Helicobacter pylori CagA mediated mitophagy to attenuate the NLRP3 inflammasome activation and enhance the survival of infected cells

Abstract

Helicobacter pylori (H. pylori) is one of the most common bacterial infections in the world, and its key virulence component CagA is the leading cause of gastric cancer. Mitophagy is a form of selective autophagy that eliminates damaged mitochondria and is essential for some viruses and bacteria to evade the immune system. However, the mechanisms by which CagA mediates H. pylori-induced mitophagy and NLRP3 inflammasome activation remain elusive. In this study, we reported that H. pylori primarily uses its CagA to induce mitochondrial oxidative damage, mitochondrial dysfunction, dynamic imbalance, and to block autophagic flux. Inhibition of mitophagy led to an increase in NLRP3 inflammasome activation and apoptosis and a decrease in the viability of H. pylori-infected cells. Our findings suggested that H. pylori induces mitochondrial dysfunction and mitophagy primarily via CagA. It reduces NLRP3 inflammasome activation to evade host immune surveillance and increases the survival and viability of infected cells, potentially leading to gastric cancer initiation and development. Our findings provide new insights into the pathogenesis of H. pylori-induced gastric cancer, and inhibition of mitophagy may be one of the novel techniques for the prevention and treatment of this disease.

Keywords: Helicobacter pylori; Autophagy flux; Gastric cancer; Mitophagy; NLRP3 inflammasome; Survival and viability.

Fig. 1

H. pylori CagA induces mitochondrial dysfunction, apoptosis and mitochondrial dynamic imbalance of gastric epithelial cells. GES-1 and AGS cells infected with H. pylori for 48 h at an MOI of 60, respectively. (A) Morphological observation of GZ7/cagA and GZ7/Δ cagA after Gram staining, and western blotting was used to detect the expression of intracellular CagA protein.Scale bar: 200 μm. The membrane strips were cut before hybridization with antibodies, which resulted in the inability to provide the original full-length blot images. (B) Effect of H. pylori infection on cellular ATP production the ATP production levels were determined using an ATP assay kit. (C) Effect of H. pylori infection on Mitochondrial Membrane Potential, the Mitochondrial Membrane Potential were determined using the JC-1 kit, (D) Effect of H. pylori infection on cell apoptosis, the rates of cell apoptosis were measured by flow cytometry. The figure presents the average of three independent experiments. Data are presented as the means ± SD. Error bars represent the standard deviation. control, the uninfected cells; GZ7/ΔcagA, the GZ7/ΔcagA strains infected cells; GZ7/cagA, the GZ7/cagA strains infected cells; *p < 0.05, **p < 0.01; GZ7/cagA vs control, GZ7/cagA vs GZ7/ΔcagA.

Fig. 2

H. pylori CagA induces mitochondrial dynamic imbalance in gastric epithelial cells. GES-1 and AGS cells infected with H. pylori for 48 h at an MOI of 60, respectively. (A) The expression of proteins related to mitochondrial dynamics of H. pylori-infected AGS cells were detected by western blotting; (B) The expression of proteins related to mitochondrial dynamics of H. pylori-infected GES-1 cells were detected by western blotting. The membrane strips were cut before hybridization with antibodies, which resulted in the inability to provide the original full-length blot images.The figure presents the average of three independent experiments. Data are presented as the means ± SD. Error bars represent the standard deviation. control, the uninfected cells; GZ7/ΔcagA, the GZ7/ΔcagA strains infected cells; GZ7/cagA, the GZ7/cagA strains infected cells; *p < 0.05, **p < 0.01; GZ7/cagA vs control, GZ7/cagA vs GZ7/ΔcagA.

Fig. 3

H. pylori CagA induces mitophagy in infected gastric epithelial cells. GES-1 and AGS cells infected with H. pylori for 48 h at an MOI of 60, respectively. (A) The expression of mitophagy- related proteins PINK1, PARKIN, P62, LC3, VDAC1and TOM20 were detected by western blotting. The membrane strips were cut before hybridization with antibodies, which resulted in the inability to provide the original full-length blot images.; (B) The immunofluorescence signal accumulation of LC3 co-localization with mitochondria were observed by confocal microscopy, Scale bar: 10 μm; (C) The accumulation of autophagosomes and autolysosomes was performed by TEM, Scale bar: 5 μm and 1 μm. The figure presents the average of three independent experiments. Data are presented as the means ± SD. Error bars represent the standard deviation. control, the uninfected cells; GZ7/ΔcagA, the GZ7/ΔcagA strains infected cells; GZ7/cagA, the GZ7/cagA strains infected cells; *p < 0.05, **p < 0.01; GZ7/cagA vs control, GZ7/cagA vs GZ7/ΔcagA.

