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Gut 1997; 40: 25-30 25

Synthesis and activity of Helicobacterpyloni and at low pH Gut: first published as 10.1136/gut.40.1.25 on 1 January 1997. Downloaded from

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

Abstract and UreB-62 kDa) in a one:one molar ratio.R 9 Background- pylori pro- The low Michaelis constant (K1m) of H pylori duces large amounts ofurease presumably urease permits this to be catalytically to be prepared for the rare event of a efficient even at low concentrations.8 9 sudden acid exposure. The hypothesis that Urease is clearly central to the pathogenesis of Hpyloni is acid sensitive and pro- Hpylori infection. This activity is necessary for duction is inhibited by low pH was the survival of H pylori in acidic environment examined. in vitro.'0 Although urease negative H pylori Methods-Hpyloni or its soluble mutants were unable to colonise the gastric were incubated buffered or unbuffered at mucosa in an animal model," this was pH a pH ranging from 2-7 in the presence independent. Thus, the role of urease in vivo of 5 mM urea for 30 minutes. After is probably not only its acid neutralising exposure, urease and catalase activities capacity. of whole cells, supernatants, and soluble Nevertheless, protection against acidity seems enzyme preparations were measured at sufficiently important, that H pyloni produces pH 6-8. Newly synthesised enzyme was urease in large quantities. This is even more quantified by immunoprecipitation of surprising because H pyloni is usually found [35S]-methionine labelled protein. underneath the mucous layer where pH Results-Exposure to buffer below pH 4 approaches neutrality.'2 Thus, exposure to low resulted in loss of intracellular urease acidity seems to be a very rare event occurring activity. In soluble enzyme preparations only if the mucous layer is damaged mech- and supernatant, no urease activity was anically or by chemical agents such as aspirin'3

measurable after incubation at pH<5. or during first infection. However, the route of http://gut.bmj.com/ In contrast, catalase in whole cells, infection is unknown and it might happen supernatant, and soluble enzyme prep- during food intake, when gastric pH is already arations remained active after exposure to raised.'4 Thus, although neutralisation of acid pH>3. Exposure below pH 5 inhibited is probably rarely necessary, large amounts of synthesis of total protein including urease is synthesised even in a situation where nascent urease and catalase. At pH 6 or 7, no acid is present.

urease represented 10% of total protein, Based on these findings we hypothesised that on September 29, 2021 by guest. Protected copyright. catalase 1/5%. Exposure of H pylon to H pylori is very sensitive to acid exposure and unbuffered HCI (pH>2) resulted in an the large stock of urease is necessary to protect immediate neutralisation; urease and Hpylori against a sudden drop in pH where no catalase activities and synthesis were time is available to up regulate production of unchanged. the enzyme. Conclusion-Low surrounding pH reduces In contrast, in salivarius a urease Division ofInfectious activity of urease and synthesis of nascent positive bacterium found in dental plaque, Diseases, and of most regulation of urease levels by pH has been University ofMaryland urease, catalase, presumably School ofMedicine, other . This suggests that Hpyloni demonstrated."' Other species regulate urease Baltimore, is not acidophilic although it tolerates synthesis in response to ureal618 or available Maryland, USA short-term exposure to low pH. concentration.'9 In H pylori and P Bauerfeind R Gamer (Gut 1997; 40: 25-30) containing cloned H pyloni H L T Mobley urease genes, neither nitrogen limitation nor Keywords: Helicobacterpylori, pH, urease, catalase. addition of urea seen to stimulate synthesis of Pathology and Laboratory Medicine the urease structural subunits.8 20 21 Service, The aim of this study was to evaluate the Clement J Zablocki VA Helicobacterpylori causes chronic active gastritis, effect of exposure ofHpylori cells to pH values, Medical Center, Milwaukee, and is closely associated with the development which vary within the physiological gastric Wisconsin, USA of .' 2 The bacterium range from 2 to 7, on the synthesis and activity B E Dunn exclusively colonises gastric mucosa including of urease. The direct effect of different pH Correspondence to: areas with and without acid secretion. In vivo values on the activity of crude enzyme was Dr P Bauerfeind, Department of Internal and in vitro, Hpylori produces large quantities tested by exposing fresh preparations of soluble Medicine, of urease,36 which converts urea into am- H pylon enzymes to the same pH values as Division of Gastroenterology, University ofZurich, monium permitting neutralisation of gastric intact H pylori cells. Experiments were con- Rdmistrasse 100, acid.7 Native urease ofHpylori has a molecular ducted in buffered and unbuffered solutions CH-8091 Zurich, Switzerland. mass of approximately 550 kilodalton (kDa) both containing urea. Exposure to buffered Accepted for publication and is a containing protein consisting of solutions allowed us to evaluate the effect of 28 August 1996 six copies each of two subunits (UreA-30 kDa exposure to a constant pH even in the presence 26 Baue,fiWnd, Gamer, Dunn, Mobley

