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

doi:10.21203/rs.3.rs-2978545/v1

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

https://doi.org/10.21203/rs.3.rs-2978545/v1

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Background

H. pylori is 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 of H.pylori CagA in the crosstalk between mitophagy and NLRP3 inflammasome is not completely known.

Objective

In this study, we aimed to understand the impact of H. pylori and its CagA on the induction of mitochondrial dysfunction and mitophagy and the interactions between mitophagy induction and NLRP3 inflammasome activation in the survival of H. pylori-infected cells.

Methods

We co-cultured gastric epithelial cells (GES)-1 and human gastric adenocarcinoma cell line (AGS) with H. pylori CagA 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 of H. pylori-infected mice. Next, we used green fluorescent protein-mCherry-LC3 as a tandem reporter to explore the effect of H. pylori infection 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 in H. pylori-infected cells.

Results

We discovered that H. pylori primarily 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 by H. pylori infection. Conversely, mitophagy inhibition can increase NLRP3 inflammasome activation caused by H. pylori infection. CagA plays an evident role in these processes. Moreover, inhibiting mitophagy can also increase apoptosis and reduce the viability of H. pylori-infected cells.

Conclusion

Our findings suggested that H. 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.)

Autophagy flux

gastric cancer

Helicobacter pylori

mitophagy

NLRP3 inflammasome

survival and viability

Helicobacter pylori (H. pylori), the most common cause of bacterial infection in the world, is a gram-negative bacterium that is specifically colonized in the human stomach. It is estimated that roughly half of the world’s population is infected with this pathogen, with some developing countries reporting prevalence rates as high as 90%^[1]. According to the 1994 World Health Organization (WHO) classification, H. pylori is a primary carcinogen of gastric cancer since its infection can cause chronic gastritis, peptic ulcer, gastric adenocarcinoma, and mucosa-associated lymphoid tissue lymphoma^[2–3]. Currently, the mechanism of gastric pathological injury caused by H. pylori may be related to immune reaction, the implantation of H. pylori in the gastric wall, and the virulence proteins of H. pylori, however, the exact causative mechanism remains elusive^[4–5]. CagA protein, a major virulence factor of H. pylori, is translocated into host cells by the T4SS after bacterial attachment and has been implicated in the occurrence and progression of gastric cancer^[6]. Although CagA status is not the only contributing factor to further gastrointestinal impairments caused by H. pylori, CagA-positive strains are associated with more severe inflammation, higher atrophy steps, and a higher risk of progression to gastric adenocarcinoma when compared to cagA-negative strains^[7]. Consequently, further research into the pathogenesis and carcinogenic mechanism of H. pylori and its CagA protein is warranted.

Our previous research discovered that H. pylori CagA can increase reactive oxygen species (ROS) production and activate the nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing 3 (NLRP3) inflammasome, thereby, promoting the inflammatory factor interleukin-1β secretion and cell pyrogenesis^[8]. Excessive NLRP3 inflammasome activation is associated with the development of inflammatory diseases and cancer^[9]. High levels of ROS play an oxidative role, which can gradually oxidize and damage mitochondria, endoplasmic reticulum, and other cell organelles. Moreover, mitochondrial damage can cause a dynamic imbalance of mitochondrial fusion/division, a decrease in mitochondrial membrane potential, an increase in apoptosis rate, and the occurrence of mitophagy^[10–11]. Mitophagy is a selective autophagy pathway that maintains mitochondrial quality control by targeting and destroying mitochondria; studies have also reported that mitophagy protects against several inflammatory diseases associated with the NLRP3 inflammasome^[12]. Mitophagy can not only remove endogenous NLRP3 inflammasome signals to regulate the NLRP3 inflammasome activation, avoid changes in mitochondrial membrane permeability, and maintain cell homeostasis but also remove damaged and dysfunctional mitochondria, which is crucial for inhibiting the release of pro-apoptotic proteins to restore cell viability and destruction of intracellular pathogens^[13]. Consequently, mitophagy is widely considered a major regulatory mechanism in the mitochondrial quality control process, involved in the regulation of host immune responses. However, further research into the role of H. pylori in mitophagy and its effect on the activation of NLRP3 inflammasomes will provide a better understanding of H. pylori pathogenesis and help in designing new strategies for the prevention and treatment of gastric cancer.

