Helicobacter pylori CagA Protein Negatively Regulates Autophagy and Promotes Inflammatory Response via c-Met-PI3K/Akt-mTOR Signaling Pathway

Abstract

Cytotoxin-associated-gene A (CagA) of Helicobacter pylori (H. pylori) is a virulence factor that plays critical roles in H. pylori-induced gastric inflammation. In the present study, gastric biopsies were used for genotyping cagA and vacA genes, determining the autophagic activity, and the severity of gastric inflammation response. It was revealed that autophagy in gastric mucosal tissues infected with cagA⁺H. pylori strains was lower than the levels produced by cagA^-H. pylori strains, accompanied with accumulation of SQSTM1 and decreased LAMP1 expression. In vitro, deletion mutant of cagA gene resulted in increased autophagic activity, and decreased expression of SQSTM1 and cytokines, whereas over-expression of CagA down-regulated the starvation-induced autophagy, and induced more production of the cytokines. Moreover, the production of the cytokines was increased by inhibition of autophagy, but decreased by enhancement of autophagy. Deletion of CagA decreased the ability to activate Akt kinase at Ser-473 site and increased autophagy. c-Met siRNA significantly affected CagA-mediated autophagy, and decreased the level of p-Akt, p-mTOR, and p-S6. Both c-Met siRNA and MK-2206 could reverse inflammatory response. H. pylori CagA protein negatively regulates autophagy and promotes the inflammation in H. pylori infection, which is regulated by c-Met-PI3K/Akt-mTOR signaling pathway activation.

Keywords: CagA; Helicobacter pylori; SQSTM1; autophagy; c-Met.

Figure 1

The inflammatory response on gastric biopsies from patients infected with cagA⁻/vacA^s1m2 or cagA⁺/vacA^s1m2 strains of H. pylori. (A) Histological scores of inflammation (H&E staining) in the gastric mucosa of patients without H. pylori infection and those infected with cagA⁻/vacA^s1m2 or cagA⁺/vacA^s1m2 strains of H. pylori. The intensity of staining is shown in the right graph and the data are expressed as mean±SEM. (B) mRNA expression of proinflammatory cytokines in gastric mucosa of patients without H. pylori (n = 8), patients infected with cagA⁻/vacA^s1m2 (n = 7), and those infected with cagA⁺/vacA^s1m2 (n = 8). All real-time PCR data are normalized to β-actin and expressed as fold change. Experiments performed in triplicate showed consistent results. ^*P < 0.05, or ^**P < 0.01.

Figure 2

Autophagy is down-regulated in human gastric mucosa of patients infected with CagA positive H. pylori strains. (A) Immunohistochemistry showing SQSTM1 expression in the gastric mucosa of patients without H. pylori infection and those infected with cagA⁻/vacA^s1m2 or cagA⁺/vacA^s1m2 strains of H. pylori. The intensity of staining is shown in the right graph and the data are expressed as mean ± SEM. (B) Western blot assay showing the protein levels of MAP1LC3B-II, SQSTM1 and LAMP1 in the gastric mucosa of patients of normal control (patients 1–4), cagA⁻/vacA^s1m2(patients 5–8), and cagA⁺/vacA^s1m2 (patients 9–12) with the rates to β-actin being illustrated in the graphs in which the data are expressed as mean ± SEM. (C) Transmission electron microscopy showing autophagosomes in gastric biopsy sections of patients without H. pylori infection and those infected with cagA⁻/vacA^s1m2 or cagA⁺/vacA^s1m2 strains of H. pylori. Normal controls are patients without H. pylori infection. The white arrows indicate the autophagosomes. The numbers of autophagic vacuoles per cell in each TEM section (n = 35 cells) are shown in the right graph and the data are expressed as mean ± SEM. Experiments performed in triplicate showed consistent results. ^*P < 0.05, or ^**P < 0.01.