of urea . In experiments using the Concentrated proteins were solubilised in unbuffered solutions, bacterial was 20 mM sodium phosphate buffer, pH 6-8. expected to increase the pH gradually. These Enzyme activities were determined for whole latter experiments allowed us to study the cells, soluble enzyme preparations, and super- effect of a transient exposure to low pH as it natants as described below immediately after Gut: first published as 10.1136/gut.40.1.25 on 1 January 1997. Downloaded from may occur in the stomach. each experiment. For comparison with urease, catalase was used in this study to control for non-specific effects of low surrounding pH on enzyme synthesis Exposure ofH pylori whole cells to various pH and activity. By analogy to other , values and measurement ofurease and catalase catalase is assumed to protect H pylori against synthesis by immunoprecipitation the damaging effect of hydrogen peroxide22 by In a second series of experiments that converting H202 to H20+02. The native involved radiolabelling of proteins, H pylori catalase of H pylori has a molecular mass of cells were deprived of L-methionine by pre- 165-200 kDa; subunits have a molecular mass paring suspensions of the bacteria (OD600=2-5) of 50-52 kDa.22 No evidence exists that in a L-methionine free medium (Difco) and catalase production is regulated by the sur- incubating the suspensions for 90 minutes rounding pH in H pylori or other bacterial at 37°C under microaerobic conditions. After species. incubation of the cells in the various pH solutions, incubations were continued after addition of 63 ,uCi of [35S]-methionine (>600 Methods Ci/mmol; Dupont, New England Nuclear, Boston, MA). After incubation for 30 minutes, Bacterial strains labelling was stopped by addition of unlabelled H pylori strain UMAB41 was isolated from L-methionine to a final concentration of 8 mg/ gastric biopsy specimens from a patient under- ml. Cells and supematant were separated; cells going endoscopy at the University of Maryland, were washed twice and resuspended in 200 RI School ofMedicine, Baltimore, Maryland. The of 50 mM TRIS-HCI buffer containing 1% strain has been characterised as reported SDS, pH 8, and lysed by boiling for 10 earlier.8 23 minutes. Soluble protein was used for im- munoprecipitation as described below. Culture conditions andpreparation ofbacterial suspensions Exposure ofH pylori cytosolic enzymes to