The effects of H. pylori and CagA on mitophagy, NLRP3 inflammasome activation, and their mutual regulation are currently unknown. This study aimed to determine how H. pylori and its CagA affect the induction of mitochondrial oxidative damage, mitochondrial dynamic imbalance, mitochondrial dysfunction, and mitophagy and the effects on the interaction between mitophagy induction and NLRP3 inflammasome activation. We infected GES cells and C57BL/6 mice with the H. pylori CagA positive strain (GZ7/CagA) and CagA gene knockout strain (GZ7/ΔCagA), respectively, to examine the mitochondrial damage and mitophagy induced by H.pylori and whether CagA is mainly involved in the process. To further explore the role of mitophagy in NLRP3 inflammasome activation and the survival and viability of H. pylori-infected cells, we demonstrated that H. pylori mainly uses its CagA to cause mitochondrial oxidative damage, mitochondrial dynamic imbalance, and mitochondrial dysfunction, increase the level of mitophagy, and block the autophagic flux. Furthermore, mitophagy regulates the activation of NLRP3 inflammasome by H. pylori to escape host immune surveillance and enhance the survival and viability of infected cells. Our findings for selective mitophagy and the NLRP3 inflammasome in H. pylori-infected cells provide a novel perspective on gastric cancer pathogenesis caused by H. pylori infection.

Cultural conditions and cell lines

The Chinese Academy of Sciences Cell Bank provided human GES-1 cells (Shanghai, China). American Type Culture Collection provided the human gastric epithelial adenocarcinoma cell line (AGS) (ATCC, USA). All cell lines were grown in Roswell Park Memorial Institute 1640 medium supplemented with 10% fetal bovine serum (Gibco, Rockville, USA) and 100 U/mL penicillin and streptomycin (Gibco, USA) at 37°C in a 10% CO₂ humidified atmosphere.

H. pylori culture and infection

In our previous research^[14], we identified H. pylori strain GZ7/CagA (CagA⁺, GenBank accession ID: KR154737.1) as a typical East Asian strain by sequencing, which was isolated from clinical gastric cancer tissue with informed consent and approved by the Ethics Committee of Guizhou Medical University. GZ7/ΔCagA, CagA gene knockout from the GZ7/CagA was constructed and identified by Sangon Biotech (Shanghai, China). GZ7/CagA and GZ7/CagA were grown in microaerophilic conditions at 37°C on a Columbia agar plate (Oxoid Ltd, Basingstoke, UK) containing 10% sheep blood (BD Diagnostics) and Dent antibiotics supplement (Oxoid). After growing GES-1 and AGS cells in six-well plates with antibiotic-free medium and infecting them with H. pylori (multiplication of infection (MOI), 60:1) for 6 h, the cell culture medium was changed to complete medium, and the cell samples of each group were collected after 42 h for subsequent experiments. The non-infected groups served as a negative control.

Measurement of Adenosine Triphosphate (ATP)

After corresponding treatment, cells were collected and the ATP levels were determined using an ATP assay kit (Beyotime, China, S0026B). The fluorescence intensity was measured using a luminometer (ELX800UV, Bio-tec, USA). The ATP levels in the samples were determined according to the established standard curve.

Mitochondrial Membrane Potential (MMP) determination

MMP levels in differentially treated cells were measured using the JC-1 kit (Beyotime, China, C2006). After collecting the cells, 1000 µL of JC-1 dye solution was added. The cells were stained for 20 min in the dark at 37 ºC, and further, 10 µL of the stained cell mixture was collected and analyzed using a fluorescence microscope. ImageJ (Rawak Software Inc., Stuttgart, Germany) was used to calculate JC-1 red/JC-1 green, and five microscopy fields were included in each group for image analysis.

Measurement of cell apoptosis

Cells were collected and stained using the Annexin V-APC/7-AAD apoptosis kit (MultiSciences Biotech Co, China) following the manufacturer's instructions after corresponding treatment, and quantification was performed using flow cytometry (FACS verse, BD Bioscience, USA).

