Gut 1997; 40: 25-30

Synthesis and activity of Helicobacter pyloni ureaseand catalase at low pH

P Bauerfeind, R Garner, B E Dunn, H L T Mobley

AbstractBackground-Helicobacter pylori pro-duces large amounts ofurease presumablyto be prepared for the rare event of asudden acid exposure. The hypothesis thatHpyloni is acid sensitive and protein pro-duction is inhibited by low pH wasexamined.Methods-H pyloni or its soluble enzymeswere incubated buffered or unbuffered ata pH ranging from 2-7 in the presenceof 5 mM urea for 30 minutes. Afterexposure, urease and catalase activitiesof whole cells, supernatants, and solubleenzyme preparations were measured atpH 6-8. Newly synthesised enzyme wasquantified by immunoprecipitation of[35S]-methionine labelled protein.Results-Exposure to buffer below pH 4resulted in loss of intracellular ureaseactivity. In soluble enzyme preparationsand supernatant, no urease activity wasmeasurable after incubation at pH<5.In contrast, catalase in whole cells,supernatant, and soluble enzyme prep-arations remained active after exposure topH>3. Exposure below pH 5 inhibitedsynthesis of total protein includingnascent urease and catalase. At pH 6 or 7,urease represented 10% of total protein,catalase 1/5%. Exposure of H pylon tounbuffered HCI (pH>2) resulted in animmediate neutralisation; urease andcatalase activities and synthesis wereunchanged.Conclusion-Low surrounding pH reducesactivity of urease and synthesis of nascenturease, catalase, and presumably of mostother proteins. This suggests that Hpyloniis not acidophilic although it toleratesshort-term exposure to low pH.(Gut 1997; 40: 25-30)

Keywords: Helicobacterpylori, pH, urease, catalase.

Helicobacterpylori causes chronic active gastritis,and is closely associated with the developmentof peptic ulcer disease.' 2 The bacteriumexclusively colonises gastric mucosa includingareas with and without acid secretion. In vivoand in vitro, H pylori produces large quantitiesof urease,36 which converts urea into am-monium permitting neutralisation of gastricacid.7 Native urease of Hpylori has a molecularmass of approximately 550 kilodalton (kDa)and is a nickel containing protein consisting ofsix copies each of two subunits (UreA-30 kDa

and UreB-62 kDa) in a one:one molar ratio.R 9The low Michaelis constant (K1m) of H pyloriurease permits this enzyme to be catalyticallyefficient even at low urea concentrations.8 9Urease is clearly central to the pathogenesis ofHpylori infection. This activity is necessary forthe survival of H pylori in acidic environmentin vitro.'0 Although urease negative H pylorimutants were unable to colonise the gastricmucosa in an animal model," this was pHindependent. Thus, the role of urease in vivois probably not only its acid neutralisingcapacity.

Nevertheless, protection against acidity seemssufficiently important, that H pyloni producesurease in large quantities. This is even moresurprising because H pyloni is usually foundunderneath the mucous layer where pHapproaches neutrality.'2 Thus, exposure to lowacidity seems to be a very rare event occurringonly if the mucous layer is damaged mech-anically or by chemical agents such as aspirin'3or during first infection. However, the route ofinfection is unknown and it might happenduring food intake, when gastric pH is alreadyraised.'4 Thus, although neutralisation of acidis probably rarely necessary, large amounts ofurease is synthesised even in a situation whereno acid is present.Based on these findings we hypothesised that

H pylori is very sensitive to acid exposure andthe large stock of urease is necessary to protectHpylori against a sudden drop in pH where notime is available to up regulate production ofthe enzyme.

