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 Helicobacterpylori survives for several hours at pH 1 with concomitant cytoplasmicpH homeostasis. To study this effect in detail, the transmembraneproton motive force and cytoplasmic urease activity of H. pyloriwere determined at various pH values. In the absence of urea,the organism maintained a close-to-neutral cytoplasm and aninternally negative membrane potential at external pH valuesgreater than 4 to 5. In the presence of urea, H. pylori accomplishedcytoplasmic pH homeostasis down to an external pH of 1.2. Atthis external pH, the cytoplasmic pH was 4.9 and the membranepotential was slightly negative inside. The latter finding isin contrast to the situation in acidophiles, which develop inside-positivemembrane potentials under similar conditions. Measurements ofthe time course of the membrane potential confirmed that additionof urea to the cells led to hyperpolarization. Most likely,this effect was due to electrogenic export of ammonium cationsfrom the cytoplasm. The urease activity of intact cells increasednearly exponentially with decreasing external pH. This activationwas not due to enhanced gene expression at low external pH values.In cell extracts the pH optimum of urease activity was dependenton 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 activationof the urease at low external pH values. The mechanism by whichurease activity leads to cytoplasmic pH homeostasis in H. pyloriis discussed.

INTRODUCTION

Top

Introduction

As a neutrophilic bacterium capable of growth at pH values of>5.5 (21), Helicobacter pylori is unique with respect toits acid tolerance and long-term persistence in the human stomach.Mechanisms enabling H. pylori to cope with fluctuating pH mustbe essential, particularly during primary infection, in orderto overcome the gastric acid barrier. Recently, we have shownthat in the presence of urea and without any previous adaptation,growing cells of H. pylori are capable of survival and cytoplasmicpH (pH_in) homeostasis for several hours after a shift of themedium pH (pH_out) to pH 1 (30), a physiologically relevant conditionfrequently found in the gastric lumen. At this pH, acidophilesexhibit a positive inside membrane potential ( ${Delta}$ ${Psi}$ ) (2, 22, 33).For H. pylori, the sign and value of ${Delta}$ ${Psi}$ at low pH_out values isstill a matter of debate, and data are lacking for pH_out valuesof <3. An inside positive ${Delta}$ ${Psi}$ has been reported at pH 3, whichwas, however, not sensitive to addition of a protonophore (14).In other studies ${Delta}$ ${Psi}$ remained negative inside down to a pH of 3(28). Because of these conflicting data, we reexamined the ${Delta}$ ${Psi}$ of H. pylori cells at low pH_out values and extended the studiesto pH 1 to 2. We observed that in the presence of urea ${Delta}$ ${Psi}$ remainedinside negative at all pH_out values between 1.2 and 7. Therefore,we propose that this phenomenon is associated with the electrogenicexport of ammonium cations from the cytoplasm.

Urease is a virulence factor of H. pylori, and this enzyme isthought to confer acid resistance to H. pylori by cleavage ofurea and elevation of the microenvironmental pH (12). However,the mechanism by which urease contributes to survival underacidic conditions is highly controversial. Originally, it wasassumed that the enzyme activity is extracytoplasmic and thatin the stomach protection occurs due to the creation of a cloudof ammonia around the cells (9). This hypothesis, that externalurease activity protects H. pylori from acid stress, was recentlyput forward again, based on the observation that in a nonstirredsolution urease exhibits some residual activity at pH 3 (8).However, it has been shown that urease is cytoplasmic (28) andthat the urea porter UreI influences urease activity by mediatingacid-triggered urea uptake (24, 27, 36). According to the authorsof these papers the NH₃ product of the urease reaction leavesthe cytoplasm in its neutral form and neutralizes the periplasmicpH by binding protons in that environment. Finally, our preliminarydata suggest that urease activity leads to cytoplasmic ratherthan periplasmic pH homeostasis of H. pylori cells and thatthis process is sufficient for survival at pH 1 (30). The resultsof the experiments reported here fully support this notion,and a hypothesis for the mechanism of this process (30, 37)is discussed.