Transmission Electron Microscopy (TEM)

After co-culturing GES-1 and AGS cells with GZ7/ΔcagA and GZ7/cagA for 48 h at an MOI of 60, respectively, cells were collected and immersed in specimen preservation solution and sent to the Servicebio company (Wuhan, China) for examination using electron microscopy and obtaining digital images.

Generation of a c57bl/6 model

Eighteen SPF male c57bl/6 mice, 4–5-week-old, weighing 60–100 g, were purchased from Zhejiang Provincial Laboratory Animal Center, Zhejiang, China (license number: SYXK 2018-0001). C57bl/6 mice were orally inoculated with the GZ7/CagA and GZ7/ΔCagA, respectively, thrice with 1 mL each (15×10⁸/mL) at 48-h intervals. c57bl/6 mice orally inoculated with saline were used as a negative control and observed for 3 months. These mice were sacrificed by cervical dislocation, after fasting for 24 h and deprivation from water for 12 h, and the gastric tissue was removed and divided into two parts, one of which was fixed in 4% formaldehyde and the other in liquid nitrogen. The data represented the average of five mice per group. The Guizhou Medical University Committee on Animal Care approved all animal experiments (No: 1900801, February 25, 2019).

Immunochemistry

Tissue sections from c57bl/6 mice were deparaffinized, rehydrated, and incubated for 15 minutes in 3% H₂O₂ and methanol. To perform antigenic repair, the slides were placed in 0.01 M sodium citrate buffer (pH 6.0) and incubated at 95°C for 20 minutes. After cooling in 3% H₂O₂ for 10 minutes, sections were washed in phosphate-buffered saline (PBS) for 10 minutes before being incubated with the primary antibody overnight at 4°C. Sections were further washed thrice with PBS at 10-minute intervals before being incubated for 1 h at room temperature with the secondary antibody labeled with horseradish peroxidase (1:10,000; Proteintech Group, Inc.; Wuhan Sanying). After incubating with 3,3′-diaminobenzidine, immunostaining was performed, and the sections were counter-stained with hematoxylin. Using an optical microscope, the level of protein expression in each sample was assessed based on the number of stained cells and the depth of staining. The average integrated optical density of active areas in each group was calculated using Image-Pro Plus 6.0 software (Media Cybernetics, Inc.USA), and the average optical density was used for analysis.

LC3 Immunofluorescence assay

We co-cultured GES-1 and AGS cells with GZ7/ΔCagA and GZ7/CagA for 48 h at an MOI of 60, respectively. Cells in the wells were fixed with 4% paraformaldehyde for 15 minutes at 4°C before being permeabilized for 15 minutes with 0.5% Triton X-100 in PBS at room temperature. The cells were then blocked for 2 h with 1% bovine serum albumin, incubated overnight at 4°C with LC3 primary antibodies (1:200, 14600-1-AP, Proteintech, USA), and washed thrice with PBS. Afterward, the fluorescein-isothiocyanate-labeled secondary antibodies (1:300, F-2765, Proteintech, USA) were incubated at room temperature for 2 h before being washed with PBS and stained with DAPI for nuclear counterstaining. The stained slides were photographed and analyzed using a confocal microscope (Carl Zeiss LSM 880) and Image J software.

Observation of the autophagic flux

The AGS and GES-1 cells were transfected with a monomeric red fluorescent protein (mRFP)-green fluorescent protein (GFP)-LC3 lentivirus (Hanbio Co. LTD, HB-LP2100001) for 48 h and further infected with H. pylori (MOI, 60:1) for an additional 24 h. Following cell collection, a 100-µL Mito-Tracker Green and LTR dye solution was added to stain the cells for 15 minutes in the dark, and 10 µL of the stained cell mixture was collected and analyzed using a confocal microscope and ImageJ Software. Early autophagosomes were represented by yellow puncta (merge of GFP and RFP signals), whereas late autolysosomes were represented by red puncta (RFP signal alone). The number of yellow and red puncta was counted in five fields, with each group exhibiting no fewer than 10 cells.

