INTRODUCTION

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Nitric oxide (NO) is known to play an important role in host defense against a variety of microbes (1, 12, 15, 20, 36, 37), although NO itself does not show sufficient antimicrobial activity (24, 55). Some metabolites of NO, such as peroxynitrite (ONOO), are considered to be responsible for the antimicrobial as well as the pathogenic effects of NO. NO and superoxide (O₂) react in a diffusion-limited manner, forming ONOO (5), a strong oxidant and nitrating agent (4, 5, 23) that exhibits potent bactericidal activity (22, 57) as well as cytotoxicity for mammalian cells in vitro and in vivo (4, 5). It has been reported that both NO and O₂ that are simultaneously produced in local areas of infection are critically involved in antimicrobial defense in murine salmonellosis (Salmonella enterica serovar Typhimurium infection), possibly through formation of ONOO (49).

Helicobacter pylori can infect human gastric mucosa chronically; such infection is known to be associated with gastritis, peptic gastric ulcer, duodenal ulcer, and an increased risk for gastric cancer (3, 6, 21, 45, 52). A unique feature of H. pylori infection is its persistence, which causes prolonged active inflammation, including infiltration of neutrophils and monocytes in gastric mucosa (11, 39). Increased expression of the inducible type of NO synthase (iNOS) (16-18, 30, 42, 47) and elevated formation of nitrotyrosine (17, 30) are also observed in the gastric mucosae of patients with H. pylori infection. However, the mechanism of the persistent infection of H. pylori, despite the production of highly bactericidal ONOO and other reactive nitrogen species, is not clear.

Several investigations have suggested a role for H. pylori urease in the survival and pathogenesis of the bacteria (29, 31, 35, 46). Urease catalyzes the hydrolysis of urea to form carbon dioxide (CO₂) and ammonia (NH₃). It is reported that urease functions in H. pylori infection to neutralize gastric acid by producing NH₃ (31). Enhanced production of NH₃ also may facilitate the formation of NH₃-derived compounds, such as monochloramine, which shows cytotoxic effects on host cells (46). Enhancement of bacterial motility (35) and inhibition of phagocytic clearance of bacteria (29) were also reported as functions of urease. The pathogenic potential of urease is so far mainly attributed to NH₃ produced by the enzymatic reaction. In contrast, little attention has been paid to the roles of CO₂/HCO₃ produced in the same process. It is noteworthy that the chemical reactivity of ONOO is reported to be modulated by CO₂/HCO₃ (26, 28, 54). Specifically, ONOO reacts rapidly with CO₂, and through the formation of ONOOCO₂, not only is isomerization of ONOO to NO₃ accelerated (27, 50), but also the nitration potency of ONOO is significantly enhanced and the oxidation potential is markedly attenuated (54, 56). For example, CO₂/HCO₃ facilitates ONOO-induced nitration of aromatic compounds, such as tyrosine and guanine (guanosine); however, it suppresses their oxidation (26, 54, 56). In addition, the in vitro bactericidal activity of ONOO on Escherichia coli was reduced by the addition of NaHCO₃ (22, 57).

Therefore, the purpose of this study was to clarify the role of urease in persistent colonization of H. pylori, especially to examine the protective effects of CO₂ produced by urease against the bactericidal activity of ONOO in vitro.

	MATERIALS AND METHODS

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Bacteria. H. pylori ATCC 43504 was obtained from the American Type Culture Collection (Manassas, Va.). H. pylori HPK5 and its isogenic ureB mutant HPT209 (lacking urease), which was produced by allelic exchange mutagenesis, were generously provided by T. Nakazawa, Department of Microbiology, Yamaguchi University School of Medicine, Ube, Japan (35). Campylobacter jejuni isolated from a clinical source was also used in this study. These bacteria were routinely grown in brucella broth (Becton Dickinson & Co., Cockeysville, Md.) supplemented with 10% fetal calf serum (Intergen Co., Purchase, N.Y.) in the presence (H. pylori HPT209) or absence (H. pylori ATCC 43504, H. pylori HPK5, and C. jejuni) of 7 µg of kanamycin sulfate/ml under microaerobic conditions maintained in a GasPak jar (Becton Dickinson & Co.) with an H₂- and CO₂-generating agent, CampyPak (Becton Dickinson & Co.).

