COMMUNICATION
Helicobacter pylori Generates Superoxide Radicals and
Modulates Nitric Oxide Metabolism*
Kumiko
Nagata
,
Hidenori
Yu§,
Manabu
Nishikawa§,
Misato
Kashiba§,
Akihiro
Nakamura§,
Eisuke F.
Sato§,
Toshihide
Tamura
, and
Masayasu
Inoue§¶
From the
Department of Bacteriology, Hyogo College of
Medicine, 1-1 Mukogawa, Nishinomiya, Hyogo 663 and
§ Department of Biochemistry, Osaka City University Medical
School, 1-4-54 Asahimachi, Abeno, Osaka 545, Japan
 |
ABSTRACT |
During studies of the bactericidal action of
nitric oxide (NO), we found that it reversibly inhibited the
respiration of Escherichia coli and irreversibly inhibited
the respiration of Helicobacter pylori. Peroxynitrite, a
reaction product of NO and superoxide, irreversibly inhibited the
respiration of both H. pylori and E. coli. H. pylori, but not E. coli, generated substantial
amounts of superoxide radicals. These results suggest that NO directly inhibits the respiration of E. coli whereas it rapidly
reacts with endogenously generated superoxide radicals in H. pylori. The resulting peroxynitrite inactivates the respiration
of H. pylori.
 |
INTRODUCTION |
Nitric oxide (NO)1
is a multifunctional gaseous free radical produced by NO
synthase in various types of cells, such as endothelial cells, neurons,
neutrophils, and macrophages (1). Nitric oxide is also generated from
nitrite in saliva and from food by microorganisms in the oral cavity as
well as by nonenzymatic mechanisms under acidic conditions, such as in
gastric juice (12). Because NO synthase is also present in gastric
mucosa (13, 14), physiological concentrations of NO in gastric juice
are fairly high.
We have shown that the biological activity of NO is augmented
significantly by physiologically low levels of oxygen tension (2-5).
Although NO plays important roles in defense mechanisms against enteric
bacteria (6, 7), few studies have explored the mechanism of its
bactericidal action at physiological levels of oxygen tension.
Helicobacter pylori is a Gram-negative and
microaerophilic bacterium that resides in the mucus layer overlying the
gastric epithelium of the human stomach. This organism is thought to
play essential roles in the pathogenesis of gastric inflammation,
ulceration, and carcinogenesis (8-11). Although H. pylori
is exposed to fairly high concentrations of NO in gastric juice, which
has low oxygen tension, the effect of NO on the metabolism of H. pylori remains to be defined. We therefore studied the effects of
NO on the respiration of H. pylori and Escherichia
coli under physiologically low levels of oxygen tension.
 |
MATERIALS AND METHODS |
Reagents--
Peroxynitrite solution and
carboxy-2-phenyl-4,4,5,5-tetramethylimidazolin-1-oxyl-3-oxide (cPTIO)
were obtained from Dojin Co. (Kumamoto, Japan). Mn-type superoxide
dismutase (SOD) from E. coli and
2-methyl-6-[p-methoxyphenyl]-3,7-dihydroimidazol[1,2-
]pyra zin-3-one (MCLA) were obtained from Sigma and Tokyo Kasei Kogyo Co.
(Tokyo, Japan), respectively. Nitric oxide solution was prepared as
described previously (5).
Bacterial Strains and Their Culture--
Two types of enteric
bacteria, E. coli K-12 JM109 and H. pylori
NCTC-11637, were used in all experiments. E. coli was
cultured at 37 °C with shaking in nutrient broth (Difco) containing
0.5% NaCl. H. pylori was cultured in Brucella
broth containing 5% horse serum under a microaerophilic atmosphere,
produced with the use of a gas pack BBL CampyPak (Becton, MD), at
37 °C for 20 h as described previously (15). The cultured
E. coli and H. pylori were harvested at the
logarithmic growth phase, suspended in 10 mM HEPES buffer
(pH 7.0) containing 0.9% NaCl, and used in the experiments.