Western blotting analyses

Cells were harvested and lysed in radioimmunoprecipitation assay lysis buffer containing fresh protease and phosphate inhibitor mixture and further boiled for denaturation to determine protein concentrations using the Bicinchoninic Acidic Assay Protein Assay Kit (Beyotime, China). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to separate the cell lysate, which was then transferred to polyvinylidene difluoride membranes (Millipore Corporation, MA, USA). Membranes were blocked for 1 h with 5% non-fat milk before being incubated overnight at 4°C with primary antibodies. Primary antibodies were as follows: anti-P62 (1:800, 18420-1-AP), anti-LC3 (1:800, 14600-1-AP), anti-phosphatase and tensin homolog-induced kinase 1 (PINK1) (1:1000, 23274-1-AP), anti-Parkin (1:1000, 14060-1-AP), anti-mitofusin 1 (Mfn1) (1:1000, 13798-1-AP), anti-Mfn2 (1:1000, 12186-1-AP), anti-dynamin-related protein 1 (Drp1) (1:800, 12957-1-AP); anti-mitochondrial fission 1 (Fis1) (1:1000, 10956-1-AP), anti-NLRP3 (1:1000, 19771-1-AP), anti-Caspase-1 (1:1000, 22915-1-AP), and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:5000,10494-1-AP) were obtained from Proteintech, USA. Antibodies against CagA (1:500, sc-28368) were obtained from Santa Cruz Biotechnology. Following three washes with Tris-buffered saline with 0.1% Tween-20 detergent, the membranes were incubated for 1 h with peroxidase-conjugated secondary antibody (1:10,000 dilution, Santa Cruz, CA, USA). Bands were imaged using a chemiluminescence imager (Amersham Biosciences, USA) and ECL Plus chemiluminescence (Thermo Fisher Scientific, MA, USA). The western blot band intensities were quantified using ImageJ software, with GAPDH protein serving as a loading control.

Cell Counting Kit (CCK)-8 assay

The cell viability was determined using the CCK-8 assay (Beyotime, China, C0038). Cell suspensions were prepared and seeded into 96-well culture plates. The cells were pretreated with Bafilomycin A1 (BafA1) (10 M, MCE, HY-100558) for 24 h before being infected with GZ7/c\CagA and GZ7/CagA (MOI, 1: 60) at four different time points (0, 6, 24, and 48 h). Further, 10 µL of CCK-8 reagent was added to each well and incubated in the dark for 2 h at 37°C with 5% CO₂. After shaking for 10 seconds, the optical density (O.D.) at 450 nm was measured with a microplate reader to determine the relative number of surviving cells (ELX800UV, Bio-tec, USA).

Statistical analysis

The data from at least three independent experiments were presented as mean ± SD, and statistical analyses were performed using GraphPad Prizm 5.0. (GraphPad Software Inc., San Diego, CA, USA). Unpaired t-tests were used to compare two groups, whereas a one-way analysis of variance was used to analyze differences between groups. P-values of 0.05 were considered statistically significant.

H. pylori CagA induces mitochondrial dysfunction and apoptosis of infected cells

We previously discovered that H. pylori CagA can increase ROS production and activate the NLRP3 inflammasome. High levels of ROS can perform oxidation and cause oxidative damage to cell mitochondria, thus, leading to cell dysfunction and apoptosis^[15]. To elaborate the effect of H. pylori and its CagA on mitochondrial function and cell apoptosis in GES, we co-cultured GES-1 and AGS cells with H. pylori CagA mutant strain (GZ7/ΔCagA) and CagA-positive wild-type strain (GZ7/CagA ) for 48 h at an MOI of 60, respectively, followed by detection of mitochondrial membrane potential, ATP production levels, and cells apoptosis. GZ7/ΔCagA and GZ7/CagA strains were cultured and observed under a microscope following Gram staining and exhibited a short rod-shared structure and round blunt Gram-negative bacteria, respectively. Western blotting was performed to confirm CagA protein expression in GZ7/CagA-infected cells, however, no expression in GZ7/ΔCagA infected cells was observed (see Fig. 1A). The results demonstrated that GZ7/CagA infection significantly decreased the mitochondrial membrane potential and ATP production levels and promoted cell apoptosis compared with the control cells and GZ7/ΔCagA-infected cells (see Fig. 1B, C, and D). These data indicated that CagA mediates H. pylori to induce mitochondrial dysfunction and apoptosis of GES.