Cultures were grown on 10% sheep blood agar solutions ofvarious and measurement of http://gut.bmj.com/ supplemented with vancomycin (10 p,g/ml), urease and catalase acitivity polymyxin (2.5 IU/ml), and trimethoprim H pylori cells were harvested from plates and lactate (5 pug/ml).24 Cultures were incubated at lysed by passage through a French pressure cell 37°C in a microaerobic environment generated at 20 000 psi. After centrifugation (12 000 rpm, by an activated Campypak (Becton-Dickinson, 5 min, 4°C), 50 [lI of soluble enzyme (approxi- Baltimore, MD). Hpylori were cultured for 72 mately 150 pug protein) were added to 2 ml of citrate adjusted hours before use in experiments. each HCI, phosphate, or buffer on September 29, 2021 by guest. Protected copyright. to various pH values; solutions were incubated for 30 minutes as described in the first experi- Exposure ofH pylori cells to various pH steps and ment. After incubation, cellular proteins were measurement ofurease and catalase activity in separated from the buffer solutions as described cells and supemnatant above. Enzyme activities were determined Bacteria were suspended to an OD600 of 2-5 in as described below immediately after each 2 ml HCI adjusted to an initial pH of 2, 3, 4 experiment. or 5, or in 0 1 M sodium phosphate at pH 6 or 7 or in 0 1 M citrate-phosphate buffer at a pH of 2, 3, 4, 5, 6 or 7. All solutions contained Urease activity 5 mM urea (Sigma) and had an osmolarity of Urease activity was measured using the phenol 300 mmol. The pH was controlled and red assay of Hamilton-Miller and Gargan25 as corrected if necessary before each experiment. calibrated for quantitative determination as H pylori cells were incubated in the various described previously.26 Rates ofurea hydrolysis solutions for 30 minutes under microaerobic are expressed as mmol NH3/min/mg protein. conditions at 37°C. After incubation, samples For each experiment, protein concentration were taken for determination of viable counts was determined by the BCA method according (CFU/ml) by direct plating of 10-fold to the instructions ofthe manufacturer (Pierce, dilutions. Cells and supematant were separated Rockford, IL) using bovine serum albumin as by centrifugation (5000 rpm, 20 min, 4°C). a standard. The pH of the supematant was determined using a pH meter (Coming, model 320). Cells were washed twice and lysed in a French Catalase activity pressure cell at 20000 psi (SLM Aminco, Catalase activity was determined by observing Urbana, IL). Proteins in the supernatant were the disappearance of H202 as measured by its concentrated by filtration using a centrifugal absorbance at 240 nm.27 The 2 ml reaction concentrator (Centrisart I, Sartorius, Goettingen, mixture contained 11 mM H202 in 10 mM Germany) with a cut off size of 20 kDa. potassium phosphate, pH 6-8. The reaction Effect ofpH on urease and catalase in H pylori 27

was started by adding 5 RI of soluble enzyme Statistical analysis preparation or concentrated supernatants. Results for enzyme activities and results of Absorption was monitored for one minute at immunoprecipitation studies were compared 23°C. Based on the initial rate, catalase activity by the non-parametric Kruskal-Wallis test.30 was expressed as the disappearance of H202 in If this test showed statistical significance Gut: first published as 10.1136/gut.40.1.25 on 1 January 1997. Downloaded from mmol/min/mg protein. The calculation was (p<0 05), individual comparisons were per- based on a standard curve determined from the formed using the test procedure described by absorption of 0-11 mM H202 under the same Nemenyi.30 conditions. The results were normalised for the protein concentration of the enzyme preparations. Results Duration ofacid exposure to H pylori Antiserum Conditions were established whereby H pylori Polyclonal antisera were raised against the was either briefly exposed to acidic conditions small (UreA) and large (UreB) subunits of or where acidic conditions were held constant the H pyloni urease in using purified despite active urea hydrolysis. When H pylori urease subunits as previously described.28 For was incubated in unbuffered HC1 of pH 2 immunoprecipitation of catalase, polyclonal containing 5 mM urea, pH increased during goat antiserum against H pylori catalase was five minutes to values around 4 (Fig 1). used. The specificity of the antisera was con- Incubation in HC1 of pH 3, 4 or 5 containing firmed by western blot. Pre-immune sera were 5 mM urea increased the pH to values above used as controls. 7 within the first minute. The increase in pH was assumed to be due to neutralisation of unbuffered HC1 by ammonia produced by Immunoprecipitation hydrolysis of urea. The pH of citrate or phos- H pylori bacterial cells, labelled with [35S]- phate buffers, however, remained unchanged methionine as described above, were used for during 30 minutes of incubation with Hpylori. immunoprecipitation. Cells were washed twice Although all buffers contained 5 mM urea, and lysed by boiling for 10 minutes. neutralisation by ammonia did not change the Immunoprecipitation was performed accord- pH. ing to a standard protocol.29 Briefly, 10 [lI ofcell lysate or supernatant were counted to deter- mine total counts per pl in each sample. For Viability ofH pylori after incubation at various

immunoprecipitation the different amounts pHs http://gut.bmj.com/ of total cpms were standardised by using Survival of H pylori after incubation for 100 000 cpms in each set up. [35S]-labelled 30 minutes in citrate buffer, phosphate buffer, soluble proteins were incubated for one hour or unbuffered HC1 is shown in Figure 2. at 4°C in 400 ml of 50 mM TRIS-HCI With a starting inoculum of approximately (pH 8-0), 2% (v/v) Triton X 100, 0-15 M 2 X 109 cfu/ml, only exposure to pH 2 NaCl, 0 1 M EDTA containing urease antisera citrate buffer reduced viability significantly