In contrast, in Streptococcus salivarius a ureasepositive bacterium found in dental plaque,regulation of urease levels by pH has beendemonstrated."' Other species regulate ureasesynthesis in response to ureal618 or availablenitrogen concentration.'9 In H pylori andEscherichia coli containing cloned H pyloniurease genes, neither nitrogen limitation noraddition of urea seen to stimulate synthesis ofthe urease structural subunits.8 20 21The aim of this study was to evaluate the

effect of exposure ofHpylori cells to pH values,which vary within the physiological gastricrange from 2 to 7, on the synthesis and activityof urease. The direct effect of different pHvalues on the activity of crude enzyme wastested by exposing fresh preparations of solubleH pylon enzymes to the same pH values asintact H pylori cells. Experiments were con-ducted in buffered and unbuffered solutionsboth containing urea. Exposure to bufferedsolutions allowed us to evaluate the effect ofexposure to a constant pH even in the presence

Division of InfectiousDiseases,University ofMarylandSchool ofMedicine,Baltimore,Maryland, USAP BauerfeindR GamerH LT Mobley

Pathology andLaboratory MedicineService,Clement J Zablocki VAMedical Center,Milwaukee,Wisconsin, USAB E DunnCorrespondence to:Dr P Bauerfeind,Department of InternalMedicine,Division of Gastroenterology,University of Zurich,Rdmistrasse 100,CH-8091 Zurich,Switzerland.Accepted for publication28 August 1996

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of urea hydrolysis. In experiments using theunbuffered solutions, bacterial ammonia wasexpected to increase the pH gradually. Theselatter experiments allowed us to study theeffect of a transient exposure to low pH as itmay occur in the stomach.For comparison with urease, catalase was used

in this study to control for non-specific effectsof low surrounding pH on enzyme synthesisand activity. By analogy to other bacteria,catalase is assumed to protect H pylori againstthe damaging effect of hydrogen peroxide22 byconverting H202 to H20+02. The nativecatalase of H pylori has a molecular mass of165-200 kDa; subunits have a molecular massof 50-52 kDa.22 No evidence exists thatcatalase production is regulated by the sur-rounding pH in H pylori or other bacterialspecies.

Methods

Bacterial strainsH pylori strain UMAB41 was isolated fromgastric biopsy specimens from a patient under-going endoscopy at the University of Maryland,School ofMedicine, Baltimore, Maryland. Thestrain has been characterised as reportedearlier.8 23

Culture conditions andpreparation of bacterialsuspensionsCultures were grown on 10% sheep blood agarsupplemented with vancomycin (10 p,g/ml),polymyxin (2.5 IU/ml), and trimethoprimlactate (5 pug/ml).24 Cultures were incubated at37°C in a microaerobic environment generatedby an activated Campypak (Becton-Dickinson,Baltimore, MD). Hpylori were cultured for 72hours before use in experiments.

Exposure ofH pylori cells to variouspH steps andmeasurement of urease and catalase activity incells and supemnatantBacteria were suspended to an OD600 of 2-5 in2 ml HCI adjusted to an initial pH of 2, 3, 4or 5, or in 0 1 M sodium phosphate at pH 6or 7 or in 0 1 M citrate-phosphate buffer at apH of 2, 3, 4, 5, 6 or 7. All solutions contained5 mM urea (Sigma) and had an osmolarity of300 mmol. The pH was controlled andcorrected if necessary before each experiment.H pylori cells were incubated in the varioussolutions for 30 minutes under microaerobicconditions at 37°C. After incubation, sampleswere taken for determination of viable counts(CFU/ml) by direct plating of 10-folddilutions. Cells and supematant were separatedby centrifugation (5000 rpm, 20 min, 4°C).The pH of the supematant was determinedusing a pH meter (Coming, model 320). Cellswere washed twice and lysed in a Frenchpressure cell at 20000 psi (SLM Aminco,Urbana, IL). Proteins in the supernatant wereconcentrated by filtration using a centrifugalconcentrator (Centrisart I, Sartorius, Goettingen,Germany) with a cut off size of 20 kDa.

Concentrated proteins were solubilised in20 mM sodium phosphate buffer, pH 6-8.Enzyme activities were determined for wholecells, soluble enzyme preparations, and super-natants as described below immediately aftereach experiment.

