Unique host iron utilization mechanisms of Helicobacter pylori revealed with iron-deficient chemically defined media

Abstract

Helicobacter pylori chronically infects the gastric mucosa, where it can be found free in mucus, attached to cells, and intracellularly. H. pylori requires iron for growth, but the sources of iron used in vivo are unclear. In previous studies, the inability to culture H. pylori without serum made it difficult to determine which host iron sources might be used by H. pylori. Using iron-deficient, chemically defined medium, we determined that H. pylori can bind and extract iron from hemoglobin, transferrin, and lactoferrin. H. pylori can use both bovine and human versions of both lactoferrin and transferrin, contrary to previous reports. Unlike other pathogens, H. pylori preferentially binds the iron-free forms of transferrin and lactoferrin, which limits its ability to extract iron from normal serum, which is not iron saturated. This novel strategy may have evolved to permit limited growth in host tissue during persistent colonization while excessive injury or iron depletion is prevented.

FIG. 1.

H. pylori utilizes hemoglobin as an iron source. Hemoglobin was added to TT18 at the concentrations indicated. Inocula containing the same amount of H. pylori strain 26695m were added to triplicate wells. Growth was measured by analysis of ATP after overnight incubation. rlu, relative light units. The error bars indicate standard deviations. *, P ≤ 0.01 compared with the control.

FIG. 2.

Growth on heme iron in the presence of deferoxamine. Deferoxamine mesylate was added to TT18 at a final concentration of 1 μM. Hemin chloride was added at a starting concentration of 62.5 nM, and serial 2-fold dilutions were prepared to obtain the concentrations shown. Inocula containing the same amount of strain 26695m were added to triplicate wells. Growth was assessed by measuring the ATP content after 30 h. rlu, relative light units. The error bars indicate standard deviations. *, P < 0.05 compared with the culture containing no heme.

FIG. 3.

Acquisition of iron from transferrin and lactoferrin. Iron-saturated human Tf (hTf), human Lf (hLf), bovine Tf (bTf), and bovine Lf (bLf) were added to TT18 at a concentration of 6 μg/ml. As a positive control, 10 μM FeCl₃ was added. Inocula containing the same amount of H. pylori strain 26695m were added to quadruplicate wells. Growth was measured by analysis of ATP after overnight incubation. rlu, relative light units. The error bars indicate standard deviations. *, P < 0.01 compared with the TT18 control.

FIG. 4.

Effect of lactoferrin iron saturation on H. pylori growth. Ferri-lactoferrin and apo-lactoferrin were added to TT18 at ratios that resulted in the saturation levels indicated. The total lactoferrin concentration was 9 μg/ml. Inocula containing the same amount of strain 26695m were added to duplicate wells. Growth was assessed by measuring the ATP content. rlu, relative light units.

FIG. 5.

Binding of FITC-labeled proteins to H. pylori strain SS1. Three micrograms of each protein was incubated with 1 ml of H. pylori suspended in PBS for 1 h at 37°C. Samples were centrifuged and resuspended in PBS to remove unbound protein. The transferrin and lactoferrin were iron saturated. hTf, human Tf; hLf, human Lf; bTf, bovine Tf; bLf, bovine Lf; hHb, human Hb.

FIG. 6.

Characteristics of transferrin and lactoferrin binding. H. pylori strain 26695m was incubated with FITC-labeled proteins at various concentrations (C and D) or in the presence or absence of excess unlabeled protein (A and B). The fluorescence of bacteria was measured by flow cytometry. (A) A 50-fold excess of transferrin does not prevent binding of lactoferrin. (B) A 50-fold excess of lactoferrin blocks binding by transferrin. (C) Effect of apo-Tf concentration on fluorescence distribution. (D) Effect of apo-Lf concentration on fluorescence distribution. hTf, human Tf; hLf, human Lf; bLf, bovine Lf.

FIG. 7.

Percentages of bacteria displaying levels of fluorescence greater than the background level following incubation with different amounts of protein. Bacteria were incubated with FITC-labeled apo-transferrin or apo-lactoferrin at concentrations ranging from 1 to 150 μg/ml. Gating was set to exclude 99.5% of the unstained population. hTf, human Tf; bLf, bovine Lf.

FIG. 8.

Competition between ferri-Tf or ferri-Lf and apo-Tf or apo-Lf. H. pylori strain 26695m (A to D) or N. gonorrhoeae (E and F) was incubated with FITC-labeled protein in the presence or absence of excess unlabeled protein. The fluorescence of bacteria was measured by flow cytometry. (A) FITC-ferri-Lf with or without a 50-fold excess of unlabeled apo-Lf. (B) FITC-apo-Lf with or without a 50-fold excess of unlabeled ferri-Lf. (C) FITC-ferri-Tf with or without a 50-fold excess of unlabeled apo-Tf. (D) FITC-apo-Tf with or without a 50-fold excess of unlabeled ferri-Tf. (E) N. gonorrhoeae incubated with FITC-ferri-Lf with or without a 50-fold excess of unlabeled apo-Lf. (F) N. gonorrhoeae incubated with FITC-apo-Lf with or without a 50-fold excess of unlabeled ferri-Lf. hTf, human Tf; bLf, bovine Lf.

FIG. 9.

Effect of iron restriction on binding of H. pylori to host iron-containing proteins. Binding of FITC-labeled Lf, Tf, and Hb (40 μg/ml) to wild-type strain 26695m was measured by flow cytometry following growth in iron-replete medium (F-12 with 1% FBS) or iron-restricted medium (TT18 with 1 μM DFO) for 18 h. The data are the percentages of the fluorescent population (P2) in the total population (50,000 events). Fe+, iron-replete medium; Fe−, iron-restricted medium; hTf, human Tf; hLf, human Lf; bTf, bovine Tf; bLf, bovine Lf; hHb, human Hb.

FIG. 10.

Free iron in the gastric mucosa. Lf, lactoferrin; Tf, transferrin; Hb, hemoglobin. Blue hexagons indicate iron-free Lf; red hexagons indicate iron-saturated Lf; curved blue bars indicate H. pylori cells.