Fig 1.
Quantification of transferrin receptor mRNA and protein in AGS cells infected with H. pylori.
Cells were infected with wild type H. pylori strain 60190 (MOI, 10:1) or supplemented with 100 μm iron sulphate, with (A) transferrin receptor (TfR) and (B) H-ferritin mRNA (dotted line) and protein levels (solid line) quantitated throughout 12 h of infection. Levels are expressed as the amount of RNA and protein relative to endogenous controls HPRT and GAPDH, respectively, with representative immunoblots shown below the graphs. Data points for mRNA levels represent the average of three experiments each done in triplicate, whereas data points for protein represent ±SEMs for three independent immunoblotting experiments. *, levels over time are statistically different (P<0.05), as determined by one-way ANOVA with Tukey’s test for multiple comparisons.
Fig 2.
Distribution of transferrin receptor and H-ferritin in H. pylori-infected AGS cells.
AGS cells infected with H. pylori strain 60190 (MOI, 10:1) or supplemented with iron sulphate (100 μm) for 15 h were fixed and stained with Alexa Fluor 488-labelled transferrin receptor (green), Texas Red-conjugated phalloidin-stained actin (red) and Hoechst 33342-stained nuclei (blue) in non-permeabilized cells; Streptavidin-Phycoerythrin-stained H-ferritin (orange) and Hoechst 33342-stained nuclei (blue) in Triton X-100 permeabilized cells, and lysosomal iron using sulphide-silver (left, middle and right columns, respectively). The images, which are representative of three independent experiments, denote cells with no bacteria (Uninfected), infected with H. pylori strain 60190 (Wildtype) or supplemented with 100 μm iron sulphate (Iron) for 15 h. All images were taken with the same exposure time for each channel. Bar = 50μm. (B) The mean pixel intensity (MPI) of sulphide-silver stained AGS cells following infection with H. pylori or supplemented with iron sulphate calculated using ImageJ software, with levels of lysosomal iron (evidenced by MPI) expressed as a percentage of that observed in uninfected (Ctrl) cells. (C) H. pylori-infected and iron-supplemented AGS cells treated with calcein, with and without the addition of SIH to measure the cytosolic labile iron pool. Results, which show an increase in fluorescence (ΔF) in infected and iron-supplemented cells, are presented as a percentage of uninfected cells. (B) Results are ± SEM of five images taken from three independent experiments and (C), ± SEM of 3 independent repeats. *, ****, results are statistically different from those of untreated controls (P<0.05 and <0.0001, respectively), as determined by one-way ANOVA with Tukey’s test for multiple comparisons.
Fig 3.
H. pylori co-localise with ferritin in Rab7-rich compartments within AGS cells.
(A) Phagosomes were collected from lysed AGS cells following infection with H. pylori strain 60190 (MOI, 10:1) for 15 h (top) or not (bottom) by sucrose density gradient centrifugation. Ten (top down) fractions were blotted onto PVDF membrane and probed with antibodies to H. pylori Lpp20, H-ferritin and Rab7. Antibody binding was visualised using a chemiluminescent detection of HRP-conjugated secondary antibody binding. Images are representative for three independent experiments. (B) AGS cells infected with DiO-labelled H. pylori strain 60190 and stained with LysoTracker Red are shown individually and in a merged image. Bar = 20μm.
Fig 4.
H. pylori uptake by AGS cells.
(A) Flow cytometry was used to assess internalization of DiO-labelled bacteria following infection of AGS cells with H. pylori (MOI, 10:1) for 15 h. (B) Infected cells were treated with gentamycin for 2 h to kill any remaining extracellular bacteria. Quantitative assessment of viable H. pylori in AGS cells was by serial dilutions of cells lysates. Results are ± SEM of three independent experiments. *, results are statistically different from those of cells infected with H. pylori wildtype strain 60190 (P<0.05), as determined by one-way ANOVA with Tukey’s test for multiple comparisons.
Fig 5.
Labile iron pool in AGS cells infected with H. pylori CagA and VacA mutants.
AGS cells infected with H. pylori (MOI, 10:1) for 15 h were (A) fixed and stained with sulphide-silver to detect lysosomal iron or (B), treated with calcein with and without the addition of SIH to measure the cytosolic labile iron pool. (A) The mean pixel intensity (MPI) of each image was calculated using ImageJ software and evidence of lysosomal iron in infected cells (indicated by MPI) was expressed as a percentage of that observed in uninfected (Ctrl) cells. (B) Results reflect the increase in fluorescence (ΔF) in cells infected with the wildtype, and VacA and CagA mutant strains of H. pylori strain 60190 presented as a percentage of uninfected cells. (A) Results are ± SEM of five images taken from three independent experiments and (B), ± SEM of 3 independent repeats. *, **, ****, results are statistically different from those of untreated controls (P<0.05, <0.01 and <0.0001, respectively), as determined by one-way ANOVA with Tukey’s test for multiple comparisons.