Exposure ofH pylori whole cells to various pHvalues and measurement of urease and catalasesynthesis by immunoprecipitationIn a second series of experiments thatinvolved radiolabelling of proteins, H pyloricells were deprived of L-methionine by pre-paring suspensions of the bacteria (OD600=2-5)in a L-methionine free medium (Difco) andincubating the suspensions for 90 minutesat 37°C under microaerobic conditions. Afterincubation of the cells in the various pHsolutions, incubations were continued afteraddition of 63 ,uCi of [35S]-methionine (>600Ci/mmol; Dupont, New England Nuclear,Boston, MA). After incubation for 30 minutes,labelling was stopped by addition of unlabelledL-methionine to a final concentration of 8 mg/ml. Cells and supematant were separated; cellswere washed twice and resuspended in 200 RIof 50 mM TRIS-HCI buffer containing 1%SDS, pH 8, and lysed by boiling for 10minutes. Soluble protein was used for im-munoprecipitation as described below.

Exposure ofH pylori cytosolic enzymes tosolutions of various pHs and measurement ofurease and catalase acitivityH pylori cells were harvested from plates andlysed by passage through a French pressure cellat 20 000 psi. After centrifugation (12 000 rpm,5 min, 4°C), 50 [lI of soluble enzyme (approxi-mately 150 pug protein) were added to 2 ml ofeach HCI, phosphate, or citrate buffer adjustedto various pH values; solutions were incubatedfor 30 minutes as described in the first experi-ment. After incubation, cellular proteins wereseparated from the buffer solutions as describedabove. Enzyme activities were determinedas described below immediately after eachexperiment.

Urease activityUrease activity was measured using the phenolred assay of Hamilton-Miller and Gargan25 ascalibrated for quantitative determination asdescribed previously.26 Rates of urea hydrolysisare expressed as mmol NH3/min/mg protein.For each experiment, protein concentrationwas determined by the BCA method accordingto the instructions of the manufacturer (Pierce,Rockford, IL) using bovine serum albumin asa standard.

Catalase activityCatalase activity was determined by observingthe disappearance of H202 as measured by itsabsorbance at 240 nm.27 The 2 ml reactionmixture contained 11 mM H202 in 10 mMpotassium phosphate, pH 6-8. The reaction

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was started by adding 5 RI of soluble enzymepreparation or concentrated supernatants.Absorption was monitored for one minute at23°C. Based on the initial rate, catalase activitywas expressed as the disappearance of H202 inmmol/min/mg protein. The calculation wasbased on a standard curve determined from theabsorption of 0-11 mM H202 under the sameconditions. The results were normalised forthe protein concentration of the enzymepreparations.

Statistical analysisResults for enzyme activities and results ofimmunoprecipitation studies were comparedby the non-parametric Kruskal-Wallis test.30If this test showed statistical significance(p<0 05), individual comparisons were per-formed using the test procedure described byNemenyi.30

Results

AntiserumPolyclonal antisera were raised against thesmall (UreA) and large (UreB) subunits ofthe H pyloni urease in rabbits using purifiedurease subunits as previously described.28 Forimmunoprecipitation of catalase, polyclonalgoat antiserum against H pylori catalase wasused. The specificity of the antisera was con-firmed by western blot. Pre-immune sera wereused as controls.

ImmunoprecipitationH pylori bacterial cells, labelled with [35S]-methionine as described above, were used forimmunoprecipitation. Cells were washed twiceand lysed by boiling for 10 minutes.