(1:50 v/v of anti-UreB (recognises the large (p<0 05). A tendency for reduced viability on September 29, 2021 by guest. Protected copyright. subunit of urease)), 1:25 v/v of anti-UreA was also found after exposure to pH 3 citrate (recognises the small subunit of urease), anti- buffer or pH 2 unbuffered HC1 compared with catalase serum (1:50 v/v), or pre-immunise viability after exposure to pH 7. However, control sera. After one hour, 50 pI protein A these differences were not statistically sig- (10% w/v) (Gibco BRL, Gaithersburg, MD) nificant. was added and incubation was continued at 4°C for 30 minutes. For immunoprecipitation of catalase, protein G (Gibco BRL, Gaithersburg, MD) was used because previous tests showed 9 a higher yield for precipitation of the catalase 8 antiserum than protein A. After incubation, precipitated antibody-antigen complex and 7 supernatant were separated by centrifugation 6 (5000 rpm, 5 min, 23°C), washed twice with I 5 -pH5 the Triton X-100 containing buffer, re- 4 pH4 suspended in 100 pl of 50 mM TRIS-HCl, pH 8-0 containing 1% SDS, boiled for 10 3 -pH3 minutes, and counted by liquid scintillation. 2 -pH2 Counts obtained from incubation with pre- immunised control sera were subtracted from " the counts obtained for specific anti-urease or 0 1 2 3 4 5 30 anti-catalase sera. Values for immunoprecipi- Time (min) tated urease and catalase were then expressed Figure 1: pH ofunbuffered HCI solutions ofpH 2, 3, 4, as percentage of total counts. Thus, the results and 5 during incubation ofH pylon cels. H pylon was represent the percentage of protein as urease suspended in HCI solutions ofvarious pH containing S mM or catalase from total protein produced by the urea. pH was measured over time; the initialpH is given at the start ofeach curve. Each point represents the mean H pylori cells during the period of incubation (SD) offour experiments, unless SD is smaller than the in the solutions ofvarious pH. point. 28 Bauerfeind, Garner, Dunn, Mobley

10 ; 10° activity, catalase activity was not affected by 3 S @ | incubation at pH 3 or after incubation in HCl

