage by enhancing epithelial internalization of H. pylori virulence factors through activation of endocytosis. Increased transmembrane diffusion of ammonia could also contribute to this process.     

Partial characterization of a nuclear proteolytic activity from fertilized sea urchin eggs

The nuclei from fertilized sea urchin eggs, obtained 80 min after fertilization, contains a neutral proteolytic activity. Optimal action on casein was observed at pH 7-8 and a Km value of 1.2 mg/ml was determined for this substrate. The proteolytic activity was stimulated 1.5 fold by the addition of 3 M urea and decreased at higher urea concentrations. NaCl and CaCl2 were inhibitory whereas MgCl2 increased the enzyme activity. Isolated histones from sea urchin sperms, and especially histones H1, H2A, H2B and H3, were degraded by the nuclear activity. A partial inhibition of histones degradation was caused by sodium bisulfite and NaCl. The proteolytic activity was found associated to the chromatin of fertilized sea urchin eggs.     

Helicobacter pylori environmental interactions: effect of acidic conditions on H. pylori-induced gastric mucosal interleukin-8 production

To explore the interactions between the host, environment and bacterium responsible for the different manifestations of Helicobacter pylori infection, we examined the effect of acidic conditions on H. pylori-induced interleukin (IL)-8 expression. AGS gastric epithelial cells were exposed to acidic pH and infected with H. pylori[wild-type strain, its isogenic cag pathogenicity island (PAI) mutant or its oipA mutant]. Exposure of AGS cells to acidic pH alone did not enhance IL-8 production. However, following exposure to acidic conditions, H. pylori infection resulted in marked enhancement of IL-8 production which was independent of the presence of the cag PAI and OipA, indicating that H. pylori and acidic conditions act synergistically to induce gastric mucosal IL-8 production. In neutral pH environments H. pylori-induced IL-8 induction involved the NF-kappaB pathways, the extracellular signal-regulated kinase (ERK)-->c-Fos/c-Jun-->activating protein (AP-1) pathways, JNK-->c-Jun-->AP-1 pathways and the p38 pathways. At acidic pH H. pylori-induced augmentation of IL-8 production involved markedly upregulated the NF-kappaB pathways and the ERK-->c-Fos-->AP-1 pathways. In contrast, activation of the JNK-->c-Jun-->AP-1 pathways and p38 pathways were pH independent. These results might explain the clinical studies in which patients with duodenal ulcers had higher levels of IL-8 in the antral gastric mucosa than patients with simple H. pylori gastritis.     

Urease-mediated destruction of bacteria is specific for Helicobacter urease and results in total cellular disruption

Abstract The survival of Helicobacter mustelae, Proteus mirabilis, Escherichia coli and Campylobacter jejuni in the presence of urea and citrate at pH 6.0 was examined. H. mustelae , which has urease activity similar to H. pylori , had a markedly reduced survival, median 2.5% (0–78%) ( P <0.001) when incubated nder these conditions. Only 7% of the ammonia produced by H. mutelae urease activity was recovered from the buffer, a similar percentage to that previously reported with H. pylori . None of the other organisms, all of which had lower urease activity, had impaired survival under these conditions. Electron microscopical studies demonstrated extensive structural damage to H. pylori following exposure to urea and citrate at pH 6.0. This structural damage to the organisms makes it unlikely that the low recovery of ammonia was due to retention of ammonia within the bacteria and suggests that the ammonia may have been incorporated into glutamate or other amino acids. Incorporation of ammonia into these compounds would deplete the cell of the key metabolic intermediate α-ketoglutarate and could thus explain the mechanism of the urease-dependent destruction of the organism.     

Effect of the carbonic anhydrase inhibitor, acetazolamide, on Helicobacter pylori infection in vivo: a pilot study

BACKGROUND: Carbonic anhydrase inhibitors have been successfully used to treat peptic ulcers. Although carbonic anhydrase restriction does not inhibit Helicobacter pylori in vitro, recent studies suggest that carbonic anhydrase inhibition reduces the ability of H. pylori to survive in an acid environment as present in the stomach. METHODS: In a pilot study, we examined the effect of acetazolamide 500 mg as twice a day for 4 days in volunteers with active H. pylori infection. Effectiveness was judged by changes in the results of the urea breath test. RESULTS: Eight H. pylori infected volunteers completed the test. No urea breath test reverted to negative and there was a trend for the urea breath test value to increase [e.g. delta over baseline (DOB) mean +/- SE increased from 50.9 +/- 13 at baseline to 64.9 +/- 13 at day 5] during treatment with acetazolamide. CONCLUSION: The potential effect of carbonic anhydrase inhibitors on acid secretion may prevent effect on H. pylori in vivo and/or the sites of infection at the surface of the stomach may have a pH higher for any postulated acid-dependent effect to have an effect clinically.     

Purification and characterization of Helicobacter pylori arginase, RocF: unique features among the arginase superfamily.