Analysis of Bacterial Respiration--
Respiration of E. coli and H. pylori was monitored polarographically at
37 °C in 10 mM HEPES buffer (pH 7.0) containing 0.9% NaCl and 5 mM succinate. A Clark-type oxygen electrode was
used as described previously (5).
Assay of Superoxide and SOD Activity--
Superoxide was
determined by the cytochrome c method (16) and the
chemiluminescence method (17) with the use of a Bio-Orbit 1251 luminometer in buffer solution containing 1 µM MCLA and
0.5% Triton X-100. After disrupting cells by sonication, the samples were centrifuged at 10,000 × g and 4 °C for 20 min.
SOD activity in the supernatant was determined spectrophotometrically
with an SOD assay kit (Calbiochem). The assay is based on the
SOD-mediated increase in the rate of autooxidation of
5,6,6a,11b-tetrahydro-3,9,10-trihydroxybenzo[c]fluorene in aqueous alkaline solution (18). Protein concentrations in the
samples were determined by the method of Lowry et al.
(19).
 |
RESULTS AND DISCUSSION |
Fig. 1 shows the
succinate-dependent respiration of E. coli and
H. pylori. Nitric oxide reversibly inhibited the respiration of E. coli. Consistent with our previous observations with
mitochondria (2-4) and E. coli (5), the inhibitory effect
of NO increased with a decrease in oxygen concentration of the medium.
Although NO also inhibited the respiration of H. pylori in
an oxygen concentration-dependent manner, it was
significantly less active than with E. coli, and inhibition
persisted throughout the observation period. Washing the NO-treated
cells did not restore respiratory activity of H. pylori
(data not shown).

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Fig. 1.
Inhibition of cellular respiration by
NO. Respiration of E. coli and H. pylori (1 × 108 cells/ml) was monitored polarographically with a
Clark-type oxygen electrode at 37 °C in 10 mM HEPES-NaOH
(pH 7.0) containing 0.9% NaCl and 5 mM succinate. At the
indicated times (arrows), NO was added to give a final
concentration of 5 µM. Dotted
lines, control experiments without NO.
|
|
Because NO is rapidly metabolized into various compounds, such as
nitrite and nitrate, these metabolite(s) might act to inhibit cellular
respiration. To test this hypothesis, we studied the effects of NO
metabolites and NO-trapping agents on respiration. Fairly high
concentrations of nitrite and nitrate (~100 µM) did not
inhibit the respiration of E. coli or H. pylori
(Fig. 2). In contrast, both cPTIO and
oxyhemoglobin, NO-trapping agents (20, 21), instantaneously abolished
the inhibitory effect of NO on the respiration of E. coli.
Thus, NO, rather than its metabolites, might directly interact with
E. coli and thereby inhibit cellular respiration. In the
case of H. pylori, neither cPTIO nor oxyhemoglobin abolished
the inhibitory effect of NO. Thus, some metabolite(s) of NO other than
nitrite and nitrate might irreversibly inhibit the respiration of
H. pylori.

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Fig. 2.
Effects of NO and related compounds on
respiration. At the indicated times, either nitrite
(NO2 ), nitrate
(NO3 ), NO, or peroxynitrite
(ONOO ) was added to reaction mixtures containing E. coli or H. pylori. In the presence of 5 (E. coli) or 35 µM (H. pylori) of NO, either
red blood cells (RBC) or cPTIO was added to the reaction
mixture (arrows). The final concentrations of cPTIO were 10 and 40 µM for experiments with E. coli and
H. pylori, respectively. Other conditions were as described
for Fig. 1. Dotted line, control experiments;
line 1, +100 µM
NO2 ; line 2,
+100 µM NO3 ;
line 3, +NO; line
4, NO + cPTIO; line 5, NO + red blood
cells (0.1% hematocrit); line 6, 10 (for
E. coli) or 45 µM (for H. pylori)
ONOO .
|
|
Because NO rapidly reacts with superoxide radicals (k = 6.9 × 109 M
1
s
1) and generates cytotoxic peroxynitrite, effects of
this metabolite on the respiration of E. coli and H. pylori were tested. When added to the incubation mixture,
peroxynitrite inhibited the respiration of both organisms (Fig. 2). The
characteristics of inhibition by peroxynitrite were similar to those of
inhibition of H. pylori by NO. Inhibited respiration of both
organisms persisted even after washing the cells with incubation medium
(data not shown). Thus, peroxynitrite rather than NO and other
metabolites might cause the irreversible inhibition of the respiration
of H. pylori. These observations suggest that H. pylori might generate superoxide radicals that rapidly react with
NO and generate peroxynitrite.