-6 at pH 2. L 6 To evaluate whether release of enzyme from

4 Hydrochloric Phosphate 4 _3 Gut: first published as 10.1136/gut.40.1.25 on 1 January 1997. Downloaded from 0 whole cells was responsible for the reduction in 2 acid buffer 2- cit IrIeIfe enzyme activity at certain pH levels, enzyme 2 3 4 5 6 7 23 4 5 6 7 activity was also measured in the supematant. pH pH Similar to the results in the intracellular Figure 2: Survival ofH pylori in citrate buffer, unbuffered enzyme activity, reduced urease activity was HCl, or phosphate buffer containing 5 mM urea a at various found after incubation in citrate buffer at pH pHs. Suspensions ofH pylori UMAB41 (109 cfulmi) were 2 and 3. Catalase activity was also reduced in incubatedfor 30 minutes in unbuffered HCl at IptH 2-5 or the supematant after incubation in citrate phosphate buffer at pH 6 and 7 (left graph) or ccitrate buffer at pH 2-7 (right graph); all solutions containeci5 mM buffer at pH 2. Under none of the conditions urea. Viability was determined by plating 1O-foid dilutions was an increase of enzyme activity found as ofthe suspensions. The x axis represents the initialpH ofthe compared with the results at pH 7. solution; they axis represents the viability after incubation for 30 minutes at 37°C given as log,o cfu. Each point represents one experiment. Survival was signific,antly reduced when H pylori was exposed to pH 2 in citrate Synthesis ofnascent urease and catalase buffer compared with pH 7 (*, p<0 01 tested b3y Wallis test, followed by Nemenyi test). There w4 Kruskal- Newly synthesised enzyme was measured tendencyfor reduced survival at pH 3 in citrate buffer and by immunoprecipitation of [35S]-methionine at pH 2 in HCl but these differences were not stiatistically labelled protein. Labelling was performed significant. during incubation in solutions of various pH. As shown in Figure 4, incubation in unbuffered Urease and catalase activity HCI or phosphate buffer of pH 6 or 7 did not Activities of urease and catalase of whole affect urease or catalase production. Urease H pylori cells incubated for 30 miinutes in and catalase represented approximately 10% buffered or unbuffered solutions c)f various and 1% respectively, of newly synthesised pHs are shown in Figure 3. ;e activity protein in unbuffered HC1 (pH 2-5) and was expressed as mmol NH3/min/mjg protein. phosphate or citrate buffer (pH 6-7). In Catalase activity was measured ass decom- contrast, incubation in citrate buffer, pH 2-5 position of H202 in mmol/min/mg pIrotein. All significantly inhibited nascent synthesis of enzyme measurements were deter mined at urease and catalase. pH 6-8 and 23°C. Urease activity waLs reduced To control for any loss of newly synthesised in the cells after incubation of H pyhwri cells in enzyme by release into the supematant, citrate buffer at or or pH 2 3 after ixncubation immunoprecipitation of urease and catalase http://gut.bmj.com/ in HCI at pH 2. For all other conditioIns, urease was also performed using the supematant. activity was similar to those of samples Although comparatively more urease and incubated in neutral citrate or Iphosphate catalase was found in the supematant at low buffers. pH in citrate buffer, the total amount of Catalase activity was reduced whein Hpylori precipitated counts at pH 2, 3, 4 and 5 in cells were incubated in citrate buffer at pH 2. citrate buffer was lower than 10% compared In contrast with the results obtained for urease with the counts at pH 6 or 7. Thus, the on September 29, 2021 by guest. Protected copyright. reduced amount of precipitated urease or 90 _ HCI catalase from the cells at low pH was not due ~~~~~~~~~1.5 to a loss of proteins into the supematant. 65 90- Citrate1.0 bul 40 -L Urease and catalase stability Phosphate 0 0.5- 1 5 Phosphate The Table shows the activity of the crude Ibuffer cytosolic enzyme after incubation in solutions 6 7 of various pH values. Enzyme activities were determined after raising the pH in all groups ~~~~~ II 50 to 6f8. Urease activity was reduced after ffer exposure ofthe enzyme to citrate buffer at a pH vzCitrate buffer A I 1.5 _ below 5. In contrast, catalase activity was only -10E q3 affected by exposure to pH 2 in HC1. Thus, exposed urease is not resistant to the effects of low pH unless it is cell associated or cytoplasmic, while catalase is relatively more JL resistant. 2 3 4 5 6 7 2 3 4 5 6 7 pH pH Figure 3: Urease and catalase activities in H pylori whole cells after incubation in HCI, Discussion phosphate buffer, or citrate buffer at different pHs. H pylori was suspended in hICI at The natural environment of H pylori is pH 2, 3, 4, 5 or phosphate buffer at pH 6, 7 (upperpanels) or in citrate buffer ,atpH 2-7 the human gastric mucosa. The bacterium is (lower panels) and incubatedfor 30 minutes. All solutions contained 5 mM urei a. Cels were lysed and activity of urease (leftpanels) and catalase (right panels) activities w ere usually found beneath the mucus layer in an measured at pH 6-8 at 23°C. Results ofsix experiments are displayed as box whiskerplots. area where the pH approaches neutrality. " The median is represented by the horizontal line; the top and bottom edge ofthe ibox Nevertheless, it could be assumed that Hpylori represent the 25 and 75 percentile; the whisker represents the 10 and 90 percentii and the circles extending away from the boxes represent minimal and maximal values might be exposed suddenly for short periods to *Significant differencefrom value obtained at pH 7. lower pH values either during disruption of Effect ofpH on urease and catalase in H pylori 29

20 r 2r seconds, we hypothesised that urease is con- HCI Phosphate HCI Phosphate stitutively produced. As the bacterium pro- buffer buffer duces large quantities of urease even during 15 - 1.5 _ periods where there is no exposure to low