H. pylori CagA induces a mitochondrial dynamic imbalance in infected cells

The proper balance of mitochondrial dynamics is considered necessary for mitochondrial homeostasis, cell stability, and cell survival ^[16]. Mitochondrial fusion can repair damaged mitochondria, whereas mitochondrial fission can activate mitophagy and regulate mitochondrial clearance^[17]. To confirm the effect of H. pylori and its CagA on mitochondrial dynamic, we co-cultured GES-1 and AGS cells with GZ7/ΔCagA and GZ7/CagA for 48 h at an MOI of 60, respectively. Western blotting was used to detect the expression of proteins related to mitochondrial fusion and fission. The findings revealed that when compared to uninfected control cells and GZ7/CagA-infected cells, GZ7/CagA-infected cells significantly reduced the expression of mitochondrial fusion-related proteins Mfn1 and Mfn2 while significantly increasing the expression of mitochondrial fission-related proteins Drp1 and Fis1 (see Fig. 2). These data indicated that CagA mediates H. pylori to induce the mitochondrial dynamic imbalance of GES, decrease mitochondrial fusion, and increase mitochondrial fission.

H. pylori CagA mediates mitophagy in infected cells

Mitochondrial damage usually occurs in response to microbial infection or cellular stress, with subsequent removal of damaged mitochondria by mitophagy to maintain mitochondrial stability^[18]. Growing evidence suggests that H. pylori or culture filtrate of H. pylori induces autophagy in cells^[19]; however, whether CagA triggers mitophagy in H. pylori-infected human GES remains elusive. In this study, we co-cultured GES-1 and AGS cells with GZ7/ΔCagA and GZ7/CagA for 48 h at an MOI of 60, respectively. The findings demonstrated that compared with the non-infected control cells and GZ7/ΔCagA-infected cells, GZ7/CagA infection significantly increased the expression of mitophagy-related proteins PINK1, Parkin, P62, LC3, VDAC1, and TOM20 in cells (see Fig. 3A). Furthermore, immunofluorescence staining was used to evaluate the localizations and expressions of LC3 in cells, LC3 were mostly observed in the cytoplasm, and its expression level in GZ7/CagA-infected cells was higher than that in non-infected control cells and GZ7/ΔCagA-infected cells (see Fig. 3B). Furthermore, TEM was performed to determine whether mitophagy occurred in GZ7/ΔCagA and GZ7/CagA-infected cells. Results revealed that there was an accumulation of autophagosomes and autolysosomes in the GZ7/ΔCagA and GZ7/CagA-infected cells, whereas the GZ7/CagA-infected cells increased more significantly (see Fig. 3C). These data indicated that CagA mediates mitophagy in H. pylori-infected cells.

Mitophagy-related proteins expression was increased in the gastric tissues of H. pylori-infected mice

To further determine the effects of mitophagy in mice during H. pylori infection, GZ7/CagA and GZ7/CagA were orally administered to C57bl/6 mice for 3 months. Gastric tissues were collected for detection of the expression of the pathological and mitophagy-related protein to verify the effects of H. pylori and its CagA on mitophagy. Pathological results demonstrated that a small amount of bleeding (represented by red arrow) and inflammatory cell infiltration (represented by yellow arrow) were observed in the submucosa of the gastric tissue from GZ7/CagA-infected mice (see Fig. 4A). Compared with the non-infected control mice and GZ7/ΔCagA-infected mice, the expression of mitophagy-related proteins P62, LC3, PINK1, and Parkin was significantly increased in GZ7/CagA-infected mice (see Fig. 4B). This indicated that H. pylori CagA can induce mitophagy in gastric tissues of infected mice, which was consistent with the experiments performed in vitro.