Immunoprecipitation was performed accord-ing to a standard protocol.29 Briefly, 10 [lI of celllysate or supernatant were counted to deter-mine total counts per pl in each sample. Forimmunoprecipitation the different amountsof total cpms were standardised by using100 000 cpms in each set up. [35S]-labelledsoluble proteins were incubated for one hourat 4°C in 400 ml of 50 mM TRIS-HCI(pH 8-0), 2% (v/v) Triton X 100, 0-15 MNaCl, 0 1 M EDTA containing urease antisera(1:50 v/v of anti-UreB (recognises the largesubunit of urease)), 1:25 v/v of anti-UreA(recognises the small subunit of urease), anti-catalase serum (1:50 v/v), or pre-immunisecontrol sera. After one hour, 50 pI protein A(10% w/v) (Gibco BRL, Gaithersburg, MD)was added and incubation was continued at 4°Cfor 30 minutes. For immunoprecipitation ofcatalase, protein G (Gibco BRL, Gaithersburg,MD) was used because previous tests showeda higher yield for precipitation of the catalaseantiserum than protein A. After incubation,precipitated antibody-antigen complex andsupernatant were separated by centrifugation(5000 rpm, 5 min, 23°C), washed twice withthe Triton X-100 containing buffer, re-suspended in 100 pl of 50 mM TRIS-HCl,pH 8-0 containing 1% SDS, boiled for 10minutes, and counted by liquid scintillation.Counts obtained from incubation with pre-immunised control sera were subtracted fromthe counts obtained for specific anti-urease oranti-catalase sera. Values for immunoprecipi-tated urease and catalase were then expressedas percentage of total counts. Thus, the resultsrepresent the percentage of protein as ureaseor catalase from total protein produced by theH pylori cells during the period of incubationin the solutions of various pH.

Duration ofacid exposure to H pyloriConditions were established whereby H pyloriwas either briefly exposed to acidic conditionsor where acidic conditions were held constantdespite active urea hydrolysis. When H pyloriwas incubated in unbuffered HC1 of pH 2containing 5 mM urea, pH increased duringfive minutes to values around 4 (Fig 1).Incubation in HC1 of pH 3, 4 or 5 containing5 mM urea increased the pH to values above7 within the first minute. The increase in pHwas assumed to be due to neutralisation ofunbuffered HC1 by ammonia produced byhydrolysis of urea. The pH of citrate or phos-phate buffers, however, remained unchangedduring 30 minutes of incubation with H pylori.Although all buffers contained 5 mM urea,neutralisation by ammonia did not change thepH.

Viability ofH pylori after incubation at variouspHsSurvival of H pylori after incubation for30 minutes in citrate buffer, phosphate buffer,or unbuffered HC1 is shown in Figure 2.With a starting inoculum of approximately2 X 109 cfu/ml, only exposure to pH 2citrate buffer reduced viability significantly(p<0 05). A tendency for reduced viabilitywas also found after exposure to pH 3 citratebuffer or pH 2 unbuffered HC1 compared withviability after exposure to pH 7. However,these differences were not statistically sig-nificant.

9

8

7

6

I 5

4

3

2

-pH5

pH4

-pH3

-pH2

"

0 1 2 3 4 5 30Time (min)

Figure 1: pH of unbuffered HCI solutions ofpH 2, 3, 4,and 5 during incubation ofH pylon cels. H pylon wassuspended in HCI solutions ofvariouspH containing S mMurea. pH was measured over time; the initialpH is given atthe start ofeach curve. Each point represents the mean(SD) offour experiments, unless SD is smaller than thepoint.

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L

0

10 ; 10°

6-6

4 Hydrochloric Phosphate 4 _32 acid buffer 2- cit

2 3 4 5 6 7 23

pHFigure 2: Survival ofH pylori in citrate buffer,HCl, or phosphate buffer containing 5 mM ureapHs. Suspensions ofH pylori UMAB41 (109 cincubatedfor 30 minutes in unbuffered HCl at Iphosphate buffer atpH 6 and 7 (left graph) or catpH 2-7 (right graph); all solutions containecurea. Viability was determined by plating 1O-foof the suspensions. The x axis represents the initsolution; they axis represents the viability afterfor 30 minutes at 37°C given as log,o cfu. Eachrepresents one experiment. Survival was significreduced when H pylori was exposed topH 2 inbuffer compared with pH 7 (*, p<0 01 tested b3Wallis test, followed by Nemenyi test). There w4tendencyfor reduced survival atpH 3 in citrateatpH 2 in HCl but these differences were not stisignificant.