Reagents. ONOO was prepared from nitrite and hydrogen peroxide in a quenched-flow reactor as previously described (5). The NO-liberating agent propylamine NONOate (CH₃N[N(O)NO](CH₂)₃NH₂⁺CH₃, 1-hydroxy-2-oxo-3-(N-methyl-3-aminopropyl)-3-methyl-1-triazine) (P-NONOate) which has a half-life of 7.6 min in aqueous solutions at a neutral pH under our experimental conditions, was obtained from Dojindo Laboratories, Kumamoto, Japan. Urea, NaHCO₃, and an aqueous solution of ammonia (NH₄OH) were from Wako Pure Chemicals Co. Ltd., Osaka, Japan. Urease from Bacillus pasteurii, 3-nitro-L-tyrosine (nitrotyrosine), and L-tyrosine were purchased from Sigma Chemical Co., St. Louis, Mo. Dihydrorhodamine 123 (DHR) was purchased from Molecular Probes, Inc., Eugene, Oreg. Two urease inhibitors, N-(diaminophosphinyl)-4-fluorobenzamide (flurofamide) and acetohydroxamic acid (AHX), were from ICN Biomedicals Inc., Aurora, Ohio, and Nacalai Tesque Inc., Kyoto, Japan, respectively. Pronase was obtained from Calbiochem-Novobiochem Co., La Jolla, Calif. All other reagents were from commercial sources.

Bactericidal assays. We used 0.5 M phosphate buffer (pH 7.6) containing 0.15 M NaCl (0.5 M phosphate-buffered saline [PBS]) for the bactericidal assays of ONOO and other effector substances to minimize changes in pH during the reaction because of infusion of the alkaline solution of ONOO (in 10 mM NaOH) and production of NH₃ and HCO₃, which might have affected the pH of the reaction mixture. Suspensions of H. pylori cultured for 36 to 48 h and of C. jejuni cultured for 24 h were washed with and resuspended in 0.5 M PBS immediately before use. Bacterial suspensions were kept on ice until use. Urease activity and bacterial motility and morphology were checked before each use. The constant-flux infusion method (7, 40) was used to treat the bacteria with steady concentrations of ONOO. In the constant-flux infusion process, the effective and constant concentration of ONOO is maintained by balancing infusion and decomposition of ONOO in the system. The concentrations of ONOO maintained constant were estimated by the DHR oxidation assay, as described earlier (8). Specifically, DHR (28 µM) was added to the reaction mixture of ONOO without bacteria; simultaneously, the ONOO infusion was stopped, and the amount of the oxidized product rhodamine was measured fluorometrically. The concentration of ONOO was then estimated by using a standard curve of the amount of rhodamine generated as a function of ONOO, which was prepared separately by reaction of DHR with a bolus of ONOO injected into 0.5 M PBS. As a result, by infusion 10, 100, and 1,000 µM ONOO in 10 mM NaOH into 0.5 M PBS (1.2 ml) at a flow rate of 240 µl/min, the concentrations of ONOO remained constant at 0.3, 1, and 3 µM, respectively. H. pylori or C. jejuni (10⁸ CFU/ml each) samples were treated constantly with 1 µM ONOO by infusing 100 µM ONOO in the absence or presence of urea, NaHCO₃, or NH₄OH as described above. Aliquots (120 µl) were removed from the reaction mixture at 30-s intervals and were immediately diluted with nutrient broth (Eiken Chemical Co. Ltd., Tokyo, Japan) and seeded on brucella agar plates containing 5% lysed horse blood (Nippon Bio-Test Laboratories Inc., Tokyo, Japan) for the colony-forming assay. After cultivation for 5 days (H. pylori) or 2 days (C. jejuni) under microaerobic conditions, the number of colonies formed was determined.

Measurement of nitrotyrosine. Suspensions of H. pylori (5 × 10⁸ CFU/ml) treated continuously with 1 µM ONOO for 3 min in the presence or absence of 20 mM NaHCO₃ or 10 mM urea, as described above, were centrifuged at 1,600 × g and then resuspended in 10 mM potassium phosphate buffer (pH 7.4) containing 0.15 M NaCl (250 µl). Aliquots (100 µl) were treated with 60 µg of pronase/ml for 18 h at 50°C. The bacterial suspension became clear and no precipitate was seen in the resultant reaction mixture even after centrifugation at 10,000 × g, indicating that the bacterial cells were completely digested by the pronase treatment. After filtration through a centrifugal filter unit (Ultrafree-MC with a 10,000 nominal molecular weight limit; Millipore Corp., Bedford, Mass.), the filtrate was processed by high-performance liquid chromatography (HPLC) coupled to electrochemical detection with 12 CoulArray electrode cells (ESA, Inc., Chelmsford, Mass.) (9, 19). Nitrotyrosine recovered from the bacterial cells was separated on a reverse-phase column (4.6 by 250 mm) (TSKgel ODS-80Ts; Tosoh Co., Tokyo, Japan) and eluted with 50 mM sodium acetate buffer (pH 4.7) containing 5% methanol at a flow rate of 0.8 ml/min. The CoulArray electrode array detector was operated with applied potentials at 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, and 750 mV. Peaks of nitrotyrosine and L-tyrosine were identified and quantified based on comigration with known concentrations of authentic standards and their electrochemical activation profiles. Identification of nitrotyrosine was confirmed by the disappearance of the peak after reduction of nitrotyrosine to aminotyrosine by 20 mM sodium dithionite. The amounts of nitrotyrosine and tyrosine were quantified from the peak areas obtained at 750 and 600 mV, respectively.

