A H+-Gated Urea Channel: The Link Between Helicobacter pylori Urease and Gastric Colonization

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Science  21 Jan 2000:
Vol. 287, Issue 5452, pp. 482-485
DOI: 10.1126/science.287.5452.482


Acidic media trigger cytoplasmic urease activity of the unique human gastric pathogen Helicobacter pylori. Deletion ofureI prevents this activation of cytoplasmic urease that is essential for bacterial acid resistance. UreI is an inner membrane protein with six transmembrane segments as shown by in vitro transcription/translation and membrane separation. Expression of UreI in Xenopus oocytes results in acid-stimulated urea uptake, with a pH profile similar to activation of cytoplasmic urease. Mutation of periplasmic histidine 123 abolishes stimulation. UreI-mediated transport is urea specific, passive, nonsaturable, nonelectrogenic, and temperature independent. UreI functions as a H+-gated urea channel regulating cytoplasmic urease that is essential for gastric survival and colonization.

The Gram-negative pathogenH. pylori is unique in its ability to colonize the human stomach. H. pylori infection is acquired during childhood, persists lifelong if not eradicated, and is associated with chronic gastritis and an increased risk of peptic ulcer disease and gastric cancer (1). An acid-tolerant neutralophile, H. pylori expresses a neutral pH–optimum urease to maintain proton motive force (PMF) and to enable gastric colonization (2).

Most urease is found in the bacterial cytoplasm, although up to 10% appears on the surface, owing to cell lysis during culture (3). Surface or free urease has a pH optimum between pH 7.5 and 8.0 but is irreversibly inactivated below pH 4.0 (4, 5). The activity of cytoplasmic urease is low at neutral pH but increases 10- to 20-fold as the external pH falls between 6.5 and 5.5, and its activity remains high down to pH ∼2.5 (5). Thus, cytoplasmic, not surface, urease is required for acid resistance. The unmodified urea permeability of the inner membrane is insufficient to supply enough urea to intrabacterial urease for urease activity to buffer the bacterial periplasm in the face of gastric acidity (the median diurnal acidity of the human stomach is pH 1.4). The data here show that H. pylori expresses a urea transport protein with unique acid-dependent properties that activates the rate of urea entry into the cytoplasm.

The permeability of urea across phospholipid bilayer membranes, 4 × 10−6 cm s−1(6), is insufficient to saturate internal urease. At neutral pH, this rate of urea entry is not able to saturate intrabacterial urease even with 100 mM external urea. In acidic media, the apparent Michaelis constant K m of internal urease becomes equal to that of free urease, ∼1 mM (5), demonstrating an accelerated urea entry. The addition of 0.01% of the nonionic detergent C12E8 permeabilizes the inner membrane, as shown by penetration of propidium iodide, without disrupting its morphology (Fig. 1). The urease of C12E8-treated intact organisms is fully active at neutral pH (intact bacteria, 0.25 ± 0.1 μmol of urea per minute per milligram of protein; bacterial homogenate, 2.76 ± 0.27 μmol of urea per minute per milligram of protein; bacteria with 0.01% C12E8, 2.65 ± 0.10 μmol of urea per minute per milligram of protein). Thus, an increase of urea permeability of intact H. pylori accounts for the activation of cytoplasmic urease in acidic media.

Figure 1

Confocal fluorescent micrograph ofH. pyloristained using the Live/Dead method (Molecular Probes, Eugene, Oregon). (A) Before C12E8 treatment. Green color is from staining with only SYTO 9, a permeant nucleic acid dye. (B) After 0.01% C12E8treatment. Red stain shows the disrupted membrane that allows entry of propidium iodide.

The urease gene cluster consists of seven genes. ureAand ureB encode the urease structural subunits, andureE, -F, -G, and -H encode accessory proteins necessary for Ni2+ insertion into the apoenzyme (7). ureI encodes a membrane protein with homology to putative amide transporters such as AmiS, AmiS2, and ORFP3 (8), and its absence impairs acid survival (9). UreI may be an acid-activated urea transporter crucial for acid resistance of H. pylori. Its function was determined in deletion mutants and by expression in Xenopusoocytes.

