In vitro and in vivo characterization of alkyl hydroperoxide reductase mutant strains of Helicobacter hepaticus.
Mutant strains in the tsaA gene encoding alkyl hydroperoxide reductase were more sensitive to O(2) and to oxidizing agents (paraquat, cumene hydroperoxide and t-butylhydroperoxide) than the wild type, but were markedly more resistant to hydrogen peroxide. The mutant strains resistance phenotype could be attributed to a 4-fold and 3-fold increase in the catalase protein amount and activity, respectively compared to the parent strain. The wild type did not show an increase in catalase expression in response to sequential increases in O(2) exposure or to oxidative stress reagents, so an adaptive compensatory mutation has probably occurred in the mutants. In support of this, chromosomal complementation of tsaA mutants restored alkyl hydroperoxide reductase, but catalase was still up-expressed in all complemented strains. The katA promoter sequence was the same in all mutant strains and the wild type. Like its Helicobacter pylori counterpart strain, a H. hepaticus tsaA mutant contained more lipid hydroperoxides than the wild type strain. Hepatic tissue from mice inoculated with a tsaA mutant had lesions similar to those inoculated with the wild type, and included coagulative necrosis of hepatocytes. The liver and cecum colonizing abilities of the wild type and tsaA mutant were comparable. Up-expression of catalase in the tsaA mutants likely permits the bacterium to compensate (in colonization and virulence attributes) for the loss of an otherwise important oxidative stress-combating enzyme, alkyl hydroperoxide reductase. The use of erythromycin resistance insertion as a facile way to screen for gene-targeted mutants, and the chromosomal complementation of those mutants are new genetic procedures for studying H. hepaticus.
Mehta
Nalini S
NS
Department of Microbiology, University of Georgia, Athens, GA 30602, USA.
Benoit
Stéphane L
SL
Mysore
Jagannatha
J
Maier
Robert J
RJ
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002374:Catalase / Q000378:metabolism; D002876:Chromosomes, Bacterial; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology; D005784:Gene Amplification; D005816:Genetic Complementation Test; D016680:Genome, Bacterial; D044923:Helicobacter hepaticus / Q000201:enzymology* / Q000235:genetics; D016254:Mutagenesis, Insertional; D010544:Peroxidases / Q000235:genetics* / Q000378:metabolism*; D054464:Peroxiredoxins; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000378:metabolism; D011995:Recombination, Genetic
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D010544:Peroxidases; D054464:Peroxiredoxins; D002374:Catalase