To test this hypothesis, the rate of superoxide generation and cellular
levels of SOD were compared between the two organisms. When incubated
in a medium containing MCLA, a chemiluminescence probe, H. pylori, elicited marked chemiluminescence that peaked after 2-3
min of incubation (Fig. 3). The MCLA
chemiluminescence was strongly inhibited by Mn-SOD, suggesting the
involvement of superoxide radicals. Under identical conditions, MCLA
chemiluminescence was negligible with E. coli. In contrast
to the superoxide generating activity, the cellular level of SOD was
significantly lower in H. pylori than in E. coli
(Table I). Based on the chemiluminescence intensity of MCLA, the steady-state level of superoxide generated by
H. pylori was calculated to be 21 ± 9 µM. Because this superoxide concentration approximates
that of NO used in the present experiments, NO might be rapidly trapped
by superoxide radicals in and around H. pylori, forming
peroxynitrite. Because peroxynitrite is cytotoxic, this metabolite,
rather than NO, might irreversibly inhibit the respiration of H. pylori. Consistent with this notion is the finding that
peroxynitrite, similar to NO, irreversibly inhibited the respiration of
H. pylori.

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Fig. 3.
Superoxide generation by H. pylori. Incubation mixtures contained in a final volume of 1 ml, 1 µM MCLA, 0.5% Triton X-100, and 1 × 108 cells/ml of either E. coli
(squares) or H. pylori (circles).
Chemiluminescence intensity was monitored continuously in the presence
(closed circles) or absence (open
symbols) of Mn-SOD (100 units/ml). Other conditions were as
described for Fig. 1. Each point represents the mean value
derived from two experiments.
|
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Table I
Superoxide generation and SOD activity in E. coli and H. pylori
Superoxide was determined by the chemiluminescence method (17) using a
Bio-Orbit 1251 luminometer in buffer solution containing 1 µM MCLA and 0.5% Triton X-100. After disrupting cells by
sonication, the homogenates were centrifuged at 10,000 × g and 4 °C for 20 min. SOD activity in the supernatants
was determined with an SOD assay kit. SOD-sensitive chemiluminescence
intensity of 106 cpm corresponds to the formation of 0.6 µM superoxide per min, as determined by the cytochrome
c method (16). Protein concentrations of 108
cells/ml corresponded to 300 and 200 µg/ml for E. coli and
H. pylori, respectively.
|
|
Because peroxynitrite reacts with metals and Fe-S clusters in various
proteins and oxidizes sulfhydryl groups of proteins (22-26), it might
affect some component(s) in the respiratory systems of bacteria. The
highly toxic nature of peroxynitrite and its metabolites (hydroxyl
radical) may also impair the structure and functions of cell
constituents, including proteins and DNA. Preliminary experiments using
anti-nitrotyrosine antibody and Western blotting after sodium dodecyl
sulfate-polyacrylamide gel electrophoresis revealed significant amounts
of immunoreactive protein bands in NO-treated H. pylori but
not in NO-treated E. coli. Helicobacter species show
considerable genomic variation (27, 28). This genomic diversity may be
one reason for their success as ubiquitous pathogens. Preliminary
polymerase chain reaction-based RFLP analysis of the gene encoding
urease showed that NO induced mutation in H. pylori. Under
identical conditions, NO had no appreciable effect on DNA samples
obtained from NO-treated E. coli. These observations are
consistent with the hypothesis that NO reacted with endogenously generated superoxide radicals and the resulting peroxynitrite reacted
with proteins and DNA of H. pylori. Reactive oxygen species, including peroxynitrite and hydroxyl radicals, have been well documented to induce gene mutations and cancer. Thus, generation of
superoxide and related metabolites might underlie the mechanisms of
their genetic diversity and their role in the pathogenesis of gastric
inflammation and cancer (29-33). However, these possibilities require
further investigation before firm conclusions can be made.