Statistical analysis. Statistical analyses were done with the two-tailed t test for unpaired data.

	RESULTS

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Bactericidal effect of ONOO on H. pylori. It is now known that ONOO is a key intermediate in the NO-dependent bactericidal effect. Induction of iNOS and formation of nitrotyrosine, an indicator of ONOO formation, in H. pylori-infected stomach have also been documented (16-18, 30, 42, 47). Therefore, we examined the bactericidal activity of authentic ONOO (5) by using the constant-flux infusion method (7, 40). The number of viable bacteria expressed as CFU declined after exposure to ONOO in a dose- and time-dependent manner (Fig. 1A). Products of ONOO decomposition, mainly nitrate anion (28), showed no bactericidal activity against H. pylori (Fig. 1A).

View larger version (19K): [in this window] [in a new window]   FIG. 1.   (A) Bactericidal effect of ONOO on H. pylori ATCC 43504. Decomposed ONOO (equivalent to 1 mM ONOO) () or ONOO at 10 µM (), 100 µM (), or 1 mM () in 10 mM NaOH was infused into 1.2 ml of bacterial suspension (108 CFU/ml) at a flow rate of 240 µl/min. Every 30 s, 120-µl aliquots were removed from the reaction mixture, and the number of viable bacteria was determined by the colony-forming assay. Concentrations of ONOO in the reaction mixture during infusion of 10 µM, 100 µM, and 1 mM ONOO were assumed to be maintained at constant 0.3, 1, and 3 µM concentrations, respectively. Data are means ± SD from three independent experiments. (B) Survival of H. pylori after exposure to the NO-donor P-NONOate. H. pylori ATCC 43504 organisms (108 CFU/ml) were incubated for the indicated periods with 1 (), 10 (), or 100 () µM P-NONOate, followed by colony-forming assay. (C and D) Effects of urea on bactericidal action of ONOO on H. pylori ATCC 43504 (C) and C. jejuni (D). (E and F) Effects of urea on the bactericidal action of ONOO on H. pylori HPK5 (E) and its isogenic ureB mutant, HPT209 (F). Bacteria (108 CFU/ml) were exposed to a constant concentration of ONOO (1 µM) in the presence () or absence () of 10 mM urea for the indicated periods, and the number of viable bacteria at each time point was determined. Data from three independent experiments are expressed as means ± SD. * and **, P < 0.05 and P < 0.01 versus control without urea, respectively.

Effect of urea on bactericidal action of ONOO on H. pylori and C. jejuni. As shown in Fig. 1C, in the presence of a physiological concentration (10 mM) of urea, survival of H. pylori was significantly increased. Because urea did not affect the decomposition rate of ONOO at the pH range 7.0 to 10.0, as assessed by measuring absorbance at 302 nm (data not shown), direct detoxification of ONOO by urea itself was not plausible. When clinically isolated C. jejuni, another microaerophilic bacteria lacking urease activity, was treated with ONOO in the same experimental settings in the presence or absence of urea, the susceptibility of C. jejuni to ONOO was not affected by the addition of urea (Fig. 1D), suggesting that the contribution of urease produced by H. pylori to the suppression of the cytotoxicity of ONOO was required. To further verify this notion, the bactericidal action of ONOO against H. pylori HPK5 and its isogenic mutant HPT209, lacking urease, was examined with or without the addition of 10 mM urea. The strains showed similar sensitivities to ONOO in the absence of urea (Fig. 1E and F). In contrast, urea attenuated the bactericidal effect of ONOO on the wild-type strain, HPK5 (Fig. 1E), but it did not affect the bacterial killing of ONOO for the mutant with the urease gene disruption, HPT209 (Fig. 1F). This result indicates again that urease activity is required for urea-dependent attenuation of the ONOO cytotoxicity.