A further controversial issue concerns the pH optimum of theH. pylori urease. In cell extracts diluted with citrate-phosphatebuffer the enzyme exhibits a pH optimum of 7.4 (28). In wholecells urease activity is maximal at a low pH_out (28). This phenomenonis attributed to a controlled urea supply determined by acidactivation of UreI. However, at a pH_out of 3 urease activityin the cytoplasm is expected to be severely inhibited, sinceunder these conditions the pH_in is only 5.5 to 5.7 (30), a valuesubstantially different from the pH optimum of the urease (28).The urease activities of Yersinia enterocolitica and Morganellamorganii have a pH optimum of 5.5 in citrate-based buffers (37).Moreover, the activity of the urease from Klebsiella aerogeneshas been reported to be inhibited by acid forms of phosphate(31). These conflicting data led us to reexamine the pH optimumof urease activity in H. pylori cell extracts. Various buffersystems were used, since it is known that at acidic pH valuesthe pH optimum of an enzyme may depend strongly on the bufferused (15). We observed that in sodium citrate buffer the pHoptimum of H. pylori urease is around pH 5. This finding wascombined with the results of determinations of pH_in and cytoplasmicurease activity of cells suspended at pH_out values between 1.2and 7. We concluded that lowering the pH_in to values around5 after an acid shift of H. pylori cells may contribute to theactivation of urease activity observed under these conditions.

MATERIALS AND METHODS

Top

Materials and Methods

Strain and growth conditions. H. pylori wild-type strain DSM 4867 was cultivated as describedbefore (30).

Buffers. Citrate-phosphate buffers were prepared at 37°C as describedpreviously (16, 18). For pH 3 to 7 buffers 100 mM citric acidand 200 mM Na₂HPO₄ were mixed to obtain the appropriate pH.For the pH 1 buffer, the pH of 100 mM citric acid was adjustedwith concentrated HCl. For pH 2, the 100 mM citric acid bufferwas used without any further addition. Buffer containing 100mM MES [2-(N-morpholino)ethanesulfonic acid] or 100 mM citricacid was titrated with concentrated NaOH to obtain the appropriatepH. Sodium phosphate buffer was composed of a mixture of 100mM Na₂HPO₄ and 100 mM NaH₂PO₄.

Urease activity. The in vivo urease activity of H. pylori cells was monitoredby determining the decrease in the urea concentration in APM(acid-precipitated brucella broth supplemented with 5% fetalcalf serum, as described previously [30]). Samples (200 µl)were withdrawn at different times. Fluorofamide (10 µM)was added immediately, and the samples were stored frozen inliquid nitrogen. Subsequently, samples were thawed in a sonicationbath for 3 min and centrifuged at a low speed (5 min, 16,000x g). The concentration of urea in the supernatant was determinedas described by Rahmatullah and Boyde (23).

For determination of urease activity in cell extracts, cellsfrom 2 ml of an H. pylori culture were harvested by centrifugation(16,000 x g) and washed twice with 500 µl of 350 mM Tris-HCl(pH 8.0). The cell pellet was frozen in liquid N₂. After thawing,the cells were resuspended in 500 µl of 20 mM Tris-HCl(pH 8.0) and sonicated three times (30 s each) on ice by usinga microtip (Branson cell disrupter B 15; output control 3, 50%pulsed). After centrifugation at 16,000 x g for 5 min, the ureaseactivity in the supernatant was determined by measuring thereleased ammonia by the phenol-hypochlorite assay (35). Theassay was started by addition of 1 µg of protein to 1ml of buffer supplemented with 10 mM urea. After 5, 10, and15 min of incubation at 37°C, a 20- to 40-µl aliquotwas added to 400 µl of 100 mM sodium phosphate buffer(pH 7.4) plus 200 µl of 3% phenol-0.003% sodium nitroprusside;this was followed by addition of 200 µl of 2% NaOH-0.05%NaClO. After incubation for 45 min in the dark at room temperature,absorption was determined at 635 nm. NH₄Cl was used as a standard.

Protein concentration determination. Protein concentrations in cell extracts were determined by themethod of Bradford (5) by using the Bio-Rad protein assay (Bio-Rad,Munich, Germany) and bovine serum albumin as a standard.

