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

AbstractBackgroundH. pyloriis the most prevalent bacterial infection in the world, and its crucial virulence component CagA is the primary 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 potential impact ofH.pyloriCagA in the crosstalk between mitophagy and NLRP3 inflammasome is not completely known.ObjectiveIn this study, we aimed to understand the impact ofH. pyloriand its CagA on the induction of mitochondrial dysfunction and mitophagy and the interactions between mitophagy induction and NLRP3 inflammasome activation in the survival ofH. pylori-infected cells.MethodsWe co-cultured gastric epithelial cells (GES)-1 and human gastric adenocarcinoma cell line (AGS) withH. pyloriCagA mutant strain (GZ7/ΔCagA) and CagA-positive wild-type strain (GZ7/CagA) for 48 h at the multiplication of infection (MOI) of 60, respectively. Afterward, mitochondrial membrane potential, adenosine triphosphate production levels, and cell apoptosis detection were performed. Furthermore, western blotting was used to detect the expression of mitochondrial fusion and fission proteins, mitophagy-related proteins, and NLRP3 inflammasome-related proteins; immunofluorescence staining was used to assess the localization and expression of LC3; transmission electron microscope (TEM) was used to obtain digital images of mitophagy. Additionally, immunochemistry was used to identify the expression of mitophagy-related proteins in the gastric tissues ofH. pylori-infected mice. Next, we used green fluorescent protein-mCherry-LC3 as a tandem reporter to explore the effect ofH. pyloriinfection on autophagic flux. Furthermore, the expression of associated proteins for mitophagy and the NLRP3 inflammasome in each group of cells was examined after pretreatment with mitophagy inducer (Olaparib), mitophagy inhibitor (BafA1), and NLRP3 inflammasome inhibitor (MCC950) for 24 h and subsequent infection with GZ7/ΔCagA and GZ7/CagA at an MOI of 60–48 h, respectively. Finally, we assessed the effect of mitophagy inhibition on apoptosis and viability inH. pylori-infected cells.ResultsWe discovered thatH. pyloriprimarily used its CagA to cause mitochondrial oxidative damage, induce mitochondrial dysfunction, dynamic imbalance, and mitophagy, and impede the autophagic flux. Although NLRP3 inflammasome inhibition hinders the induction of mitophagy, mitophagy activation can reduce NLRP3 inflammasome activation caused byH. pyloriinfection. Conversely, mitophagy inhibition can increase NLRP3 inflammasome activation caused byH. pyloriinfection. CagA plays an evident role in these processes. Moreover, inhibiting mitophagy can also increase apoptosis and reduce the viability ofH. pylori-infected cells.ConclusionOur findings suggested thatH. pylori, primarily via CagA, is required for the induction of mitochondrial dysfunction and mitophagy, which not only reduced NLRP3 inflammasome activation to evade host immune surveillance and increased infected cell survival and viability but also caused abnormal mitochondrial accumulation, possibly leading to the occurrence and development of gastric cancer.)