 |
FOOTNOTES |
*
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed. Tel. and Fax:
81-6-645-2025; E-mail: masainoue{at}msic.med.osaka-cu.ac.jp.
1
The abbreviations used are: NO, nitric oxide;
cPTIO, carboxy-2-phenyl-4,4,5,5-tetramethylimidazolin-1-oxyl-3-oxide;
SOD, superoxide dismutase; MCLA,
2-methyl-6-[p-methoxyphenyl]-3,7-dihydroimidazol[1,2-
]pyrazin-3-one.
 |
REFERENCES |
-
Nathan, C.,
and Xie, Q.
(1994)
Cell
78,
915-918[Medline]
[Order article via Infotrieve]
-
Tekehara, Y.,
Kanno, T.,
Yoshioka, T.,
Inoue, M.,
and Utsumi, K.
(1995)
Arch. Biochem. Biophys.
323,
27-32[CrossRef][Medline]
[Order article via Infotrieve]
-
Inai, Y.,
Takehara, Y.,
Yabuki, M.,
Sato, E. F.,
Akiyama, J.,
Inoue, M.,
Horton, A. A.,
and Utsumi, K.
(1996)
Cell Struct. Funct.
21,
151-157[Medline]
[Order article via Infotrieve]
-
Nishikawa, M.,
Sato, E. F.,
Utsumi, K.,
and Inoue, M.
(1996)
Cancer Res.
56,
4535-4540[Abstract]
-
Yu, H.,
Sato, E. F.,
Nagata, K.,
Nishikawa, M.,
Kashiba, M.,
Arakawa, T.,
Kobayashi, K.,
Tamura, T.,
and Inoue, M.
(1997)
FEBS Lett.
409,
161-165[CrossRef][Medline]
[Order article via Infotrieve]
-
Lin, J. Y.,
and Chadee, K.
(1992)
J. Immunol.
148,
3999-4005[Abstract/Free Full Text]
-
Beckerman, K. P.,
Rogers, H. W.,
and Corbett, J. A.
(1993)
J. Immunol.
150,
888-895[Abstract/Free Full Text]
-
Blaser, M. J.
(1990)
J. Infect. Dis.
161,
626-633[Medline]
[Order article via Infotrieve]
-
Parsonnet, J.,
Friedman, G. D.,
Vandersteen, D. P.,
Chang, Y.,
Vogelman, J. H.,
Orentreich, N.,
and Sibley, R. K.
(1991)
N. Engl. J. Med.
325,
1127-1131[Abstract]
-
Parsonnet, J.,
Hansen, S.,
Rodriguez, L.,
Gelb, A. B.,
Warnke, R. A.,
Jellum, E.,
Orentrich, N.,
Vogelman, J. H.,
and Friedman, G. D.
(1994)
N. Engl. J. Med.
330,
1267-1271[Abstract/Free Full Text]
-
Rabeneck, L.,
and Ransohoff, D. F.
(1991)
Am. J. Med.
91,
566-572[Medline]
[Order article via Infotrieve]
-
Lundberg, J. O. N.,
Weitzberg, E.,
Lundberg, J. M.,
and Alving, K.
(1994)
Gut
35,
1543-1546[Abstract]
-
Brown, J. F.,
Tepperman, B. L.,
Hanson, P. J.,
Whittle, B. J. R.,
and Moncada, S.
(1992)
Biochem. Biophys. Res. Commun.
184,
680-685[Medline]
[Order article via Infotrieve]
-
Akiba, Y., Nakamura, M., Ito, T., Morikawa, A., Oda, M., and Tsuchiya,
M. (1993) Gastroenterology 104, (suppl.) A31
-
Nagata, K.,
Satoh, H.,
Iwahi, T.,
Shimoyama, T.,
and Tamura, T.