Determination of pH_in. For the acid shock experiments, cells were first collected bycentrifugation (10 min, 5,000 x g, 37°C), resuspended toan optical density at 578 nm (OD₅₇₈) of about 2 in 150 mM NaCl,and diluted 10-fold with citrate-phosphate buffer at the appropriatepH supplemented with 20 mM urea, as indicated below. The pH_inwas calculated from the distribution of [¹⁴C]salicylate acrossthe cytoplasmic membrane (14, 25). Separation of the supernatantand the pellet was performed by centrifugation through siliconeoil as described by Michels and Bakker (19). For nonenergizedcells, 1 mM 2,4-dinitrophenol (2,4-DNP) was added. After 5 minof incubation in the appropriate citrate-phosphate buffer, 5ml of the cell suspension was incubated with 2 µCi of³H₂O (1 mCi/g) per ml and 50 nCi of [7-¹⁴C]salicylic acid (55.5mCi/mmol; New England Nuclear Corp.) per ml for 3 min at 37°Cwith shaking. Cells were separated from the medium by centrifugationin a 12-ml conical polypropylene centrifuge tube (Sarstedt)through 0.5 ml of a 1:2 mixture of AR20 and AR200 silicon oils(Serva) for 10 min at 5,400 x g and room temperature, followedimmediately by centrifugation for 10 min at 9,500 x g and 4°Cto improve phase separation. A 50-µl aliquot of the supernatantwas added to 0.5 ml of 0.4 M NaOH in a 6-ml miniature scintillationvial (Canberra-Packard). The remainder of the supernatant wasdiscarded. The bottom part of the centrifuge tube containingthe complete cell pellet and some oil was separated with a scalpeland added to 0.5 ml of 0.4 M NaOH in a scintillation vial, andthis was followed by overnight incubation at room temperature.Subsequently, 5 ml of Ultima Gold scintillation fluid (Canberra-Packard)was added to each sample. The radioactivity of the two isotopesin each sample was determined with a Tri-Carb 2300TR liquidscintillation counter (Canberra-Packard) in the dual-label disintegrationsper minute mode with automatic correction for spill-over andquenching with the aid of an external standard. pH_in valueswere calculated from the measured distribution of [¹⁴C]salicylatebetween the sediment and the supernatant by using the equation:A^T_in/A^T_out = C_r (1 + V_r) - V_r = (1/K_a + 1/H⁺_in)/(1/K_a + 1/H⁺_out),where A^T_in and A^T_out are the total ¹⁴C concentrations in thecells and in the medium, respectively; C_r is the ¹⁴C/³H accumulationratio between the sediment and the supernatant; V_r is the ratioof the external water space of the cell pellet to the internalwater space of the cell pellet, which was taken to be 1.0, avalue observed for many other prokaryotes with this method (3,4, 19); and 1/K_a is 933 for salicylic acid (7). Binding of [¹⁴C]salicylateto cellular components was neglected for apparent values. Correctedvalues were corrected for binding by the exponential mean method(38).

Fluorimetric determination of ${Delta}$ ${Psi}$ . Cells of H. pylori (OD₅₇₈, $~$ 0.2) were harvested by centrifugationand resuspended in 0.1 volume of prewarmed 0.9% NaCl. Continuousfluorimetric measurements were obtained with citrate-phosphatebuffers at the appropriate pH values by using 1 µM 3,3'-dipropylthiadicarbocyanineiodide (Disk₃) (Molecular Probes Inc.) (5) as a ${Delta}$ ${Psi}$ -sensitive cationicdye (18, 28). The bacterial suspension was added to the dyesolution at a final OD₅₇₈ of about 0.03. Fluorescence was measuredat 37°C at an excitation wavelength of 600 nm and an emissionwavelength of 665 nm. ${Delta}$ ${Psi}$ was eliminated by addition of 150 nM3,3',4',5-tetrachlorosalicylanilide (TCS) or 2% n-butanol.

Determination of ${Delta}$ ${Psi}$ with ¹⁴C-labeled lipophilic ions. ${Delta}$ ${Psi}$ was calculated from the distribution of [¹⁴C]tetraphenylphosphonium([¹⁴C]TPP⁺) or S¹⁴CN^- across the cytoplasmic membrane of cellscentrifuged through silicone oil as described above for thedetermination of pH_in. For estimation of binding of the radiochemicalsto cellular components, 2% butanol was added. Each cell suspensionwas incubated with 2 µCi of ³H₂O (1 mCi/g) per ml and50 nCi of [¹⁴C]TPP⁺ (20 mCi/mmol; American Radiolabeled ChemicalsInc.) per ml or 100 nCi of S¹⁴CN^- (4.2 mCi/mmol; Sigma) perml for 3 min at 37°C with shaking. ${Delta}$ ${Psi}$ was determined by usingthe equations: A^T_in/A^T_out = C_r x (1 + V_r) - V_r and ${Delta}$ ${Psi}$ = -ln(A^T_in/A^T_out)x (RT/zF). Binding of [¹⁴C]TPP⁺ to cellular components was neglectedfor apparent values. Corrected values were corrected for bindingby the exponential mean method (38).

Sign of bioenergetic parameters. Transmembrane parameters are determined by subtracting outsidevalues from inside values. Hence, ${Delta}$ ${Psi}$ , ${Delta}$ pH (pH_in - pH_out), and theproton motive force (PMF) are negative when the values insideare negative.