In contrast to the large acid-induced increase in urease activity of wild-type organisms, no increase of activity in acidic medium was observed in the intact ureI mutant (10), DW504UreI (Fig. 2). However, urease activity of detergent-treated ureI cells (3.0 ± 0.25 μmol of urea per minute per milligram of protein) or cell lysate resulted in urease activity equal to that of wild-type organisms. The normal level of urease shows that deletion of ureI did not affect the expression of the downstream urease accessory genes essential for its biosynthesis. The mutation is therefore nonpolar. Abolition of acid activation of urease in intact organisms byureI deletion and full activation of intrabacterial urease by C12E8 suggest that UreI-mediated transport of urea determines the pH dependence of cytoplasmic urease.

Figure 2

Comparison of the pH profiles of cytoplasmic urease activity in wild-type (WT) and UreI H. pylori ATCC 43504 with that ofureI lysate (n = 3) (22). pHmedium, pH of the medium. Error bars indicate ± SEM.

Because activity of the H. pylori cytoplasmic urease maintains an inward urea gradient, uptake does not need energy from adenosine triphosphate (ATP) or ion gradients. Measurement of passive urea uptake in prokaryotes, with their small volume-to-surface ratio, is precluded by the endogenous permeability of phospholipid bilayers. Oocytes have a volume-to-surface ratio several hundred times that of prokaryotes and express neither endogenous urea transporters nor urease activity (11). UreI was therefore expressed inXenopus oocytes by injection of ureIcomplementary RNA (cRNA) (Fig. 3). Over 30 min, urea uptake in UreI oocytes was accelerated 6- to 10-fold at pH 5.0 compared with pH 7.5 and was the same as in noninjected oocytes at either pH (12) (Fig. 4, A and C). Control oocytes equilibrated to the same level as oocytes expressing UreI, but required 48 hours to reach equilibrium as compared with 1 hour for ureI-injected oocytes. No increase in internal concentration was found above the increase from equalization of the concentration gradient. Accumulation was consistent with acid-dependent UreI facilitation of urea transport into the 0.4 to 0.6 μl of internal oocyte water space (13).

Figure 3

Western blot analysis showing the absence of UreI in the ureI mutants and its presence in the inner but not outer membrane of wild-type H. pylori, as well as in oocytes injected with ureI cRNA. TM, total membranes; IM, inner membrane; OM, outer membrane (23).

Figure 4

Uptake experiments inureI-injected and control Xenopus oocytes (n = 5 to 7). (A) Equilibration of 50 μM14C-urea at pH 5.5 and 7.5. (B) Uptake of 50 μM 14C-urea in ureI-injected oocytes as a function of pH of the medium. (C) Uptake of 50 μM14C-urea or uptake of 50 μM 14C-thiourea at pH 5.0 and 7.5 in the presence of excess unlabeled urea. (D) Effect of temperature on uptake of 14C-urea at pH 5.0 between 15° and 30°C (24). Error bars indicate ± SEM.

UreI-dependent urea uptake was activated with a pH profile nearly identical to the pH activation profile of cytoplasmic urease in H. pylori (5). Half-maximal activation of transport occurred at pH ∼6.0 (Fig. 4B). Uptake was highly selective for urea, with only trace accumulation of14C-thiourea (Fig. 4C) or 14C-mannitol (1.13 ± 0.10 and 0.41 ± 0.14 pmol per oocyte at pH 5.0, respectively). Uptake of 50 μM 14C-urea was 15.28 ± 0.27 and 13.61 ± 0.97 pmol per oocyte in the absence or presence of 100 mM unlabeled urea, respectively. Thus, saturation was not seen, even though a 2000-fold excess of urea was added (Fig. 4C). The addition of urea to voltage-clamped UreI-expressing oocytes resulted in no change in current. An inward current of 117 nA is predicted, if UreI were a proton- or cation-driven urea transporter with a stoichiometry of 1:1 (14). UreI-mediated urea uptake is therefore nonelectrogenic.

Transport at pH 5.0 was temperature independent between 15° and 30°C (Fig. 4D). This temperature insensivity and the lack of saturation of uptake suggest that, after H+ activation, urea fluxes through UreI with little interaction with the protein. Aquaporins, although also putative six transmembrane–segment channel-like water transport proteins, show substantial temperature dependence (15). Our data suggest that UreI functions as a specific, H+-activated urea channel. A channel mechanism would allow a rate of urea uptake adequate for saturation of internal urease at physiological gastric urea concentrations (1 to 3 mM).