(1993)
Antimicrob. Agents Chemother.
37,
769-774[Abstract]
-
McCord, J.,
and Fridovich, I.
(1969)
J. Biol. Chem.
244,
6049-6055[Abstract/Free Full Text]
-
Nakano, M.,
Sugioka, K.,
Ushijima, Y.,
and Goto, T.
(1986)
Anal. Biochem.
159,
363-369[Medline]
[Order article via Infotrieve]
-
Nebot, C.,
Moutet, M.,
Huet, P.,
Xu, J. Z.,
Yadan, J. C.,
and Chaudiere, J.
(1993)
Anal. Biochem.
214,
442-451[CrossRef][Medline]
[Order article via Infotrieve]
-
Lowry, O. H.,
Rosebrough, N. J.,
Farr, A. L.,
and Randall, R. J.
(1951)
J. Biol. Chem.
193,
265-275[Free Full Text]
-
Akaike, T.,
Yoshida, M.,
Miyamoto, Y.,
Sato, K.,
Kohno, M.,
Sasamoto, K.,
Miyazaki, K.,
Ueda, S.,
and Maeda, H.
(1993)
Biochemistry
32,
827-832[Medline]
[Order article via Infotrieve]
-
Doyle, M. P.,
and Hoekstra, J. W.
(1981)
J. Inorg. Biochem.
14,
351-358[CrossRef][Medline]
[Order article via Infotrieve]
-
Mayer, J.
(1981)
Arch. Biochem. Biophys.
210,
246-256[Medline]
[Order article via Infotrieve]
-
Granger, D. L.,
and Lehninger, A. L.
(1982)
J. Cell Biol.
95,
527-535[Abstract]
-
Reddy, D.,
Lancaster, J. R.,
and Cornforth, D. P.
(1983)
Science
221,
769-770[Medline]
[Order article via Infotrieve]
-
Drapier, J. C.,
and Hibbs, J. B., Jr.
(1986)
J. Clin. Invest.
78,
690-797
-
Radi, R.,
Rodriguez, M.,
Castro, J.,
and Telleri, R.
(1994)
Arch. Biochem. Biophys.
308,
89-95[CrossRef][Medline]
[Order article via Infotrieve]
-
Jorgensen, M.,
Daskalopoulos, G.,
Warburton, V.,
Mitchell, H. M.,
and Hazell, S. L.
(1996)
J. Infect. Dis.
174,
631-635[Medline]
[Order article via Infotrieve]
-
Blaser, M. J.
(1996)
in
Genetic Basis for Heterogeneity of Helicobacter pylori: Helicobacter pylori Basic Mechanisms to Clinical Cure (Hunt, R. H., and Tygat, G. N. J., eds), pp. 33-39, Kluwer Academic Publishers, Dordrecht, The Netherlands
-
Perchellet, J. P.,
and Perchellet, E. M.
(1989)
Free Radical Biol. Med.
7,
377-408[CrossRef][Medline]
[Order article via Infotrieve]
-
Basu, A. K.,
Lechler, E. L.,
Leadon, S. A.,
and Essigmann, J. M.
(1989)
Proc. Natl. Acad. Sci. U. S. A.
86,
7677-7681[Abstract]
-
Salgo, M. G.,
Stone, K.,
Squadrito, G. L.,
and Pryor, W. A.
(1995)
Biochem. Biophys. Res. Commun.
210,
1025-1030[CrossRef][Medline]
[Order article via Infotrieve]
-
Yermilov, V.,
Rubio, J.,
and Oshima, H.
(1995)
FEBS Lett.
376,
207-210[CrossRef][Medline]
[Order article via Infotrieve]
-
Lie, J. L.,
Okada, S.,
Hamazaki, S.,
Ebina, Y.,
and Midorikawa, O.
(1987)
Cancer Res.
47,
1867-1869[Abstract]
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.