RESULTS

Top

Results

pH_in in the presence of urea. Figure 1 shows the profiles for pH_in (Fig. 1A and B) and ${Delta}$ pH(Fig. 1C) of H. pylori cells suspended in citrate-phosphatebuffers having pH values between 1 and 7.2. The data were obtainedby measuring the distribution of the anionic form of the weakacid [¹⁴C]salicylic acid across the cell membrane by an establishedprocedure (19, 25) adapted for H. pylori cells (30). In thepresence of urea, the pH_out increased by 0.1 to 0.8 pH unitduring the experiment depending on the cell urease activityand the buffering capacity of the medium at each pH (see Table2). These changes in pH_out were taken into account for calculationof all data. Figure 1A shows that the apparent pH_in of urea-degradingcells decreased from 7.5 to 5.4 when the pH_out decreased from7.2 to 1.2. At a pH_out of 2 the apparent pH_in (pH 5.5) was thesame as the value previously determined after 1 h of incubationin the same buffer (30), indicating that after 8 min of acidstress a steady-state pH_in was reached. In the presence of 1mM 2,4-DNP (a protonophore) at a low pH_out, the apparent pH_indecreased to around 4 (Fig. 1A). A residual pH_in of 4 has alsobeen reported for nonenergized acidophilic organisms (19, 39)and has been attributed to several phenomena (13, 19), includingnonspecific binding of the radiochemical to cellular components(11, 19). Support for the notion that the latter phenomenonalso occurs in H. pylori comes from the observation that permeabilizationof the cells by treatment with 2% n-butanol gave values virtuallyidentical to those obtained after treatment with 1 mM 2,4-DNP(data not shown). By using data obtained in the presence of2,4-DNP, pH_in values were corrected for binding by the exponentialmean method (Fig. 1B) (38). At pH_out values of 1.2 and 2.4,a corrected pH_in of 4.9 was maintained with urea, whereas thepH_in was 5.2 for a pH_out of 3.3. At pH_out values of >4 andin the presence of urea, ${Delta}$ pH decreased continuously to zero ata neutral pH_out (Fig. 1C).

View larger version (18K):
[in this window]
[in a new window]
FIG. 1. pH_in of H. pylori measured after 8 min of incubation in citrate-phoshate buffer at the appropriate pH_out. (A) Apparent pH_in; (B) pH_in after correction for binding as described by Zaritsky et al. (38); (C) ${Delta}$ pH (corrected values). Open circles, cells in the absence of urea; solid circles, cells in the presence of 20 mM urea; gray circles, cells with urea and 1 mM 2,4-DNP. The ratio of accumulation of ¹⁴C in the cells to accumulation of ¹⁴C in the medium (A^T_in/A^T_out) was 280 ± 67 for pH 1 in the presence of urea, while it was only 10 ± 1 at pH 1 in the presence of urea and 2,4-DNP. At pH 7 A^T_in/A^T_out was around 2 ± 0.5, irrespective of the presence of urea or 2,4-DNP, indicating that the pH_in approximately matched the pH_out.

View this table:
[in this window]
[in a new window]
TABLE 2. ${Delta}$ pH, ${Delta}$ ${Psi}$ , and PMF determined for H. pylori at different pH_out values in the presence and absence of urea^a

pH_in in the absence of urea. In the absence of urea, pH_out remained constant regardless ofthe starting pH of the buffer used. At pH_out values of $>=$ 4 thecells maintained an alkaline inside ${Delta}$ pH across the cytoplasmicmembrane, indicating that they performed pH homeostasis (Fig.1). In contrast, at pH_out values between 1 and 3 the cells didnot accomplish pH homeostasis, since the pH_in was close to thatof cells treated either with the protonophore 2,4-DNP (Fig.1A) or with n-butanol (data not shown). Hence, these cells didnot maintain a ${Delta}$ pH across the cytoplasmic membrane at pH_out valuesof <4 (Fig. 1C). At pH_out values of 4 to 7, the pH_in waslower in the presence of urea than in its absence. Possibleexplanations for this effect are discussed below.

Taken together, the data in Fig. 1 indicate that urease activityis essential for pH homeostasis at pH_out values of <4 andthat at pH_out values of >4, at which urease activity apparentlywas not essential for the pathogen, the pH_in increased graduallyfrom 5.8 to 7.5 with increasing pH_out, irrespective of the presenceof urea.