Figure 3

CagA could inhibit the generation of autophagosomes in AGS cells. (A) Confocal microscopy showing AGS cells transfected with GFP-MAP1LC3B without H. pylori infection (UI), and transfected AGS cells with the wild type H. pylori (Hp-WT), the cagA-knockout H. pylori (Hp-ΔcagA) or the cagA-knockout complementation mutant H. pylori (Hp-c-cagA) (MOI = 100:1) infection for 6 h (left) and the indicated periods of time (right). Scale bars: 10 μm. The number of GFP-MAP1LC3B puncta in each cell (n ≥ 200 cells) was counted. (B) Representative transmission electron microscopy showing AGS cells without H. pylori infection and those infected with Hp-WT, Hp-ΔcagA, or Hp-c-cagA (MOI = 100:1) for 6 h. The white arrows indicate autophagosomes, and the black arrows indicate autolysosomes, and the white triangle indicate H. pylori. The numbers of autophagic vacuoles per cell in each TEM section (n = 35 cells) are shown in the lower left graph and the data are expressed as mean ± SEM. (C,D) Flow cytometry showing MDC and AO staining of AGS cells 6 h after infection with Hp-WT, Hp-ΔcagA, or Hp-c-cagA (MOI = 100:1). (E) Western blotting showing the protein levels of CagA, SQSTM1, and MAP1LC3B-II with the rates of SQSTM1 and MAP1LC3B-II to β-actin in AGS cells infected with Hp-WT, Hp-ΔcagA, or Hp-c-cagA (MOI = 100:1) for 6 h. (F) Measurement of MAP1LC3B-II conversion and SQSTM1 in AGS cells infected with Hp-WT or Hp-ΔcagA (MOI = 100:1) for 6 h in the presence of Baf-A1 (10 nM). (G) AGS cells were transfected with GFP-CagA, and then infected with Hp-WT or Hp-ΔcagA (MOI = 100:1) for 6 h in the presence of Baf-A1 (10 nM). Results shown are representative of three independent experiments. ^*P < 0.05, ^**P < 0.01.

Figure 4

CagA down-regulates starvation-induced autophagy in AGS cells. (A) Transmission electron microscopy showing autophagic vacuoles in AGS cells transfected with GFP-CagA, GFP-CagA-Mut, or a control (pEGFP-C1) plasmid before pretreatment of normal media or subjected to 4 h starvation. The white arrows indicate autophagosomes, and the black arrows indicate autolysosomes. The numbers of autophagic vacuoles per cell in each TEM section (n = 35 cells) are shown in the lower graph and the data are expressed as mean ± SEM. (B) Western blot assay showing MAP1LC3B-II conversion and expression of p-CagA, CagA, AMPK, and SQSTM1 in AGS cells transfected with GFP-CagA, GFP-CagA-Mut, or a control (pEGFP-C1) plasmid in the nutrient rich medium or 4 h starvation. (C) Confocal microscopy showing AGS cells co-transfected with RFP-MAP1LC3B and GFP-CagA, GFP-CagA-Mut, or a control (pEGFP-C1) plasmid in the nutrient rich medium or 4 h starvation. Scale bars: 5 or 10 μm. The number of RFP-MAP1LC3B puncta in each cell (n ≥ 200 cells) was counted. Experiments were performed in triplicate, and all replicates showed similar results. ^*P < 0.05, ^**P < 0.01.

Figure 5

Inhibition of autophagy enhances cytokines production induced by the cagA-knockout H. pylori. (A,B) Production of IL-8, IL-1β and TNF-α in AGS cells infected Hp-WT, Hp-ΔcagA or Hp-c-cagA at MOI of 100 for the indicated periods of time (A) or at different MOIs (10, 50, 100, and 200) for 12 h (B), as assessed by enzyme-linked immunosorbent assay (ELISA). (C) After pretreatment of SC (solvent control, 0.1% DMSO), 3-MA (2 mM), Baf-A1 (10 nM) or Rapa (100 nM), AGS cells were infected with Hp-WT or Hp-ΔcagA (MOI = 100:1) for 6 h. Supernatants were assessed by ELISA for levels of IL-8, IL-1β, and TNF-α. (D) Production of IL-8, IL-1β, and TNF-α in AGS cells transfected with siRNA specific for ATG5 or ATG12 (50 nM) for 24 h and infected with Hp-WT or Hp-ΔcagA (MOI = 100) for 6 h, as assessed by ELISA. Data are presented as the mean ± SEM of three experiments. ^*P < 0.05, ^**P < 0.01.