Urease and catalase activityActivities of urease and catalaseH pylori cells incubated for 30 m

buffered or unbuffered solutions c

pHs are shown in Figure 3. Ureaswas expressed as mmol NH3/min/mjCatalase activity was measured asposition of H202 in mmol/min/mg pIenzyme measurements were deterpH 6-8 and 23°C. Urease activity wain the cells after incubation of H pyhcitrate buffer at pH 2 or 3 or after ixin HCI at pH 2. For all other conditioactivity was similar to those ofincubated in neutral citrate or I

buffers.Catalase activity was reduced whei

cells were incubated in citrate bufferIn contrast with the results obtained

90 _ HCI~~~~~~~~~1.5

65

90- Citrate1.0 bul40-L

Phosphate 0 0.5-15

vzCitrate buffer A I 1.5 _

~~~~~ II 50

-10E

2 3 4 5 6 7 2 3 4 5pH pH

Figure 3: Urease and catalase activities in H pylori whole cells after incubationphosphate buffer, or citrate buffer at different pHs. H pylori was suspended in hpH 2, 3, 4, 5 orphosphate buffer atpH 6, 7 (upper panels) or in citrate buffer ,

(lower panels) and incubatedfor 30 minutes. All solutions contained 5 mM ureilysed and activity of urease (left panels) and catalase (right panels) activities wmeasured atpH 6-8 at 23°C. Results ofsix experiments are displayed as box whThe median is represented by the horizontal line; the top and bottom edge of the irepresent the 25 and 75 percentile; the whisker represents the 10 and 90 percentiithe circles extending awayfrom the boxes represent minimal and maximal value*Significant differencefrom value obtained atpH 7.

activity, catalase activity was not affected by3 S @ | incubation at pH 3 or after incubation in HCl

at pH 2.To evaluate whether release of enzyme from

whole cells was responsible for the reduction inIrIeIfe enzyme activity at certain pH levels, enzyme4 5 6 7 activity was also measured in the supematant.pH Similar to the results in the intracellularunbuffered enzyme activity, reduced urease activity was

a at various found after incubation in citrate buffer at pHfulmi) were 2 and 3. Catalase activity was also reduced inptH 2-5 or the supematant after incubation in citratecitrate bufferi5 mM buffer at pH 2. Under none of the conditionsid dilutions was an increase of enzyme activity found asialpH of the compared with the results at pH 7.incubationpoint,antlycitrate Synthesis of nascent urease and catalasey Kruskal- Newly synthesised enzyme was measuredbuffer and by immunoprecipitation of [35S]-methionineatistically labelled protein. Labelling was performed

during incubation in solutions of various pH.As shown in Figure 4, incubation in unbufferedHCI or phosphate buffer ofpH 6 or 7 did not

of whole affect urease or catalase production. Ureaseiinutes in and catalase represented approximately 10%)f various and 1% respectively, of newly synthesised;e activity protein in unbuffered HC1 (pH 2-5) andg protein. phosphate or citrate buffer (pH 6-7). Ins decom- contrast, incubation in citrate buffer, pH 2-5rotein. All significantly inhibited nascent synthesis ofmined at urease and catalase.Ls reduced To control for any loss of newly synthesisedwri cells in enzyme by release into the supematant,ncubation immunoprecipitation of urease and catalaseIns, urease was also performed using the supematant.samples Although comparatively more urease and

phosphate catalase was found in the supematant at lowpH in citrate buffer, the total amount of

n Hpylori precipitated counts at pH 2, 3, 4 and 5 inat pH 2. citrate buffer was lower than 10% compared

for urease with the counts at pH 6 or 7. Thus, thereduced amount of precipitated urease orcatalase from the cells at low pH was not dueto a loss of proteins into the supematant.

Urease and catalase stabilityPhosphate The Table shows the activity of the crudeIbuffer cytosolic enzyme after incubation in solutions6 7 of various pH values. Enzyme activities were

determined after raising the pH in all groupsto 6f8. Urease activity was reduced after

ffer exposure ofthe enzyme to citrate buffer at a pHbelow 5. In contrast, catalase activity was only

q3 affected by exposure to pH 2 in HC1. Thus,exposed urease is not resistant to the effectsof low pH unless it is cell associated orcytoplasmic, while catalase is relatively more

JL resistant.6 7