Continuous fluorimetric determination of ${Delta}$ ${Psi}$ . The potentiometric fluorescent probe DiSC₃(5) can be used forqualitative, continuous detection of ${Delta}$ ${Psi}$ of H. pylori (18, 28).This cationic fluorophore accumulates on hyperpolarized membranesand is translocated into the lipid bilayer, resulting in a decreasein fluorescence and absorption shifts (6). At pH 7, additionof H. pylori cells (final OD₅₇₈, $~$ 0.03) led to a 75% quenchingof fluorescence intensity caused by uptake of the fluorescentdye driven by ${Delta}$ ${Psi}$ (Fig. 2A). Addition of 5 mM urea resulted ina marginal further decrease in fluorescence (i.e., increasein ${Delta}$ ${Psi}$ ). The ${Delta}$ ${Psi}$ was stable until its collapse was induced by theaddition of 150 nM TCS, a protonophore. For determination of ${Delta}$ ${Psi}$ of H. pylori cells at lower pH_out values, the fluorimetricmethod turned out to have limited value. At pH 3, addition ofurea did result in a significant quenching of fluorescence,suggesting that there was hyperpolarization (internally negative)(Fig. 2B). However, fluorescence intensity was not stable andincreased with time to a value hardly lower than that priorto addition of urea, whereas the pH_out remained constant atthe low cell density used. Collapse of ${Delta}$ ${Psi}$ was implemented with2% butanol, since TSC was not suitable for uncoupling the cellsat a pH of <4 (data not shown). n-Butanol caused only a smallfurther increase in fluorescence intensity. Similar resultswere obtained at pH_out values of 4 and 5, whereas at pH_out valuesof 1 to 2 no reproducible data were obtained (results not shown).As a control, cells of the acidophilic gram-positive bacteriumAlicyclobacillus acidocaldarius were used, which do not possesssignificant urease activity. Addition of urea to these cellsat pH 3 had no effect on ${Delta}$ ${Psi}$ when the fluorimetric method was used(results not shown). Taken together, these data confirm thataddition of urea to H. pylori cells at a low pH_out leads torapid and significant hyperpolarization (28). We discuss thiseffect below.

View larger version (23K):
[in this window]
[in a new window]
FIG. 2. Fluorimetric determination of ${Delta}$ ${Psi}$ of H. pylori at pH 7 (A) and pH 3 (B). Cells were added at an OD₅₇₈ of $~$ 0.03 to citrate-phosphate buffer containing 1 µM DiSC₃(5). The times when 5 mM urea, 150 nM TCS, and 2% n-butanol were added are indicated. Excitation was at 600 nm, and emission was at 665 nm.

Determination of ${Delta}$ ${Psi}$ via distribution of the lipophilic ions TPP⁺ and SCN^-. A more quantitative analysis of ${Delta}$ ${Psi}$ was performed by using theradioactive lipophilic ions [¹⁴C]TPP⁺ and S¹⁴CN^- (19, 25, 33).The principle for this measurement is the movement of a permeableion across the membrane in response to ${Delta}$ ${Psi}$ until electrochemicalequilibrium is established. An additional advantage over thefluorimetric method is the possibility of obtaining simultaneousmeasurements for several samples, which is especially importantfor H. pylori as aging of cells rapidly leads to a loss of viability.

Figure 3 shows that when [¹⁴C]TPP⁺ was the ${Delta}$ ${Psi}$ probe, H. pylorimaintained a negative internal ${Delta}$ ${Psi}$ in the presence of urea overthe whole pH_out range used, pH 1 to 7. Binding of the radiochemicalwas estimated after addition of n-butanol, which permeabilizescells. By using the data obtained, corrected ${Delta}$ ${Psi}$ values were calculated(Fig. 3B) (38). At a pH_out of 1, ${Delta}$ ${Psi}$ was -26 mV. ${Delta}$ ${Psi}$ increased to-64 mV at a pH_out of 3 (Fig. 3B), remained approximately constantat pH_out values between 3 and 5, and increased further to -140mV at a pH_out of 7. In the absence of urea, ${Delta}$ ${Psi}$ was close to zeroat pH_out values between 1 and 5. At pH_out values of >5 ${Delta}$ ${Psi}$ increasedsteeply to values similar to those observed in the presenceof urea (Fig. 3B).

View larger version (21K):
[in this window]
[in a new window]
FIG. 3. ${Delta}$ ${Psi}$ of H. pylori determined by the distribution of the lipophilic cation [¹⁴C]TPP⁺ across the cytoplasmic membrane after incubation for 8 min in citrate-phosphate buffer at the appropriate pH_out. (A) Apparent values of ${Delta}$ ${Psi}$ ; (B) values after correction for binding as described by Zaritsky et al. (38). Open circles, cells in the absence of urea; solid circles, cells in the presence of 20 mM urea; gray circles, cells in the presence of 2% n-butanol.