Autophagic Flux blocked during H. pylori infection

The autophagosome, such as the phagosome, culminates in fusion with the lysosome to form a degradative autolysosome, and the entire autophagy process is referred to as autophagy flux^[20]. Autophagosome-lysosome fusion is required for autophagic flux, and inhibiting this process impairs autophagic degradation^[21]. Therefore, we used GFP-mCherry-LC3 as a tandem reporter to explore whether H. pylori infection affected autophagic flux. We transfected AGS and GES-1 cells with mRFP-GFP-LC3 for 24 h and further infected them with GZ7/ΔCagA and GZ7/CagA (MOI, 60:1) for an additional 48 h, respectively. To assess autophagic flux, confocal microscopy was employed to observe the co-localization of mitochondria and lysosomes. As indicated in Fig. 5, GZ7/CagA and GZ7/CagA infection increased both green and red dot formation, and co-localization caused more yellow-only puncta, whereas yellow-only puncta increased more dramatically in GZ7/CagA-infected cells. Our findings demonstrated that H. pylori infection increased autophagosome generation without fusion with lysosomes, indicating a block in autophagy flux.

H. pylori CagA induced mitophagy to attenuate NLRP3 inflammasome activation

As demonstrated in our previous study, H. pylori CagA can activate the NLRP3 inflammasome and promote the invasion and migration of gastric cancer cells^[7]. Recent evidence suggests that mitophagy is associated with immune response and can suppress NLRP3 inflammasome overactivation and subsequent immune response^[22]. However, the role of H. pylori-induced mitophagy in NLRP3 inflammasome activation remains elusive. Therefore, we examined the interaction between the two processes of mitophagy and NLRP3 inflammasome activation induced by H. pylori. We pre-treated AGS and GES-1 cells with mitophagy inducer (Olaparib, 2µM, MCE, Ku-0059436), mitophagy inhibitor (BafA1, 10µM, MCE, HY-100558 ), and NLRP3 inflammasome inhibitor (MCC950, 5µM, MCE, CP-456773) for 24 h, separately, and further infected the cells with GZ7/ΔCagA and GZ7/CagA at an MOI of 60 for 48 h, respectively. Western blotting was used to detect the expression of related proteins of mitophagy and the NLRP3 inflammasome in each cell group. The results revealed that NLRP3 and Caspase-1 protein expression was reduced in Olaparib-treated cells. In comparison to H. pylori-infected cells alone, Olaparib pretreatment significantly decreased the expression of NLRP3 and Caspase-1, implying that mitophagy induction may inhibit NLRP3 inflammasome activation in H. pylori-infected cells (see Fig. 6A,6B). NLRP3 and Caspase-1 protein expression were increased in BafA1-treated cells. In comparison to H. pylori-infected cells alone, BafA1 pretreatment significantly increased the expressions of NLRP3 and Caspase-1, implying that mitophagy inhibition may cause the over-activation of the NLRP3 inflammasome in H. pylori-infected cells (see Fig. 7A,7B). It was also observed that, in MCC950-treated cells, LC3, P62, and Parkin protein expressions were decreased. Compared with H. pylori-infected cells alone, MCC950 pretreatment significantly decreased the expressions of proteins LC3, P62, and Parkin, suggesting that NLRP3 inflammasome inhibition reduced mitophagy induced by H. pylori (see Fig. 8A, 8B). Based on these data, GZ7/CagA-infected cells produced more evident results than GZ7/ΔCagA-infected cells. Our findings confirmed that H. pylori CagA induced mitophagy to attenuate the NLRP3 inflammasome activation to escape host immune surveillance.

Inhibition of mitophagy can increase apoptosis and decrease the viability of H. pylori-infected cells

Mitophagy disruption can cause the progressive accumulation of damaged mitochondria, which activates apoptosis^[23]. We pretreated AGS and GES-1 cells with mitophagy inhibitor (BafA1, 10µM) for 24 h and further infected them with GZ7/ΔCagA and GZ7/CagA at an MOI of 60 for 6 h, 24 h, and 48 h, respectively. Flow cytometry and the CCK8 assay were used to detect apoptosis and cell viability in H. pylori-infected cells. It was observed that compared with H. pylori-infected cells alone, BafA1 pretreatment significantly increased the apoptosis rate of H. pylori-infected cells (see Fig. 9A) and decreased the viability of H. pylori-infected cells (see Fig. 9B). We confirmed that inhibition of mitophagy can increase the apoptosis rate and decrease the cell viability of H. pylori-infected cells. Thus, contrarily, our study indicated that CagA mediates H. pylori to induce mitophagy, which attenuated the activation of NLRP3 inflammasome to escape host immune surveillance and enhanced the survival and viability of infected cells. Mitophagy is believed to play an important role in the immune escape and survival of H. pylori-infected cells, and inhibition of mitophagy can be one of the new strategies for the prevention and treatment of H. pylori-induced gastric cancer.