in HCI, DiscussionICI at The natural environment of H pylori isatpH 2-7 the human gastric mucosa. The bacterium isa. Cels wereere usually found beneath the mucus layer in anisker plots. area where the pH approaches neutrality. "box Nevertheless, it could be assumed that Hpyloris and

might be exposed suddenly for short periods tolower pH values either during disruption of

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Phosphatebuffer

0

0

0 cE 1

00

0

C.

* 0

+,o 2

0

m0.15

a)0 0

Co

,o* a 1.5

a)

*ux

00

HCI

1.5 _

0 * .

0

0

00

2 3 4 5

Citrate bufferH

seconds, we hypothesised that urease is con-Phosphate stitutively produced. As the bacterium pro-

buffer duces large quantities of urease even duringperiods where there is no exposure to lowpH, we hypothesised that survival and its

* S vital functions depend on the availability ofurease and its protective function. Thus, we

* * hypothesised that H pyloni's protein synthesisand urease activity are very sensitive to low pH.To test this hypothesis, H pyloni cells wereexposed to pH values ranging from 2 to 7, the

6 7 same pH range that is likely to occur in thehuman stomach.'4 We used buffered and

* unbuffered solutions to obtain conditions ofstable pH as well as gradually increasing pH,

* respectively. Exposure to unbuffered hydro-chloric acid mimicked the probable physio-logical events in the stomach where the pHmay be quickly changed by neutralisation with

0

00.5 _

0

a I a

2 3 4 5 6 7

pH

0

I~~~t I I2 3 4 5 6 7

pH

Figure 4: Nascent enzyme synthesis during incubation ofH pylori at various pHs.Suspensions ofH pylori in HCl atpH 2, 3, 4, 5 orphosphate buffer atpH 6, 7(upper panels) or in citrate buffer atpH 2-7 (lower panels) were labelled withf S]-methionine. All solutions contained 5 mM urea. Urease and catalase synthesiswas determined by immunoprecipitation using polyclonal antiserum directed against thesmall (UreA) and large (UreB) structural subunit ofH pylori urease and against theH pylori catalase holoenzyme. Each data point represents one of three independentexperiments.

the mucous layer or during transmission fromperson to person. H pylori must survive lowsurrounding pH, at least transiently, by itsability to hydrolyse urea into ammonia, whichin turn neutralises HCI and increases the pHadjacent to the bacterium. This finding wasreported earlierl' and confirmed in this study.In contrast, urease negative H pylori does notsurvive exposure to low pH3' and is unable tocolonise the stomach in an animal model."As urease activity seems crucial for the

survival and transmission of the bacterium andexposure to very low pH may happen within

Effect ofexposure to variouspH values on soluble ureaseand catalase activities

Enzyme activity*(% of activity atpH 7)

Buffer pH Urease Catalase

HCI 2 0 sig 9 sigHCI 3 65 95HCI 4 77 93HCI 5 87 82Phosphate 6 75 84Phosphate 7 100 100Citrate 2 0 sig 55Citrate 3 0 sig 42Citrate 4 0 sig 103Citrate 5 18 102Citrate 6 81 108Citrate 7 100 100

*Hpylori soluble enzyme preparations were incubated inHCI, phosphate buffer, or citrate buffer containing 5 mMurea. After 30 minutes, the pH was adjusted to pH 6-8 andenzyme activities were measured. Median results from fourexperiments are expressed as a percentage of enzyme activityobtained at pH 7 0. (sig, significantly different from the pH 7value, p<005).

ammonia.The effect of pH on the enzymes was

examined at three levels: (a) the effect on theenzyme activity when crude cytosolic enzymepreparations were exposed directly to low pH;(b) the effect on intracellular enzyme activitywhen intact Hpyloni cells were exposed to suchconditions; and (c) the effect on the synthesisof nascent enzyme during exposure of intactHpylori cells to various pH levels.The results of this study supports the initial

hypothesis. As also shown by others,3' pro-longed exposure to low pH reduces viability.Furthermore, exposure ofH pylori to low pHdecreases protein synthesis, including ureaseand catalase. Exposure to low pH alsoabolishes activity of free and cellular urease.The detrimental effect of low pH depends onthe duration of exposure and acid concen-tration. However, the inhibition of proteinproduction seems to be reversible becauseviability was not affected by exposure to pH 3,4, or 5. Free catalase seemed to be more stablethan urease when exposed directly to low pH;only exposure to pH 2 reduced catalase activitysignificantly.