The data obtained with the permeant anion S¹⁴CN^- confirmed thatin the presence of urea H. pylori does not invert its ${Delta}$ ${Psi}$ to aninternally positive value. S¹⁴CN^- uptake by the cells was notdiminished by n-butanol (Table 1), indicating that this uptakereflects binding rather than accumulation inside the cytoplasm.Moreover, addition of 10 mM KSCN, which uncouples cells of A.acidocaldarius (19), an observation which we confirmed, didnot have any effect on the pH_in of H. pylori at a pH_out of 2(results not shown). Taken together, these data indicate thatdown to a pH_out of 1 H. pylori maintains an internally negative ${Delta}$ ${Psi}$ , presumably because of the electrogenic extrusion of NH₄⁺ cationsfrom the cytoplasm (see below).

View this table:
[in this window]
[in a new window]
TABLE 1. Apparent ${Delta}$ ${Psi}$ and ratio of total ¹⁴C in the cells and to total ¹⁴C in the medium (A^T_in/A^T_out) for H. pylori determined by using S¹⁴CN^- as a potentiometric probe^a

PMF. PMF can be calculated from ${Delta}$ pH and ${Delta}$ ${Psi}$ with the equation: PMF = ${Delta}$ ${Psi}$ - 61.5( ${Delta}$ pH) (in millivolts at 37°C) (20). By using the datafor ${Delta}$ pH and ${Delta}$ ${Psi}$ corrected for probe binding (Fig. 1B and 3B, respectively),cells suspended in the presence of urea had negative PMF values(-254 to -181 mV) at pH_out values of 1.2 to 3.3. These valuesdropped to -96 and -62 mV at pH_out values of 4.8 and 5.8, respectively,and increased to -143 mV at a pH_out of 7.2. In the absence ofurea the PMF was also low at pH_out values of 4.8 and 5.8 andincreased to about -130 mV at pH_out values of $>=$ 6.3, conditionsunder which H. pylori grows (Table 2).

Urease. The urease activity of intact cells was measured after acidshock by determining the decrease in the urea concentrationin the medium, which was 20 mM at the moment of the acid shock.When the pH_out increased from 1 to 7, the urease activity ofa cell suspension at an OD₅₇₈ of $~$ 0.2 decreased approximatelyexponentially from 0.66 mM urea/min at pH 1 to 0.08 mM urea/minat pH 7 (Fig. 4). Urease activity was also measured in extractsof H. pylori cells which had been exposed to pH_out values of1 to 7 for 30 min in the presence of the concentrations of ureaindicated (Table 3). Control experiments showed that these concentrationswere saturating and not inhibitory. Irrespective of the preincubationconditions, the urease activities in H. pylori cell extractswere similar, and the average value was 57 ± 7.5 µmolmin^-1 mg of protein^-1 (Table 3). These data indicate that theincrease in cell urease activity at a low pH_out was due to activationof an enzyme-transporter function rather than to an increasein gene expression during incubation of the cells at low pH.

View larger version (15K):
[in this window]
[in a new window]
FIG. 4. Urease activity of H. pylori in APM at different pH_out values in the presence of 20 mM urea. Urease activity was measured by determination of urea in the medium as described by Rahmatullah and Boyde (23). OD, optical density.

View this table:
[in this window]
[in a new window]
TABLE 3. Urease activities of H. pylori cell extracts after 30 min of acid shock in the presence of urea^a

For comparative analysis, in vivo urease activity was also calculatedper milligram of cell protein (Fig. 4). The protein contentof a cell extract from 1 ml of a cell suspension (OD₅₇₈, $~$ 0.2)was 36 ± 9 µg (n = 6). According to this calculationthe maximal urease activity in vivo (i.e., at a pH_out of 1)was about 18 U/mg of cell protein, only one-third of the invitro activity in cell extracts ( $~$ 57 U/mg of cell protein) (Fig.4 and Table 3).

The pH optimum of urease from H. pylori cell extract was determinedin different buffer systems (Fig. 5). In sodium phosphate buffersurease activity was maximal at pH 8, which is close to the valuereported for sodium citrate/phosphate buffers (pH 7.4) (28).However, in sodium-MES buffers urease activity exhibited a broadpH maximum between pH 5.5 and 7, and in sodium citrate bufferthe pH optimum was even lower (pH 4.8 to 5). We concluded thatH. pylori urease activity strongly depends on the buffer systemused and has an acidic pH optimum in the absence of phosphate.

View larger version (31K):
[in this window]
[in a new window]
FIG. 5. pH optima of H. pylori urease from cell extracts in different buffer systems. Gray bars, 100 mM citrate-NaOH; solid bars, 100 mM MES-NaOH; cross-hatched bars, 100 mM sodium phosphate. Urease activity was determined by the phenol-hypochlorite assay (35).