0 Gut: first published as 10.1136/gut.40.1.25 on 1 January 1997. Downloaded from . pH, we hypothesised that survival and its 0 0 4 0 * . * S vital functions depend on the availability of 10 .a 0 0 cE 1 ._ I 0 urease and its protective function. Thus, we 0 0 I 0 0 * * hypothesised that H pyloni's protein synthesis 0 5 0 and urease activity are very sensitive to low pH. 0 00 0 0 To test this hypothesis, H pyloni cells were C 0 4-0 +,oC. 2 exposed to pH values ranging from 2 to 7, the 0 L 4- 2 3 4 5 0 2 3 4 5 6 7 same pH range that is likely to occur in the 1-O0 ~0 m0.15 human stomach.'4 We used buffered and * 0 20 Co * unbuffered solutions to obtain conditions of 0 stable pH as well as gradually increasing pH, Citrate buffer Citrate buffer * Cor- a) respectively. Exposure to unbuffered hydro- 15 -- 0* a0 1.5 H chloric acid mimicked the probable physio- ax a),o n logical events in the stomach where the pH *ux may be changed by neutralisation with 10 H quickly 0 ammonia. 0 0 The effect of pH on the enzymes was 5 0.5 _ 0 examined at three levels: (a) the effect on the enzyme activity when crude cytosolic enzyme 0 preparations were exposed directly to low a I a I~~~ pH; 0 0 t I I 2 3 4 5 6 7 2 3 4 5 6 7 (b) the effect on intracellular enzyme activity pH pH when intact Hpyloni cells were exposed to such conditions; and (c) the effect on the synthesis Figure 4: Nascent enzyme synthesis during incubation ofH pylori at various pHs. of nascent enzyme during exposure of intact Suspensions ofH pylori in HCl atpH 2, 3, 4, 5 orphosphate buffer atpH 6, 7 (upperpanels) or in citrate buffer at pH 2-7 (lowerpanels) were labelled with Hpylori cells to various pH levels. f S]-methionine. All solutions contained 5 mM urea. Urease and catalase synthesis The results of this study supports the initial was determined by immunoprecipitation usingpolyclonal antiserum directed against the hypothesis. As also shown by others,3' pro- small (UreA) and large (UreB) structural subunit ofH pylori urease and against the H pylori catalase holoenzyme. Each data point represents one ofthree independent longed exposure to low pH reduces viability. experiments. Furthermore, exposure of H pylori to low pH decreases protein synthesis, including urease and catalase. Exposure to low pH also the mucous layer or during transmission from abolishes activity of free and cellular urease. http://gut.bmj.com/ person to person. H pylori must survive low The detrimental effect of low pH depends on surrounding pH, at least transiently, by its the duration of exposure and acid concen- ability to hydrolyse urea into ammonia, which tration. However, the inhibition of protein in turn neutralises HCI and increases the pH production seems to be reversible because adjacent to the bacterium. This finding was viability was not affected by exposure to pH 3, earlierl' and confirmed in this 4, or 5. Free catalase seemed to be more stable reported study. on September 29, 2021 by guest. Protected copyright. In contrast, urease negative H pylori does not than urease when exposed directly to low pH; survive exposure to low pH3' and is unable to only exposure to pH 2 reduced catalase activity colonise the stomach in an animal model." significantly. As urease activity seems crucial for the Intracellular enzyme activity is defined by survival and transmission of the bacterium and the sum of the rates of new synthesis, exposure to very low pH may happen within degradation, and release. Low pH inhibited production of both urease and catalase. Thus, intracellular enzyme activity measured after Effect ofexposure to various pH values on soluble urease acid exposure was due to preformed enzyme. and catalase activities After exposure to citrate buffer at pH 2, Enzyme activity* bacterial viability and activities of both (% ofactivity atpH 7) enzymes were considerably reduced. Under Buffer pH Urease Catalase this condition, many bacterial cells were lysed and the released enzyme was inactivated by the HCI 2 0 sig 9 sig HCI 3 65 95 low pH as shown in the experiments where free HCI 4 77 93 enzymes were exposed to low pH. The most HCI 5 87 82 Phosphate 6 75 84 interesting events occurred after exposure to Phosphate 7 100 100 pH 3. Here, viability was not significantly Citrate 2 0 sig 55 decreased, and catalase activity was only mildly Citrate 3 0 sig 42 Citrate 4 0 sig 103 decreased. As only intracellular urease activity Citrate 5 18 102 was reduced, it could be assumed that the Citrate 6 81 108 Citrate 7 100 100 intracellular pH was reduced to a level where the intracellular enzyme was inactivated. As *Hpylori soluble enzyme preparations were incubated in shown in the experiments with free enzyme, HCI, phosphate buffer, or citrate buffer containing 5 mM urea. After 30 minutes, the pH was adjusted to pH 6-8 and this presumably occurs below a pH of 5. After enzyme activities were measured. Median results from four exposure of the H pyloni cells to citrate buffer experiments are expressed as a percentage of enzyme activity obtained at pH 7 0. (sig, significantly different from the pH 7 of pH 4, intracellular urease remained active value, p<005). whereas free enzyme was destroyed showing 30 Bauerfeind, Gamer, Dunn, Mobley