Our study proved that H. pylori CagA induces mitochondrial oxidative damage, mitochondrial dysfunction, dynamic imbalance, mitophagy, and autophagy flux blockage in infected cells. Mitophagy activation attenuates NLRP3 inflammasome activation caused by H. pylori infection and enhances the survival and viability of infected cells. This suggests that mitophagy activated by H. pylori CagA may be an adaptive response of cells to escape host immune surveillance against H. pylori infection. Additionally, autophagy flux blockage causes an accumulation of dysfunctional mitochondria, which may involve in the tumorigenesis caused by H. pylori infection.

Mitochondria continue the normal physiological activities of the cell as an essential energy source generator to maintain cell homeostasis only when their morphology and function are normal. Virulence factors secreted by bacteria may interact with mitochondria as a part of a key strategy for hijacking host cell physiology and promoting infection^[24]. H. pylori is a pathogen that affects the human gastric mucosa and is a major contributor to the development of gastric cancer; it has been reported to damage mucosal epithelial cells and target mitochondria, decrease the activity of oxidative phosphorylation and electron transport chain, and cause mitochondrial dysfunction and apoptosis^[25]. To date, the vacuolating cytotoxin A (VacA) is the only H. pylori protein reported to target mitochondria and decrease mitochondrial membrane potential, reducing ATP production and cytochrome c release ^[26]. Although CagA is an important virulence factor of H. pylori, its role in inducing mitochondrial function injury remains elusive. In this study, we infected AGS and GES-1 cells with GZ7/CagA and GZ7/ΔCagA strains, respectively, followed by the detection of mitochondrial membrane potential, ATP production, cell apoptosis, and mitochondrial fusion division. Results revealed that H. pylori infection reduced mitochondrial membrane potential and ATP production levels, promoted cell apoptosis, decreased the expression of mitochondrial fusion-related proteins Mfn1 and Mfn2, and increased the expression of mitochondrial fission-related proteins Drp1 and Fis1. In these processes, the results of the GZ7/CagA-infected group were more significant. It was further confirmed that H. pylori can induce mitochondrial dysfunction, mitochondrial dynamic imbalance, and apoptosis of infected cells, and CagA plays an important role in these processes.

Damaged mitochondria can induce mitophagy as a defense mechanism to eliminate damaged mitochondria and over-produced ROS, ensure the maintenance of mitochondrial homeostasis, and promote cell survival in stressful environments^[27]. Mitophagy is widely regarded as a major regulatory mechanism of mitochondrial quality control processes and is involved in the regulation of host immune responses^[28]. Previous research has suggested that H. pylori can induce defective autophagy or inhibit autophagy, allowing H. pylori to proliferate^[29]. The VacA produced by H. pylori specifically targets mitochondria, destroys mitochondrial membrane potential, causes mitochondrial damage, and induces mitophagy^[30]. Furthermore, research suggests that CagA can inhibit autophagy via the mesenchymal-epithelial transition factor-phosphatidylinositol 3-kinase/protein kinase B-mechanistic target of the Rapamycin signaling pathway^[31]. However, CagA-induced mitochondrial damage and its effect on mitophagy remain unclear. In our study, we observed little bleeding and inflammatory cell infiltration in the submucosa of the gastric tissue from GZ7/CagA-infected C57bl/6 mice in vivo, indicating an inflammatory response in the gastric mucosa of H. pylori-infected mice. Furthermore, we discovered that the expression of mitophagy-related proteins P62, LC3, PINK1, and Parkin increased significantly in the gastric mucosal tissue of GZ7/cagA-infected mice. Consistent with that in H. pylori-infected mice in vivo, the mitophagy-related proteins PINK1, Parkin, P62, LC3, VDAC1, and TOM20 expression increased significantly in H. pylori-infected cells in vitro. Immunofluorescence staining revealed that H. pylori-infected cells dispersed LC3 in the cytoplasm and exhibited increased expression as an indicator of mitophagy. Additionally, an accumulation of autophagosomes and autolysosomes in H. pylori-infected cells was detected by TEM. Furthermore, the autophagic flux observed using confocal microscopy revealed that H. pylori infection enhanced the level of mitophagy in infected cells, albeit the fusion of autophagosome and lysosome was limited. In these processes, the results of GZ7/GZ7/cagA-infected cells were more significant. Although autophagy can limit exogenous infections, it has been reported that bacterial pathogens have evolved mechanisms to interfere with autophagy initiation or autophagy flux for their benefit^[32]. Our results presented that H. pylori CagA promotes mitophagy induction but inhibits a specific stage of autophagosome maturation, causing blocked autophagy flux. Considering that the accumulation of dysfunctional mitochondria is involved in tumorigenesis, it may be one of the pathogenic mechanisms by which H. pylori and its CagA cause gastric cancer.