Intracellular enzyme activity is defined bythe sum of the rates of new synthesis,degradation, and release. Low pH inhibitedproduction of both urease and catalase. Thus,intracellular enzyme activity measured afteracid exposure was due to preformed enzyme.After exposure to citrate buffer at pH 2,bacterial viability and activities of bothenzymes were considerably reduced. Underthis condition, many bacterial cells were lysedand the released enzyme was inactivated by thelow pH as shown in the experiments where freeenzymes were exposed to low pH. The mostinteresting events occurred after exposure topH 3. Here, viability was not significantlydecreased, and catalase activity was only mildlydecreased. As only intracellular urease activitywas reduced, it could be assumed that theintracellular pH was reduced to a level wherethe intracellular enzyme was inactivated. Asshown in the experiments with free enzyme,this presumably occurs below a pH of 5. Afterexposure of the H pyloni cells to citrate bufferof pH 4, intracellular urease remained activewhereas free enzyme was destroyed showing

20 r

HCI15 -

10 .a

.

0

0I0

0

00

5

0

0

2 3 4 5

._

00

C4-0

~0

4-01-O

axn

Cor-

20

Citrate buffer15 --

10 H

5

0

29

2r

4

I

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that the cells were able to cope with thatextracellular pH and maintain the intracellularpH at a physiologically functional level.Although we did not measure intracellularpH, the comparison of intracellular ureaseactivity and activity of free urease at variouspH levels allows us to speculate indirectlyon the intracellular pH of H pyloni afterexposure to low pH. Hpylori seems to maintainan intracellular pH above 5 if exposed to apH of 4. This also explains why H pylori isable to grow at low pH values.32 It furtherseems that H pylori restores its normal intra-cellular pH after a short-term exposure topH below 4 permitting normal protein pro-duction and survival. The precise mechan-isms of maintenance of intracellular pH inH pylori are unknown. However, a H pylori Ptype ATPase has recently been characterised,33which is probably a cation transporter andmight be responsible for the regulationcytoplasmic pH.Although H pyloni can survive transient

periods of low surrounding pH, both ureaseproduction and urease stability are sensitive tolow pH. In contrast with other urease positivebacteria such as Streptococcus salivarius,'5urease production is not stimulated duringexposure to low pH. The amount of enzymeactivity in the cell is reduced when H pylori isexposed to low pH depending on duration ofexposure and level of acidity. Brief exposure tolow unbuffered acidity above pH 2 neithercompletely inhibited urease activity nor stimu-lated it. These findings are in some contrastwith the idea that the major role of H pyloriurease is its protective function against acidity.However, the extreme range of pH in itsnatural environment and the acid sensitivity ofH pyloni explains why such high amounts ofurease are prepared even without precedingacid exposure. H pyloni seems not to beacidophilic and needs the stock of urease for itsprotection against a sudden drop in pH. Assoon as the short-term episode of low pH isovercome, the bacterium is able to restore itsnormal intracellular pH and to replace thedestroyed proteins.Our findings support the concept that

H pyloni is not acidophilic and does not seemto thrive in an acidic environment. A keyenzyme of the organism, urease, is not acidstable and its synthesis is not stimulated bylow pH. In addition, the metabolism ofthe bacterium, as measured by incorporationof radiolabelled methionine into protein,functions optimally at near neutral pH and isdramatically reduced at low pH. Indeed, thepreferred environment would seem to be underthe mucous layer of the gastric mucosa wherethe pH may approach neutrality. There is noevidence that the ulcerogenic activity of acid isamplified by stimulation of the bacterialvirulence factor urease.

This work was supported by Schweizerische Stiftung furmed biol Stipendien and Public Health Service GrantsCA67497 and CA67527 and AI23328 from the NationalInstitutes of Health.

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