Western blot analysis detected the presence of UreI in purified inner but not outer membrane fractions (Fig. 3). Periodic acid–silver staining detected carbohydrate (16) in the outer but not inner membrane fraction (17), confirming the validity of the separation (18). UreI contains six hydrophobic sequences, H1 to H6. In vitro transcription/translation of various NH2-terminal lengths of UreI, fused to a glycosylatable COOH-terminal tag, was used to follow orientation of translation products in canine microsomal membranes (19). A signal anchor sequence translocates the COOH-terminus into the microsomal lumen (analogous to the bacterial periplasm), and a subsequent stop transfer sequence returns the COOH-terminus to the cytoplasmic side. Alternating signal anchor and stop transfer sequences defined (Fig. 5A) the topography of UreI.

Figure 5

Topography of UreI. (A) SDS–polyacrylamide gel electrophoresis analysis of products resulting from in vitro transcription/translation of successive UreIN-ter/H+,K+ATPase β-subunit fusion constructs containing one to six of the hydrophobic sequences of UreI, with (+) or without (–) microsomes. Glycosylation (arrow) is detected by a 12.5-kD shift in the translation product. (B) Two-dimensional model of UreI from in vitro translation results (25). Arrow, histidine 123.

The lack of a cytoplasmic retention signal in front of the first hydrophobic sequence, H1, and the presence of two positively charged amino acids in front of H2 imply a periplasmic location of the NH2-terminus. The COOH-terminus of the UreN1 construct, encoding Met1–Lys23, was glycosylated, signifying COOH-terminal “out” orientation (Fig. 5A). However, UreN1b, Met1–Lys27, had a COOH-terminal “in” orientation because of the additional positive charge (20). The product of UreN2, Met1–Thr56, showed strong glycosylation indicating that H1 and H2 are a membrane-inserted pair with the NH2- and COOH-termini oriented “out.” The translation product of UreN3, Met1–Arg102, lost the glycosylation of UreN2. H3 acts as a stop transfer sequence, yielding a COOH-terminus “in” orientation. The glycosylation of the product of UreN4 translation, Met1–Leu128, showed that H4 acted as a signal anchor, directing the COOH-terminus “out”. The translation product of UreN5, Met1–Lys162, showed no glycosylation, with H5 acting as a stop transfer sequence, whereas the product of UreN6, Met1–Val195, was glycosylated. H6 therefore behaved as a signal anchor. Hence, the inner membrane protein, UreI, has six transmembrane–inserted segments, with both NH2- and COOH-termini located in the periplasm (Fig. 5B).

The apparent pK of UreI activation implies protonation of one or more periplasmic histidines for activation of urea transport. Histidine 123, located at the boundary of H4, was mutated to arginine or glycine (21). Expression of UreI was unaffected by this mutation, but acid activation of urea uptake disappeared (1.83 ± 0.20 pmol per oocyte at pH 5.0 versus 1.75 ± 0.34 pmol per oocyte at pH 7.5). The protonated state of this histidine is important for acid activation of transport.

Acid survival of prokaryotes depends on the maintenance of suitable levels of cytoplasmic and periplasmic pH to maintain their PMF.Helicobacter pylori survives between pH 4.0 and 8.5 in the absence of urea and grows between pH 6.0 and 8.0 (2). A neutral pH–optimum urease must be shielded from gastric acidity and prevented from being active at neutral pH to avoid lethal alkalinization (5). Urea transport via UreI allows the internal urease of H. pylori to generate ammonia in an acid environment, buffering the periplasm. This allows the organism to survive and grow in the stomach in the presence of usual gastric urea concentrations. The absence of transport by UreI at neutral pH prevents high urease activity in the absence of gastric acidity, as occurs during digestion. The combination of a high level of a neutral pH–optimum urease and an acid-regulated urea channel explains whyH. pylori is unique in its ability to inhabit the human stomach. Effective inhibition of UreI would provide a means of eradicating the organism in the normal, acid-secreting stomach.

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