Fig. 4

Mitophagy-related proteins expression were increased in gastric tissues of H. pylori infected mice. C57BL/6 mice were inoculated orally with the GZ7/cagA and GZ7/ΔcagA for 3 months, respectively. (A) H. pylori colonization status was detected by immunohistochemical staining; (B) Gastric tissues were collected and the expression of P62, LC3, PINK1 and PARKIN were detected by immunochemistry, Scale bar: 100 μm. The figure presents the average of three independent experiments. Data are presented as the means ± SD. Error bars represent the standard deviation. control, the uninfected cells; GZ7/ΔcagA, the GZ7/ΔcagA strains infected cells; GZ7/cagA, the GZ7/cagA strains infected cells; *p < 0.05, **p < 0.01; GZ7/cagA vs control, GZ7/cagA vs GZ7/ΔcagA.

Fig. 5

The autophagic flux was blocked due to H. pylori infection. GES-1 and AGS cells infected with H. pylori for 48 h at an MOI of 60, respectively.,then, the co-localization of autophagosomes and lysosomes were observed by confocal microscopy. (A) AGS cells infected with GZ7/cagA and GZ7/Δ cagA strains; (B) GES-1 cells infected with GZ7/cagA and GZ7/Δ cagA strains; Scale bar: 10 μm. The figure presents the average of three independent experiments. Data are presented as the means ± SD. Error bars represent the standard deviation. control, the uninfected cells; GZ7/ΔcagA, the GZ7/ΔcagA strains infected cells; GZ7/cagA, the GZ7/cagA strains infected cells; *p < 0.05, **p < 0.01; GZ7/cagA vs control, GZ7/cagA vs GZ7/ΔcagA.

Fig. 6

Inhibition of Mitophagy can increase NLRP3 inflammasome activation of H. pylori infected cells. AGS and GES-1 cells were pretreated with mitophagy inhibitor (BafA1, 10 μM) for 24 h, respectively, and then infected with GZ7/cagA and GZ7/Δ cagA at an MOI of 60 for 48 h, respectively. (A) Western blotting analysis of the effect of BafA1treatment on the expression of mitophagy-related protein P62 and PARKIN in AGS and GES-1 cells. (B) Western blotting analysis of the effect of BafA1 treatment on the expression of NLRP3 inflammasome protein NLRP3, Caspase-1 and IL-18 in AGS and GES-1 cells. (C) Western blotting analysis of the effect of BafA1 pretreatment on the expression of NLRP3 inflammasome protein NLRP3, Caspase-1 and IL-18 in AGS and GES-1 cells with H. pylori infection. The figure presents the average of three independent experiments. Data are presented as the means ± SD. Error bars represent the standard deviation. control, the uninfected cells; GZ7/ΔcagA, the GZ7/ΔcagA strains infected cells; GZ7/cagA, the GZ7/cagA strains infected cells; *p < 0.05, **p < 0.01; GZ7/cagA vs control, GZ7/cagA vs BafA1 + GZ7/cagA, BafA1 + GZ7/cagA vs BafA1 + GZ7/ΔcagA.

Fig. 7

Inhibition of Mitophagy can increase apoptosis, and decrease the cell viability of H. pylori infected cells. AGS and GES-1 cells were pretreated with mitophagy inhibitor (BafA1, 10 μM) for 24 h, respectively, and then infected with GZ7/cagA and GZ7/Δ cagA at an MOI of 60. (A) The apoptosis rate of H. pylori-infected cells were detected by flow cytometry after H. pylori infection 48 h. (B) The cell viability of H. pylori-infected cells were detected by CCK8 assay after H. pylori infection 6 h, 24 h and 48 h. The figure presents the average of three independent experiments. Data are presented as the means ± SD. Error bars represent the standard deviation. control, the uninfected cells; GZ7/ΔcagA, the GZ7/ΔcagA strains infected cells; GZ7/cagA, the GZ7/cagA strains infected cells; *p < 0.05, **p < 0.01; GZ7/cagA vs control, GZ7/cagA vs BafA1 + GZ7/cagA, BafA1 + GZ7/cagA vs BafA1 + GZ7/ΔcagA.