that the cells were able to cope with that 1 Morris A, Nicholson G. Ingestion of pyloridis causes gastritis and raised fasting gastric pH. extracellular pH and maintain the intracellular AJf Gastroenterol 1987; 82: 192-9. pH at a physiologically functional level. 2 Warren JR, Marshall B. Unidentified curved bacilli on the gastric epithelium in active chronic gastritis. Lancet 1983; Although we did not measure intracellular i: 1273-5. pH, the comparison of intracellular urease 3 Langengberg ML, Tytgat GNJ, Schipper MEI. Gut: first published as 10.1136/gut.40.1.25 on 1 January 1997. Downloaded from Campylobacter-like organisms in the stomach of patients activity and activity of free urease at various and healthy individuals. Lancet 1984; i: 1348. pH levels allows us to speculate indirectly 4 Goodwin CS, Armstrong JA, Marshall BJ. Campylobacter pyloridis, gastritis, and peptic ulceration. J Clin Pathol on the intracellular pH of H pyloni after 1986; 39: 353-65. exposure to low pH. Hpylori seems to maintain 5 Jones DM, Lessells AM, Eldridge J. Campylobacter-like organisms on the gastric mucosa: culture, histological and an intracellular pH above 5 if exposed to a serological studies. J Clin Pathol 1984; 37: 1002-6. pH of 4. This also explains why H pylori is 6 Owen RJ, Martin SR, Borman P. Rapid urea hydrolysis by gastric . Lancet 1985; ii: 1273-5. able to grow at low pH values.32 It further 7 Marshall BJ, Barrett U, Prakash C, McCallum RW, seems that H pylori restores its normal intra- Guerrant RL. Urea protects Helicobacter (Campylo- bacter) pylori from the bactericidal effect of acid. cellular pH after a short-term exposure to Gastroenterology 1990; 99: 697-702. pH below 4 permitting normal protein pro- 8 Hu LT, Mobley HL. Purification and N-terminal analysis ofurease from . Infect Immun 1990; 58: duction and survival. The precise mechan- 992-8. isms of maintenance of intracellular pH in 9 Dunn BE, Campbell GP, Perez-Perez GI, Blaser MJ. Purification and characterization of urease from H pylori are unknown. However, a H pylori P Helicobacter pylori. JBiol Chem 1990; 265: 9464-9. type ATPase has recently been characterised,33 10 Segal ED, Shon J, Tompkins LS. Characterization of Helicobacter pylori urease mutants. Infect Immun 1992; which is probably a cation transporter and 60: 1883-9. might be responsible for the regulation 11 Eaton KA, Krakowka S. Effect of gastric pH on urease- dependent colonization of gnotobiotic piglets by cytoplasmic pH. Helicobacter pylori. Infect Immun 1994; 62: 3604-7. Although H pyloni can survive transient 12 Rawlings JW, Danesh BJ, Lucas ML, Morgan RJ, Main AN, Russell RI. Gastroduodenal mucosal surface and luminal periods of low surrounding pH, both urease pH in gastric ulcer. Dig Dis Sci 1991; 36: 1543-9. production and urease stability are sensitive to 13 Flemstrom G, Kivilaakso E. Demonstration of a pH gradient at the luminal surface of rat duodenum in vivo low pH. In contrast with other urease positive and its dependence on mucosal alkaline secretion. bacteria such as ,'5 Gastroenterology 1983; 84: 787-94. 14 Fimmel CJ, Etienne A, Cilluffo T, Von Ritter C, Gasser T, urease production is not stimulated during Rey J-P, et al. Long-term ambulatory gastric pH exposure to low pH. The amount of enzyme monitoring: validation of a new method and effect of H2-Antagonists. Gastroenterology 1985; 88: 1842-51. activity in the cell is reduced when H pylori is 15 Sissons CH, Perinpanayagam HE, Hancock EM, exposed to low pH depending on duration of Cutress TW. pH regulation of urease levels in Streptococcus salivarius. JfDent Res 1990; 69: 1131-7. exposure and level of acidity. Brief exposure to 16 Jones BD, Mobley HLT. mirabilis urease: genetic low