View larger version (14K): [in this window] [in a new window]   FIG. 2.   (A) Effects of urease inhibitors and additional urease on the bactericidal action of ONOO on H. pylori. H. pylori ATCC 43504 organisms (108 CFU/ml) were exposed to a constant concentration of ONOO (1 µM) for 3 min in the absence (a) or presence (b to e) of 10 mM urea. Reactions were performed in the presence of 10 µM flurofamide (c), 70 mM AHX (d), or 48 U of B. pasteurii urease per ml (e). (B) Effects of NaHCO3 and NH3 on bactericidal action of ONOO on H. pylori. H. pylori ATCC 43504 organisms (108 CFU/ml) were exposed to a constant concentration of ONOO (1 µM) for 3 min in the presence of 10 mM urea (b), 20 mM NH3 (c), 20 mM NaHCO3 (d), or 10 mM urea plus 20 mM NaHCO3 (e) or in the absence of all compounds (a), and the colony-forming assay was performed. Data from three independent experiments are expressed as means ± SD. *, P < 0.01 versus a; , P < 0.05 versus b; **, P < 0.005 versus b; and , P < 0.005 versus d.

Effects of NaHCO₃ and NH₃ on bactericidal action of ONOO on H. pylori. We examined the effects of the products of the urea-urease reaction, CO₂ and NH₃, on the bactericidal activity of ONOO. NaHCO₃ (20 mM) suppressed bacterial killing by ONOO to the same degree as 10 mM urea, whereas NH₄OH (20 mM) did not (Fig. 2B). Furthermore, urea (10 mM) plus NaHCO₃ (20 mM) showed an additive protective effect for the survival of H. pylori exposed to ONOO (Fig. 2B), suggesting that urease increases bacterial survival in in vivo situations in which physiological concentrations of HCO₃ and urea are close to those used in this experiment, i.e., about 20 and 10 mM, respectively (38).

Nitrotyrosine formation in H. pylori after treatment with ONOO. ONOO is known to nitrate aromatic compounds, including tyrosine (4, 23). To assess the effect of urease activity on the chemical reactivity of ONOO with the bacterial components, we quantified nitrotyrosine in H. pylori cells by using HPLC coupled to electrochemical detection with 12 electrodes (Fig. 3A). The amount of nitrotyrosine in the bacterial cells exposed to 1 µM ONOO for 3 min was 267 ± 22 pmol/10⁸ CFU, or 7.48% ± 1.2% of the total tyrosine (Fig. 3B). Nitrotyrosine was not detected (less than 0.1 pmol/10⁸ CFU) in the control bacterial cells (no exposure to ONOO). In contrast, we could not detect any appreciable amount of nitrotyrosine in the bacterial cells treated with P-NONOate (data not shown), indicating that ONOO, but not NO, exhibits a strong tyrosine-nitrating potential in H. pylori. The addition of 10 mM urea or 20 mM NaHCO₃ to the reaction mixture of ONOO lowered the formation of nitrotyrosine by 50% (Fig. 3B). Since CO₂ accelerates decomposition of ONOO (27, 50), it is plausible that CO₂/HCO₃ added or formed by bacterial urease might increase the decomposition rate of ONOO and thus suppress the reactivity of ONOO with the bacteria.

View larger version (28K): [in this window] [in a new window]   FIG. 3.   Nitrotyrosine formation in H. pylori cells after treatment with ONOO. 3-Nitro-L-tyrosine (3-Nitro-Tyr) was quantified by HPLC coupled to electrode detectors, with the pronase digests of non-ONOO-treated H. pylori ATCC 43504 cells (a) or those exposed to 1 µM ONOO (b to d) for 3 min in the absence (b) or presence of 10 mM urea (c) and 20 mM NaHCO3 (d). (A) Elution profile of the pronase digest of H. pylori exposed to 1 µM ONOO in the absence of urea or NaHCO3. (B) The amounts of 3-Nitro-Tyr formed in H. pylori were expressed as the ratio of 3-Nitro-Tyr to total L-tyrosine (Tyr) recovered from bacterial cells. ND, 3-Nitro-Tyr was not detected (<0.1 pmol/108 CFU). *, P < 0.05 versus b. Data are means ± SD of three independent experiments.

