Essential role of ferritin Pfr in Helicobacter pylori iron metabolism and gastric colonization

Abstract

The reactivity of the essential element iron necessitates a concerted expression of ferritins, which mediate iron storage in a nonreactive state. Here we have further established the role of the Helicobacter pylori ferritin Pfr in iron metabolism and gastric colonization. Iron stored in Pfr enabled H. pylori to multiply under severe iron starvation and protected the bacteria from acid-amplified iron toxicity, as inactivation of the pfr gene restricted growth of H. pylori under these conditions. The lowered total iron content in the pfr mutant, which is probably caused by decreased iron uptake rates, was also reflected by an increased resistance to superoxide stress. Iron induction of Pfr synthesis was clearly diminished in an H. pylori feoB mutant, which lacked high-affinity ferrous iron transport, confirming that Pfr expression is mediated by changes in the cytoplasmic iron pool and not by extracellular iron. This is well in agreement with the recent discovery that iron induces Pfr synthesis by abolishing Fur-mediated repression of pfr transcription, which was further confirmed here by the observation that iron inhibited the in vitro binding of recombinant H. pylori Fur to the pfr promoter region. The functions of H. pylori Pfr in iron metabolism are essential for survival in the gastric mucosa, as the pfr mutant was unable to colonize in a Mongolian gerbil-based animal model. In summary, the pfr phenotypes observed give new insights into prokaryotic ferritin functions and indicate that iron storage and homeostasis are of extraordinary importance for H. pylori to survive in its hostile natural environment.

FIG. 1.

The role of Pfr in acid-amplified iron resistance of H. pylori. The growth of H. pylori strain G27 (black) and of the pfr mutant (gray) in broth (BBF) was determined by measuring the OD₆₀₀. For high iron (Fe) and acidic conditions the medium was supplemented with 1 mM iron or titrated to pH 6, respectively. The data represent mean values from three independent determinations. Standard deviations are indicated.

FIG. 2.

The effect of the pfr mutation on iron uptake rates. The time-dependent uptake of ferrous iron (A) and of ferric citrate (B) by the wt strain G27 (filled triangles) and by the pfr mutant (filled squares) was measured with an uptake assay using the radioactively labeled compounds indicated. The data represent mean values from three independent determinations. Standard deviations are indicated.

FIG. 3.

Iron regulation of Pfr synthesis. (A) The regulation of Pfr synthesis in response to iron. The H. pylori wt strains G27 and NCTC11637 and an isogenic NCTC11637 feoB mutant, respectively (indicated on the left), were grown in BBF without supplementation and with iron at increasing concentrations (indicated on the top). Samples containing 15 μg of total protein were separated by SDS-PAGE, and the Pfr protein was detected by Western immunoblotting with the antiserum AK198. The second Pfr protein bands detected exclusively under iron-rich conditions and the position of the 20-kDa protein marker are indicated by asterisks on the right. (B) Reporter gene analysis of pfr gene expression. The H. pylori strain G27 carrying a transcriptional pfr::cat fusion was grown in broth (BBF) supplemented with iron at the concentrations indicated on the bottom, and the amount of the Cat protein (gray bars) resembling transcriptional activity of the pfr gene was monitored with a Cat-specific ELISA. The amount of Cat produced in unsupplemented BBF medium was set at 100%. The values represent means of two independent determinations. Standard deviations are indicated. The results are representative for two independent experiments. (C) Influence of iron on Fur binding to the pfr promoter. EMSA of the Fur binding activity was performed as described in Materials and Methods. In lanes 1 to 4 the DNA probe containing the intergenic region in front of the pfr gene (lane 1) was incubated with Fur (lanes 2 to 4) without additives (lane 2), with 0.2 mM iron added (lane 3), or with 0.2 mM sodium chloride added (lane 4). The picture represents a black and white image of the ethidium bromide-stained gel visualized under UV light. The positions of the DNA probe and the Fur protein-DNA complexes are indicated.

FIG. 4.

Colonization properties of the H. pylori pfr mutant in the gerbil model of infection. The animals were infected with the H. pylori wt strain Q1 or with the pfr and cagA mutants as indicated. The results for bacterial colonization obtained from reisolation and counting of colonies after 3 and 6 weeks are summarized in one column for each strain because there was no significant difference in the bacterial load after the infection periods. The data are representative of two individual experiments performed for 3 and 6 weeks, respectively. Standard deviations are indicated.