unbuffered acidity above pH 2 neither organizaiton, regulation, and expression of structural genes.JBacteriol 1988; 170: 3342-9. completely inhibited urease activity nor stimu- 17 Nicholson EB, Concaugh EA, Foxall PA, Island MD, lated it. These findings are in some contrast Mobley HLT. urease: transcriptional regulation by ureR._J Bacteriol 1993; 175: 465-73. http://gut.bmj.com/ with the idea that the major role of H pylori 18 Rosenstein IJ, Hamilton-Miller JMT, Brumfitt W. Role of urease is its protective function against acidity. urease in the formation of infection stones: comparison of from different sources. Infect Immun 1981; 32: 32-7. However, the extreme range of pH in its 19 Friedrich B, Magasanik B. Urease of aerogenes: natural environment and the acid sensitivity of control ofits synthesis by glutamine synthetase. J Bacteriol 1977; 131: 446-52. H pyloni explains why such high amounts of 20 Mobley HL, Cortesia MJ, Rosenthal LE, Jones BD. urease are prepared even without preceding Characterization of urease from Campylobacter pylori. acid exposure. H pyloni seems not to be J Clin Microbiol 1988;26: 831-6. 21 Hu LT, Mobley HLT. Expression of catalytically active on September 29, 2021 by guest. Protected copyright. acidophilic and needs the stock of urease for its recombinant Helicobacter pylori urease at wild-type levels in Escherichia coli. Infect Immun 1993; 61: 2563-9. protection against a sudden drop in pH. As 22 Hazell SL, Evans DJ Jr, Graham DY. Helicobacter pylori soon as the short-term episode of low pH is catalase.J Microbiol 1991; 137: 57-61. 23 Foxall PA, Hu LT, Mobley HL. Use of polymerase chain overcome, the bacterium is able to restore its reaction-amplified Helicobacter pylori urease structural normal intracellular pH and to replace the genes for differentiation of isolates. J Clin Microbiol 1992; 30: 739-41. destroyed proteins. 24 Skirrow MB. Campylobacter enteritis: a 'new' disease. BMJ Our findings support the concept that 1977; 2:9-11. 25 Hamilton-Miller JMP, Gargan RA. Rapid screening for H pyloni is not acidophilic and does not seem urease inhibitors. Jf Urol 1979; 16: 327-8. to thrive in an acidic environment. A key 26 Mobley HL, Jones BD, Jerse AE. Cloning of urease gene sequences from Providencia stuartii. Infect Immun 1986; enzyme of the organism, urease, is not acid 54: 161-9. stable and its synthesis is not stimulated by 27 Nelson DP, Kiesow LA. Enthalpy of decomposition of by catalase at 25'C (with molar low pH. In addition, the of exteinction coefficient ofH202 solutions in the UV). Ann the bacterium, as measured by incorporation Biochem 1972; 49: 474-8. 28 Hu LT, Foxall PA, Russell R, Mobley HL. Purification of of radiolabelled methionine into protein, recombinant Helicobacter pylori urease apoenzyme functions at near neutral and is encoded by ureA and ureB. Infect Immun 1992; 60: optimally pH 2657-66. dramatically reduced at low pH. Indeed, the 29 Harlow E, Lane D. Antibodies- a laboratory manual. Cold environment would seem to be under Spring Harbor: Cold Spring Harbor Laboratory Press, preferred 1988. the mucous layer of the gastric mucosa where 30 Sachs L. Angewandte Statistik. Berlin: Springer-Verlag, the may There is no 1983. pH approach neutrality. 31 Clyne M, Labigne A, Drunmm B. Helicobacter pylori evidence that the ulcerogenic activity of acid is requires an acidic environment to survive in the presence stimulation of the bacterial of urea. Infect Immun 1995; 63: 1669-73. amplified by 32 Kangatharalingam N, Amy PS. Helicobacter pylori comb. factor urease. nov. exhibits facultative acidophilism and obligate micro- aerophilism. Appl Environ Microbiol 1994; 60: 2176-9. 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