Figure 1.
Effects of the GFP-CagA C-ter fusion proteins expressed into gastric epithelial cells.
(A) Fluorescence of AGS cells transfected with GFP or GFP-CagA C-ter, either wt or mut. Transfected cells (green), actin staining (red). All the pictures shown represent single confocal sections. Scale bar: 10 µm. The graph: percentage of cells exhibiting the so-called “hummingbird phenotype” for each transfection condition. Mean±SEM by extensive confocal microscopy evaluation of slides from 3 independent experiments. *: P<0.05 versus non-transfected cells. (B) AGS cells transfected with GFP or GFP-CagAC-ter, either wt or mut, and then quantitatively analyzed for NF-kB p65-Rel nuclear translocation. Mean±SEM of 3 independent experiments. *: P<0.05 versus non-transfected cells. °: P<0.05 versus GFP-CagA C-ter wt. (C) AGS cells transfected with GFP or GFP-CagA C-ter, either wt or mut. After 24 h the cell culture supernatants were tested for IL-8. Mean±SEM of 3 independent experiments. *: P<0.05 versus non-transfected cells. (D) Representative blot (top) showing expression of c-Src, either total or activated form (c-Src P-Tyr 416; i.e., exhibiting tyrosine 416 phosphorylation) in AGS cells transfected with GFP or GFP-CagA C-ter, either wt or mut. Histograms (bottom) represent the quantitation of c-Src P-Tyr 416 (normalized for protein loading (α-tubulin) and shown as percentage of GFP-transfected control cells) for each transfection condition. Mean±SEM of 3 independent experiments. *: P<0.05 versus GFP. °: P<0.05 versus GFP-CagA C-ter wt. (E) Apoptosis (shown as fold increase over the non-transfected control cells) induced by 3 h treatment with 0.5 µM staurosporine in AGS cells transfected with GFP or GFP-CagA C-ter, either wt or mut. Mean±SEM of 3 independent experiments. *: P<0.05 versus paired control. °: P<0.05 versus all other staurosporine-treated conditions.
Figure 2.
Transfection of gastric epithelial cells with GFP-CagA C-ter, either wt or mut, antagonizes VacA-induced apoptosis.
(A) Apoptosis (shown as fold increase over the respective non-transfected control cells) induced by either VacA or etoposide in gastric AGS or MKN 28 cells. VacA was used at 5 µg/ml for 18 h, while etoposide at 300 µM for 6 h (AGS cells) or 24 h (MKN 28 cells). Mean±SEM of 3 independent experiments. *: P<0.05 versus paired control. (B and C) VacA internalization and its vacuolating action in transfected AGS (B) or MKN 28 (C) cells. VacA (red) and nuclei (blue). Transfected cells (green). All the pictures shown represent single confocal sections. Scale bar: 10 µm. For a more careful evaluation of VacA internalization, for each merged picture the overall z-stack profile of the cells along the depicted x-y axes were highlighted. (a), (b), and (c) are respectively zooms of the merged pictures at each x-y intersection. The graph (top right): percentage of either vacuolated cells (white columns) or cells deeply internalizing VacA (black columns) for each transfection condition. *: P<0.05 versus non-transfected cells. °: P<0.05 versus GFP-CagA C-ter mut.
Figure 3.
Cytochalasin D treatment protects gastric epithelial cells against both drop in mitochondrial transmembrane potential and apoptosis induced by VacA by preventing its mitochondrial localization.
