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Infection and Immunity, June 2001, p. 4034-4040, Vol. 69, No. 6
0019-9567/01/$04.00+0 DOI: 10.1128/IAI.69.6.4034-4040.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Superoxide Dismutase-Deficient Mutants of
Helicobacter pylori Are Hypersensitive to Oxidative Stress
and Defective in Host Colonization
Richard W.
Seyler Jr.,
Jonathan W.
Olson, and
Robert
J.
Maier*
Department of Microbiology, University of
Georgia, Athens, Georgia 30602
Received 27 November 2000/Returned for modification 12 February
2001/Accepted 13 March 2001
Superoxide dismutase (SOD) is a nearly ubiquitous enzyme among
organisms that are exposed to oxic environments. The single SOD of
Helicobacter pylori, encoded by the sodB gene,
has been suspected to be a virulence factor for this pathogenic
microaerophile, but mutations in this gene have not been reported
previously. We have isolated mutants with interruptions in the
sodB gene and have characterized them with respect to their
response to oxidative stress and ability to colonize the mouse stomach.
The sodB mutants are devoid of SOD activity, based on
activity staining in nondenaturing gels and quantitative assays of cell
extracts. Though wild-type H. pylori is microaerophilic,
the mutants are even more sensitive to O2 for both growth
and viability. While the wild-type strain is routinely grown at 12%
O2, growth of the mutant strains is severely inhibited at
above 5 to 6% O2. The effect of O2 on
viability was determined by subjecting nongrowing cells to atmospheric
levels of O2 and plating for survivors at 2-h time
intervals. Wild-type cell viability dropped by about 1 order of
magnitude after 6 h, while viability of the sodB
mutant decreased by more than 6 orders of magnitude at the same time
point. The mutants are also more sensitive to
H2O2, and this sensitivity is exacerbated by
increased O2 concentrations. Since oxidative stress has
been correlated with DNA damage, the frequency of spontaneous mutation
to rifampin resistance was studied. The frequency of mutagenesis of an
sodB mutant strain is about 15-fold greater than that of
the wild-type strain. In the mouse colonization model, only 1 out of 23 mice inoculated with an SOD-deficient mutant of a mouse-adapted strain became H. pylori positive, while 15 out of 17 mice
inoculated with the wild-type strain were shown to harbor the organism.
Therefore, SOD is a virulence factor which affects the ability of this
organism to colonize the mouse stomach and is important for the growth and survival of H. pylori under conditions of oxidative stress.
*
Corresponding author. Mailing address: Department of
Microbiology, University of Georgia, 527 Biological Sciences Building, Athens, GA 30602. Phone: (706) 542-2323. Fax: (706) 542-2674. E-mail:
rmaier{at}arches.uga.edu.
Infection and Immunity, June 2001, p. 4034-4040, Vol. 69, No. 6
0019-9567/01/$04.00+0 DOI: 10.1128/IAI.69.6.4034-4040.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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