FecA1, a bacterial iron transporter, determines the survival of Helicobacter pylori in the stomach

Helicobacter pylori encodes a single iron-cofactored superoxide dismutase (SodB), which is regulated by the ferric uptake regulator (Fur). Ferrous ion (Fe(2+)) is necessary for the activation of SodB. The activity of SodB is an important determinant of the capability of H. pylori for long-term colonization of the stomach and of the development of metronidazole (Mtz) resistance of the bacterium. This study is conducted to characterize the Fe(2+)-supply mechanisms for the activation of SodB in H. pylori, which, as mentioned above, is associated with the host-colonization ability and Mtz resistance of H. pylori. In this study, we demonstrate that fecA1, a Fe(3+)-dicitrate transporter homolog, is an essential gene for SodB activation, but not for the biogenic activity of H. pylori. H. pylori with SodB inactivation by fecA1 deletion showed reduced resistance to H(2)O(2), reduced gastric mucosal-colonization ability in Mongolian gerbils, and also reduced resistance to Mtz. Our experiment demonstrated that FecA1 is an important determinant of the host-colonization ability and Mtz resistance of H. pylori through Fe(2+) supply to SodB, suggesting that FecA1 may be a possible target for the development of a novel bactericidal drug.     

Regulation of oxidative stress response by CosR, an essential response regulator in Campylobacter jejuni

CosR (Campylobacter oxidative stress regulator; Cj0355c) is an OmpR-type response regulator essential for the viability of Campylobacter jejuni, a leading foodborne pathogen causing human gastroenteritis worldwide. Despite importance, the function of CosR remains completely unknown mainly because of cell death caused by its knockout mutation. To overcome this technical limitation, in this study, antisense technology was used to investigate the regulatory function of CosR by modulating the level of CosR expression. Two-dimensional gel electrophoresis (2DGE) was performed to identify the CosR regulon either by suppressing CosR expression with antisense peptide nucleic acid (PNA) or by overexpressing CosR in C. jejuni. According to the results of 2DGE, CosR regulated 32 proteins involved in various cellular processes. Notably, CosR negatively regulated a few key proteins of the oxidative stress response of C. jejuni, such as SodB, Dps, Rrc and LuxS, whereas CosR positively controlled AhpC. Electrophoretic mobility shift assay showed that CosR directly bound to the promoter region of the oxidative stress genes. DNase I footprinting assays identified 21-bp CosR binding sequences in the sodB and ahpC promoters, suggesting CosR specifically recognizes and binds to the regulated genes. Interestingly, the level of CosR protein was significantly reduced by paraquat (a superoxide generator) but not by hydrogen peroxide. Consistent with the overall negative regulation of oxidative stress defense proteins by CosR, the CosR knockdown by antisense rendered C. jejuni more resistant to oxidative stress compared to the wild type. Overall, this study reveals the important role played by the essential response regulator CosR in the oxidative stress defense of C. jejuni.     

Regulation of ferritin-mediated cytoplasmic iron storage by the ferric uptake regulator homolog (Fur) of Helicobacter pylori

Homologs of the ferric uptake regulator Fur and the iron storage protein ferritin play a central role in maintaining iron homeostasis in bacteria. The gastric pathogen Helicobacter pylori contains an iron-induced prokaryotic ferritin (Pfr) which has been shown to be involved in protection against metal toxicity and a Fur homolog which has not been functionally characterized in H. pylori. Analysis of an isogenic fur-negative mutant revealed that H. pylori Fur is required for metal-dependent regulation of ferritin. Iron starvation, as well as medium supplementation with nickel, zinc, copper, and manganese at nontoxic concentrations, repressed synthesis of ferritin in the wild-type strain but not in the H. pylori fur mutant. Fur-mediated regulation of ferritin synthesis occurs at the mRNA level. With respect to the regulation of ferritin expression, Fur behaves like a global metal-dependent repressor which is activated under iron-restricted conditions but also responds to different metals. Downregulation of ferritin expression by Fur might secure the availability of free iron in the cytoplasm, especially if iron is scarce or titrated out by other metals.     

Role of iron and superoxide for generation of hydroxyl radical, oxidative DNA lesions, and mutagenesis in Escherichia coli

We measured the generation of hydroxyl radical (OH(.)) and oxidative DNA lesions in aerobically grown Escherichia coli cells lacking in both superoxide dismutases (SodA SodB) and repressor of iron uptake (Fur) using electroparamagnetic resonance and gas chromatography-mass spectrometry with a selected-ion monitoring method. A specific signal corresponding to OH(.) generation and an increase in oxidative DNA lesions such as 7,8-dihydro-8-oxoguanine and 1,2-dihydro-2-oxoadenine were detected in the strain deficient in sodA sodB fur. We showed that iron metabolism deregulation in fur mutant produced a 2.5-fold iron overload. The sodA sodB fur strain was about 100-fold higher mutability than the wild-type strain. The mutation spectrum in the strain was found to induce GC --> TA and AT --> CG transversions predominantly. The hypermutability of the strain was suppressed by the tonB mutation which reduces iron transport. Thus, excess iron and excess superoxide were responsible for OH(.) generation, oxidative DNA lesion formation, and hypermutability in E. coli.