DISCUSSION

Top

Discussion

In the presence of urea, cells of H. pylori survive for severalhours at pH 1 when the pH_in is kept at a value close to neutral,indicating homeostasis of pH_in (30). This process is crucialfor the cells, since at low pH_in values cytoplasmic enzymesbecome inactive, as experiments with protonophores at low pH_outvalues with both acidophiles (19) and H. pylori (24, 30) haveshown. Urease has been proposed to play the following role inthis homeostasis of pH_in (29, 30, 37). In the cytoplasm NH₃,a product of the urease reaction, binds protons leaking in fromthe acidic medium. This process occurs because all prokaryotes,including acidophiles, possess a cytoplasmic membrane with somepermeability for protons (33, 34). The NH₄⁺ produced is thenremoved from the cytoplasm via a hypothetical NH₄⁺ efflux system.The present study was carried out to obtain more informationabout this mechanism. To do this, cells were suspended in thepresence or absence of urea in citrate-phosphate buffers ata wide range of pH values. It has been shown previously thatat a pH_out of 2 cells do not lose viability rapidly in thistype of buffer (30). At pH_out values of <4, pH homeostasisof H. pylori depended on urease activity (Fig. 1), which increasedexponentially with the inverse of pH_out (Fig. 4). This observationis consistent with the fact that H. pylori relies on the availabilityof urea for survival at pH_out values of <4 (17). A urea-dependentrise in cellular pH has previously been observed for H. pylorisuspended in buffer at pH 3 to 5 and was attributed to changesin the periplasmic pH (18). Several points argue against thenotion that the changes in cellular pH_in observed in the presentstudy reflect changes in the periplasmic pH: (i) the pH_in valuesreported here for energized and nonenergized H. pylori cellsare very similar to those of a gram-positive organism and severalarchaeal acidophiles (2, 19, 26, 33) which do not possess aperiplasmic space; (ii) protons are expected to move rapidlythrough the outer membrane porins of gram-negative bacteria,thereby restricting the possibility of building up a large pHgradient across this membrane; (iii) if the H. pylori outermembrane had limited permeability, the probes used for measuringthe ${Delta}$ ${Psi}$ and ${Delta}$ pH of H. pylori (Fig. 1, 2, and 3) (14, 18) were notexpected to reach the cytoplasmic membrane, contrary to whatwas observed; and (iv) the anionic form of [¹⁴C]salicylic acidis expected to move rapidly across the H. pylori outer membrane,making this probe unsuitable for detection of a ${Delta}$ pH across thismembrane. Since this probe did, however, detect a urea-dependentand protonophore-sensitive ${Delta}$ pH, we concluded that it detectspH differences across the cytoplasmic membrane and that we measuredpH_in rather than the periplasmic pH of H. pylori in this studyand previously (30).

The exact pH_in in H. pylori during survival at pH 1 is not known.It is between 5.8, the apparent value determined in growth medium(30), and 4.9, the value corrected for probe binding reportedhere (Fig. 1B). The latter value is close to pH 5.0, the valuefor the pH optimum of urease activity in cell extracts assayedin sodium citrate buffer (Fig. 5), and it is higher than pH4.6, the value reported for the extremely acidophilic thermophilicarchaeon Picrophilus oshimae (33). This suggests that ureaseis active under conditions under which it is most needed bythe cells (i.e., at a pH_out of 1) and that this effect contributesto the survival of the organism under extremely acidic conditions.However, the pH optimum of urease activity was strongly dependenton the type of buffer used for the assay (Fig. 5), and at presentit is not known how the ionic composition of the H. pylori cytoplasmaffects the enzyme activity and pH optimum of its urease.