Figure 6

c-Met is an important adaptor in CagA-mediated autophagy pathway. (A,B) AGS cells were infected with Hp-WT or Hp-ΔcagA, and p-c-Met and c-Met were detected by western blot. CagA was immunoprecipitated from lysates. Immunoprecipitates (IP) were subjected to SDS-PAGE and immunoblot (IB) analysis with anti-p-c-Met (top) or anti–c-Met (bottom) antibodies. (C) Confocal microscopy showing AGS cells co-transfected with GFP-MAP1LC3B plasmid and c-Met siRNAs or control siRNA for 24 h, and then infected with Hp-WT or Hp-ΔcagA for 6 h. The percentages of cells with MAP1LC3B punctas are shown in the right graph with data being expressed as means ± SEM of three experiments (n ≥ 200 cells). (D) Western blot analysis of p-c-Met, MAP1LC3B-II conversion and β-actin in AGS cells transfected with c-Met siRNA or control siRNA and infected with Hp-WT or Hp-ΔcagA for 6 h. p-c-Met and MAP1LC3B-II band intensity was normalized to β-actin. (E,F) Flow cytometry showing MDC (upper panel) and AO (lower panel) staining of AGS cells transfected with c-Met siRNA or control siRNA and then infected with Hp-WT or Hp-ΔcagA for 6 h. (G) Western blot analysis of p-c-Met, MAP1LC3B-II conversion and β-actin in CagA-expressing AGS cells (AGS cells after transfecting the CagA expression plasmid, GFP-CagA) after transfected with c-Met siRNA or control siRNA and infected with H. pylori as described above. Experiments performed in triplicate showed consistent results. ^*P < 0.05, ^**P < 0.01.

Figure 7

CagA regulates autophagy through PI3K/AKT/mTOR pathway. (A) Expression of p-Akt(Ser473), Akt, p-mTOR, mTOR, p-S6, S6, MAP1LC3B-I, and MAP1LC3B-II in AGS cells infected with Hp-WT or Hp-ΔcagA(MOI = 100) at different time points. (B) Expression of p-Akt(Ser473), Akt, p-mTOR, mTOR, p-S6, S6, MAP1LC3B-I, and MAP1LC3B-II in AGS cells transfected with GFP-CagA, GFP-CagA-Mut or control plasmid. (C) Expression of p-Akt(Ser473), Akt, p-mTOR, mTOR, p-S6, S6, MAP1LC3B-I, and MAP1LC3B-II in AGS cells transfected c-Met siRNA or control siRNA (50 nM) for 24 h and then infected with Hp-WT or Hp-ΔcagA (MOI = 100) for 6 h. (D) Expression of p-Akt(Ser473), Akt, p-mTOR, mTOR, p-S6, S6, MAP1LC3B-I, and MAP1LC3B-II in AGS cells infected with Hp-WT or Hp-ΔcagA (MOI = 100) for 6 h with or without pre-treatment of MK-2206 (8 μM). (E) Production of IL-8, IL-1β and TNF-α in AGS cells transfected c-Met siRNA or control siRNA (50 nM) for 24 h and then infected with Hp-WT or Hp-ΔcagA (MOI = 100) for 6 h, as assessed by ELISA. (F) After pretreatment of DMSO or MK-2206 (8 μM), AGS cells were infected with Hp-WT or Hp-ΔcagA (MOI = 100:1) for 6 h. Supernatants were assessed by ELISA for levels of IL-8, IL-1β, and TNF-α. Experiments performed in triplicate showed consistent results. ^*P < 0.05.