(A) Effect of cytochalasin D (CD) on mitochondrial transmembrane potential (MTP) of MKN 28 cells, treated or not with VacA, evaluated by using the ratiometric potential-sensitive dye JC-1 and confocal microscopy. All the pictures shown represent single confocal sections. Scale bar: 10 µm. The graph (right) shows the quantitation (mean±SEM) of relative MTP in the different experimental conditions by extensive confocal microscopy evaluation of slides from 3 independent experiments. *: P<0.05 versus paired control. °: P<0.05 versus the same condition in the presence of CD. (B) Morphology of mitochondria and their colocalization with VacA in AGS cells transfected with DsRed-Mito and treated or not with Cy5-labelled VacA in the absence or presence of CD. DsRed-Mito (red) and Cy5-VacA (green). All the pictures shown represent single confocal sections. Scale bar: 10 µm. (C) Apoptosis (shown as fold increase over the CD-untreated control cells) induced by VacA in MKN 28 cells treated or not with CD. Cells treated or not with CD but not exposed to VacA served as controls. Mean±SEM of three different experiments. *: P<0.05 versus paired control. °: P<0.05 versus the same condition in the presence of CD.
Figure 4.
Treatment of gastric epithelial cells with the specific SKF inhibitor PP2 impairs the arrival of VacA into endosomes.
(A and B) VacA intracellular trafficking in AGS (A) or MKN 28 (B) cells treated or not with PP2. Cells were preincubated for 30 min at 37°C in the presence or absence of 10 µM PP2 and, after a VacA binding step of 1 h at 4°C, immediately fixed (0 min) or allowed to internalize the toxin into early (30 min) or late (120 min) endocytic compartments (top left panels). VacA in green. For the 120 min time point, also LAMP1 (red; a marker of late endosomes) and its colocalization with VacA are shown (top right panels). All the pictures represent single confocal sections. Scale bar: 10 µm. The graphs (bottom panels): effect of PP2 treatment on either the percentage of cells exhibiting VacA confined in the GEECs (left) or the percentage of late endosomes (i.e., LAMP1-positive vesicles) containing VacA (right) after 2 h internalization. Mean±SEM by extensive confocal microscopy evaluation of slides from 3 independent experiments. *: P<0.05 versus PP2-untreated cells. (C) Apoptosis (shown as fold increase over the respective PP2-untreated control cells) induced by either VacA or etoposide in MKN 28 cells treated or not with PP2. Cells treated or not with PP2 but not exposed to VacA or etoposide served as controls. Mean±SEM of three different experiments. *: P<0.05 versus paired control. °: P<0.05 versus the same condition in the absence of PP2. The high cytotoxicity induced by PP2 on AGS cells after the incubation time used to measure either VacA- or etoposide-induced apoptosis did not enable us to repeat the same set of experiments in this latter cell line.
Figure 5.
Transfection of gastric epithelial cells with GFP-Bcl2 inhibits VacA-induced apoptosis while not blocking pinocytosis or intracellular trafficking of the toxin.
(A) Apoptosis (shown as fold increase over the respective non-transfected control cells) induced by either VacA or etoposide in AGS or MKN 28 cells transfected with GFP-Bcl2, GFP-Bcl-xL, GFP-Mcl1, or the GFP vector. Cells were treated with VacA or etoposide as in Figure 3A. Non-treated cells served as controls. Mean±SEM of 3 independent experiments. *: P<0.05 versus paired control. (B and C) VacA internalization in AGS (B) or MKN 28 (C) cells transfected with GFP-Bcl2, GFP-Bcl-xL, or the GFP vector. VacA (red) and nuclei (blue). Transfected cells (green). All the pictures shown represent single confocal sections. Scale bar: 10 µm. For a more careful evaluation of VacA internalization, in every merged picture the overall z-stack profile of the cells along the depicted x-y axes were highlighted. (a), (b), and (c) are respectively zooms of the merged pictures at each x-y intersection.
Figure 6.
CagA antagonizes VacA-induced apoptosis in H. pylori-infected MKN 28 gastric epithelial cells.
Apoptosis degree (shown as fold increase over the respective non-infected control cells) of MKN 28 cells infected with the wild-type CagA+/VacA+ H. pylori strain 60190 (WT) or its isogenic mutants lacking CagA (ΔcagA) or VacA (ΔvacA), respectively, in the absence or presence of 5 µg/ml dominant-negative purified VacA Δ6-27 cytotoxin. Mean±SEM of 3 independent experiments. *: P<0.05 versus paired control.