Recently, Ha et al. (8) have reported that in pH 3 buffer H.pylori urease is inactive. However, in nonbuffered, nonstirredsolutions these authors observed some residual activity of theenzyme at pH 3 (8). This observation was taken to support thealtruistic autolysis hypothesis for the survival of H. pyloriat low pH (10). According to this hypothesis, some of the H.pylori cells lyse, thereby releasing their urease into the medium,where it binds to the surface of other, still viable H. pyloricells (8, 10). At a low medium pH and in the presence of ureathis surface-bound urease is thought to create a cloud of ammoniaaround the cells, which protects the cells by binding protons,thereby increasing the local pH just outside the cells to amore neutral value (8, 10). We criticize this concept on severalgrounds. First, the data of Ha et al. do not support the conclusionsof these authors. In their nonbuffered solutions, urease activitywas completely nonproportional to enzyme concentration, reflectingill-defined conditions. Moreover, from their data it can becalculated that at the highest protein concentration the turnoverrate of the urease at pH 3 was only about 2.5% of the maximalturnover rate at neutral pH, raising doubt concerning the contentionthat at low pH the urease activity of the enzyme sticking tothe surface of nonlysed cells is sufficient for creating andmaintaining the cloud of neutralizing ammonia around the cells(8). Second, survival of H. pylori at pH 1 has been observed(30). At pH 1 to 1.3 the stomach contents are well buffered,since it takes about 99 to 29 mM OH^- ions to raise the pH to3. Hence, any results specifically obtained in nonbuffered solutionsat pH 3, such as those reported by Ha et al. (8), are irrelevantfor the survival of H. pylori at pH 1. Finally, we contend thatthe altruistic autolysis hypothesis does not apply to the low-pHconditions under which we observed survival of H. pylori cells.This argument is based on the observation that addition of aprotonophore to cells in either buffer or growth medium at pH1 to 4 eliminates all urease activity as pH_in reaches the samevalue as pH_out (24, 30), suggesting that at these low pH valuesthere is no external urease that is still active. Nevertheless,cells do not lose viability at pH_out values of 1 to 4 beforethe protonophore is added, suggesting that the presence of surface-boundurease is not essential for survival of H. pylori at low pH.

At pH 4 to 7, the pH_in of H. pylori cells was slightly lowerin the presence of urea than in the absence of urea (Fig. 1B).Moreover, in the presence of urea the cells did not developas negative a ${Delta}$ ${Psi}$ as other prokaryotes develop in this pH range(Fig. 3B) and therefore had a low PMF (Table 2). At present,the reason for this result is not clear. It may be an artifactof the methods used. In the case of ${Delta}$ pH, salicylate may not havebeen completely trapped within the cells due to the formationof a pH gradient ranging from the cytoplasm to the externalmedium rather than a steep pH change at the cytoplasmic membraneunder these conditions. However, it is noteworthy that thiseffect has nothing to do with neutralization of the microenvironmentor the periplasm, particularly at an extremely low pH, sinceproton and ammonia diffusion through the outer membrane is expectedto be some orders of magnitude greater than the production ofammonia by urease activity. In addition, it is also importantto mention that H. pylori contains several drug efflux systems(1, 32), and it may very well be that some of the probes usedin the present study for measurement of ${Delta}$ pH and ${Delta}$ ${Psi}$ are exportedfrom the cells by these systems.

A remarkable result of the present study was that unlike acidophiles(2, 33), at pH_out values of <3 H. pylori did not reversethe sign of its ${Delta}$ ${Psi}$ from internally negative to internally positive(Fig. 3 and Table 1). This conclusion disagrees with that ofprevious work, in which an internally positive ${Delta}$ ${Psi}$ was postulatedfor H. pylori at pH 3 (14). We agree with Matin et al. thatat a low pH the cells take up the lipophilic anion SCN^-. However,SCN^- uptake was not diminished by a protonophore or by permeabilizationof the cells with n-butanol (Table 1), indicating that it reflectsbinding to the cells rather than accumulation in the cytoplasm.The question that then arises is, by which mechanism is theinternally negative ${Delta}$ ${Psi}$ generated. It depends on the presence ofurea in the medium. Moreover, addition of urea to the cellscauses immediate hyperpolarization (internally negative) ofthe cells at low pH_out (28) (Fig. 2). We propose that this hyperpolarizationis caused by the electrogenic efflux of NH₄⁺ cations by a hypotheticaltransporter. However, further work, including identificationof such a transport system, is needed to show whether this proposalis correct.

Finally, our finding that even at pH 1 the urease activity ofintact cells was only one-third of the urease activity in cellextracts prepared from the equivalent number of cells (Fig.4 and Table 3) has important implications for the mechanism(s)by which urease activity is regulated. It shows that besidesthe well-documented pH-dependent activation of the urea carrierUreI (24, 27, 36) and the possible direct activation of ureaseactivity by low pH_in (this study), other still unknown factorsmay regulate urease activity in H. pylori cells.

ACKNOWLEDGMENTS

We thank L. Herrmann and K. Melchers (Byk Gulden, Konstanz,Germany) for helpful discussions.

This work was supported by the Fonds der Chemischen Industrie.

FOOTNOTES

* Corresponding author. Mailing address: Abteilung Mikrobiologie, Universität Osnabrück, D-49069 Osnabrück, Germany. Phone: 495419692867. Fax: 495419692870. E-mail: stingl{at}biologie.uni-osnabrueck.de.

REFERENCES

Top