The iron efflux protein ferroportin regulates the intracellular growth of Salmonella enterica

Abstract

We investigated the influence of the macrophage iron exporter ferroportin and its ligand hepcidin on intracellular Salmonella growth. Elevated ferroportin expression inhibited bacterial multiplication; hepcidin-induced ferroportin down-regulation enhanced it. Expression analysis of iron-responsive Salmonella genes indicated ferroportin-mediated iron deprivation. These results demonstrate a role for ferroportin in antimicrobial resistance.

FIG. 1.

Effect of FPN on intracellular survival of Salmonella in J774 macrophages. J774-GFP.RV (G) or J774-FPN1.RV2 (F) cells were infected with Salmonella, and the number of intracellular bacteria surviving at 2 or 20 h postinfection was determined. Means (plus standard deviations; n = 3) from a single experiment are shown. The results are representative of four such experiments. The upper part of the figure shows FPN expression in the two J774 clones. The numbers below the Western blots show the relative intensities of the FPN bands based on densitometry (normalized to actin).

FIG. 2.

Effect of FPN expression on survival of Salmonella in HeLa cells. HeLa cells were transiently transfected with expression plasmids encoding GFP (G), wild-type FPN (F), or the Q182H mutant of FPN (Q). The results of Western blotting for FPN and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression are shown at the top. The lower part shows the number of intracellular Salmonella surviving at 2 or 20 h postinfection. Means (plus standard deviations; n = 3) from a single experiment are shown. The results are representative of two such experiments for the GFP expression construct and at least five such experiments for the FPN and Q182H mutant constructs.

FIG. 3.

Effect of hepcidin on wild-type and Q182H FPN. HeLa cells were transfected to express either wild-type FPN or the Q182H mutant. Eighteen to 24 h later, hepcidin was added to the cells at 700 nM. For the Western blot, hepcidin was added for the indicated times after pretreatment of the cells for 60 min with 100 μM cycloheximide. The numbers below the blots indicate FPN band intensity, normalized to actin and expressed as a percentage of the control. For the gentamicin protection assay, the cells were infected with Salmonella about 18 h after the start of hepcidin treatment, and the number of intracellular bacteria surviving at 20 h postinfection was determined. Means (plus standard deviations; n = 3) from a single experiment are shown. The results are representative of three such experiments.

FIG. 4.

Effect of FPN on entF and iviXVII reporter gene expression. J774-GFP.RV (G) or J774-FPN1.RV2 (F) cells were infected with Salmonella strains carrying either the entF or iviXVII β-galactosidase reporters. β-Galactosidase expression was determined at 4 h postinfection and corrected for the number of intracellular bacteria. Means (plus standard deviations) from the results of three separate experiments are shown.