	DISCUSSION

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H. pylori produces a large quantity of urease, which amounts to 5% of the total protein of the bacterium (14). Urease genes in the H. pylori genome are composed of two gene clusters: ureAB genes and ureIEFGH genes (10). Colonization of H. pylori mutants whose ureA, ureB, ureG, or ureI gene was disrupted in experimental animals was known to be suppressed (13, 44, 48, 53). In addition, proton pump inhibitors used for treatment of H. pylori infection inhibit bacterial urease in an irreversible fashion (33). All these studies imply that H. pylori urease is essential for H. pylori colonization in the stomach.

Several studies were carried out to elucidate the roles of H. pylori urease in bacterial colonization in the stomach. Neutralization of gastric acid with NH₃ produced by the enzyme might allow the bacterium to survive in the acidic milieu (31). It is reported that the motility of H. pylori, which is known to be an important characteristic of the bacterium in the colonization of experimental animals, is enhanced by the urea-urease reaction, particularly in a viscous environment (35). Inhibition of neutrophil function by NH₃ was also proposed as a pathogenic mechanism of this enzyme (32).

In addition to these possible roles of urease, the results obtained in this study clearly demonstrate that H. pylori urease functions as a part of the defense system of the bacteria themselves against ONOO (Fig. 1 and 2).

In previous work, elevated generation of ONOO in vivo and its involvement in antimicrobial host defense were reported for a murine salmonellosis model. Results indicated that suppressing ONOO generation by inhibiting either NO or O₂ production or by scavenging these radicals accelerated the growth of S. enterica serovar Typhimurium in the liver and further augmented its pathogenicity, as evidenced by the increased mortality of infected mice (49). It was thus suggested that ONOO effectively clears bacteria from sites of infection in vivo (1, 49). In recent years, increased expression of iNOS mRNA and its product has been confirmed in H. pylori-infected gastric tissues of patients and experimental animals (16-18, 30, 42, 47). Formation of ONOO and/or other reactive nitrogen species produced by the NO₂-H₂O₂-myeloperoxidase system at sites of infection by H. pylori is also suggested by the immunohistochemical detection of nitrotyrosine (17, 30, 51). Furthermore, it has recently been reported that not only phagocytic inflammatory cells but also H. pylori itself produce O₂ (34), which indicates that ONOO may be formed in and around the bacteria in vivo, where production of NO and O₂ is simultaneously elevated as described above. Consequently, ONOO may function as a major bactericidal effector for H. pylori in the stomach. In a separate experiment, however, no significant difference was found between the number of H. pylori organisms colonizing iNOS-knockout mice and that in wild-type mice (unpublished observation). In this context, it is quite reasonable that H. pylori has evolved with the system, such as urease, that is capable of detoxifying ONOO, and hence steady and sustained colonization in the infected stomach is facilitated.

A high concentration of ONOO was used in the present study so that we could obtain reproducible results and clearly demonstrate the bactericidal action of ONOO. The bacteria were directly exposed to a 1 µM effective concentration of ONOO in vitro, which is considered to be an extremely severe condition for the bacteria compared with the in vivo setting in infected foci containing various endogenous substances that affect the reactivity of ONOO (2). Even under such conditions, the physiological concentration of urea increased the survival fractions of two strains of H. pylori (ATCC 43504 and HPK5) 3.7- to 8.4-fold after exposure to ONOO for 5 min (Fig. 1C and E). Therefore, it is conceivable that the urease could function efficiently as a protective factor of H. pylori against ONOO produced in vivo.

Although it is reported that ONOO-dependent nitration of aromatic compounds, including tyrosine, is enhanced in the presence of CO₂ (26, 54), formation of nitrotyrosine in H. pylori was suppressed by the addition of urea or NaHCO₃ (Fig. 3B). Recently, Romero et al. reported that CO₂ shortened the half-life and the diffusion distance of ONOO and hence inhibited the oxidation of oxyhemoglobin in red blood cells by ONOO (43). Therefore, the results obtained in this study suggest that CO₂ formed by bacterial urease inhibits the reactivity of ONOO with the bacterial components and accelerates its decomposition outside the bacterial cells. It is of great importance, then, that H. pylori urease is localized not only in the cytoplasm but also on the surface of the bacteria (41). In our experimental settings, surface-bound urease seemed to play an important role in the decomposition of ONOO (Fig. 2A).

In conclusion, urease of H. pylori plays a role in the defense against the toxicity of ONOO via production of CO₂, and it may confer the capacity for sustained infection in vivo. Improved understanding of the pathogenic role of urease, in view of a host-pathogen interaction, will help in the exploration of effective therapeutic treatments for H. pylori infection and its related gastric diseases, including gastric cancer.