The urea cycle enzyme arginase (EC 3.5.3.1) hydrolyzes l-arginine to l-ornithine and urea. Mammalian arginases require manganese, have a highly alkaline pH optimum and are resistant to reducing agents. The gastric human pathogen, Helicobacter pylori, also has a complete urea cycle and contains the rocF gene encoding arginase (RocF), which is involved in the pathogenesis of H. pylori infection. Its arginase is specifically involved in acid resistance and inhibits host nitric oxide production. The rocF gene was found to confer arginase activity to Escherichia coli; disruption of plasmid-borne rocF abolished arginase activity. A translationally fused His(6)-RocF was purified from E. coli under nondenaturing conditions and had catalytic activity. Remarkably, the purified enzyme had an acidic pH optimum of 6.1. Both purified arginase and arginase-containing H. pylori extracts exhibited optimal catalytic activity with cobalt as a metal cofactor; manganese and nickel were significantly less efficient in catalyzing the hydrolysis of arginine. Viable H. pylori or E. coli containing rocF had significantly more arginase activity when grown with cobalt in the culture medium than when grown with manganese or no divalent metal. His(6)-RocF arginase activity was inhibited by low concentrations of reducing agents. Antibodies raised to purified His(6)-RocF reacted with both H. pylori and E. coli extracts containing arginase, but not with extracts from rocF mutants of H. pylori or E. coli lacking the rocF gene. The results indicate that H. pylori RocF is necessary and sufficient for arginase activity and has unparalleled features among the arginase superfamily, which may reflect the unique gastric ecological niche of this organism.     

Absence of catalase reduces long-term survival of Helicobacter pylori in macrophage phagosomes

BACKGROUND: Some Helicobacter pylori strains can survive within macrophage phagosomes for up to 24 hours. The factors that play a role in this survival remain ill-defined. Therefore, the contribution of catalase in mediating the survival of H. pylori following phagocytosis was investigated in vitro. METHODS: An isogenic, catalase-deficient strain of H. pylori was generated and tested for sensitivity to hydrogen peroxide and susceptibility to macrophage-mediated killing. RESULTS: The isogenic, catalase-deficient strain of H. pylori was effectively killed by hydrogen peroxide within 3 minutes compared to wild-type H. pylori which maintained 100% survival up to 21 minutes. The catalase-deficient mutant was also significantly more susceptible to macrophage-mediated killing than the parent strain, even when the ratio of bacteria to macrophage was increased. CONCLUSION: These results indicate that although some strains of H. pylori are capable of survival within the macrophage phagosome, survival is dependent on virulence factors such as catalase for evasion of innate host defense.     

Adenosine deamination increases the survival under acidic conditions in Escherichia coli

Aims: Resistance to acidic stress contributes to bacterial persistence in the host and is thought to promote their passage through the human gastric barrier. The aim of this study was to examine whether nucleosides have a role in the survival under acidic conditions in Escherichia coli. Methods and Results: We found that adenosine has a function to survive against extremely acidic stress. The deletion of add encoding adenosine deaminase that converts adenosine into inosine and NH₃ attenuated the survival in the presence of adenosine. The addition of adenosine increased intracellular pH of E. coli cells in pH 2·5 medium. Addition of inosine or adenine did not increase the resistance to acidic conditions. Conclusions: Our present results imply that adenosine was used to survive under extremely acidic conditions via the production of NH₃. Significance and Impact of the Study: It has been proposed that amino acid decarboxylation is the major system for the resistance of E. coli to acidic stress. In this study, the adenosine deamination was shown to induce the survival under acidic conditions, demonstrating that bacteria have alternative strategies to survive under acidic conditions besides amino acid decarboxylation.     

Transport kinetics and selectivity of HpUreI, the urea channel from Helicobacter pylori

Helicobacter pylori's unique ability to colonize and survive in the acidic environment of the stomach is critically dependent on uptake of urea through the urea channel, HpUreI. Hence, HpUreI may represent a promising target for the development of specific drugs against this human pathogen. To obtain insight into the structure-function relationship of this channel, we developed conditions for the high-yield expression and purification of stable recombinant HpUreI. Detergent-solubilized HpUreI forms a homotrimer, as determined by chemical cross-linking. Urea dissociation kinetics of purified HpUreI were determined by means of the scintillation proximity assay, whereas urea efflux was measured in HpUreI-containing proteoliposomes using stopped-flow spectrometry to determine the kinetics and selectivity of the urea channel. The kinetic analyses revealed that urea conduction in HpUreI is pH-sensitive and saturable with a half-saturation concentration (or K(0.5)) of ~163 mM. The extent of binding of urea by HpUreI was increased at lower pH; however, the apparent affinity of urea binding (~150 mM) was not significantly pH-dependent. The solute selectivity analysis indicated that HpUreI is highly selective for urea and hydroxyurea. Removing either amino group of urea molecules diminishes their permeability through HpUreI. Similar to urea conduction, diffusion of water through HpUreI is pH-dependent with low water permeability at neutral pH.