Mitophagy is important in limiting inflammasome activation to prevent mitochondrial damage and is used by viruses and bacteria to avoid host immune clearance^[33]. Mitophagy, a crucial component in the preservation of mitochondrial homeostasis, develops through the elimination of damaged mitochondria, thereby, preventing hyperinflammation triggered by NLRP3 inflammasome activation and destruction of intracellular pathogens^[34]. Similarly, the NLRP3 inflammasome signaling pathway can regulate autophagy and help maintain a balance between the necessary host defense inflammatory response and the prevention of excessive and harmful inflammation^[35]. Recent studies have revealed that some pathogens can directly or indirectly trigger mitophagy and regulate the process of mitophagy via various strategies, thereby, weakening the innate immune response and allowing pathogens to promote persistent infection, which impacts the development of infectious diseases. Pseudomonas aeruginosa can disrupt mitochondrial homeostasis in host cells, thus, activating mitophagy, which enhances the host defense against the toxic responses caused by its virulence factor Pyoverdin^[36]. Mycobacterium bovis (M. bovis) infection induces mitophagy in murine macrophages, and induction of mitophagy improves M. bovis growth, whereas inhibition of mitophagy improves growth restriction^[37]. We infected AGS and GES-1 cells with GZ7/CagA and GZ7/ΔCagA strains combined with mitophagy inducer (Olaparib), mitophagy inhibitor (BafA1), and NLRP3 inflammasome inhibitor (MCC950), respectively. The results demonstrated that olaparib-stimulated mitophagy can inhibit the activation of NLRP3 inflammasomes induced by H. pylori CagA; inhibition of mitophagy by BafA1 may cause the over-activation of NLRP3 inflammasome in H. pylori-infected cells, whereas inhibition of NLRP3 inflammasome by MCC950 reduced mitophagy induced by H. pylori. Our results confirmed that H. pylori CagA promoted mitophagy and was involved in NLRP3 inflammasome activation against H. pylori infection, thereby, inhibiting the antibacterial innate immune response, which may enhance pathogen intracellular survival and cause persistent infections.

Mitophagy is important in cancer cell homeostasis and tumor progression, acting as a pro-cancer or anticancer factor, and its failure promotes oncogenic stress and tumorigenesis^[38–40]. The normal level of mitophagy is the body's defense and protection mechanism against various environmental pressures; however, excessive abnormal mitophagy may cause an abnormal mitochondrial network and energy metabolism disorder, which leads to cancer occurrence and progression^[41]. Although H. pylori is an extracellular pathogen, it can also survive intracellularly in GES, causing chronic infection and even the development of gastric cancer. The potential of mitophagy as a mechanism and therapeutic strategy remains elusive in this process. In our study, we confirmed that inhibition of mitophagy can increase the apoptosis rate and decrease the cell viability of H. pylori-infected cells, suggesting that CagA mediates H. pylori to induce mitophagy, which attenuated the activation of NLRP3 inflammasome to escape host immune surveillance and enhanced the survival and viability of infected cells from a contrasting perspective. It eventually causes chronic H. pylori infection and promotes the development of gastric cancer. Furthermore, mitophagy inhibition can be considered one of the new strategies for the prevention and treatment of H. pylori-caused gastric cancer.

In conclusion, our findings revealed that H. pylori causes mitochondrial oxidative damage, mitochondrial dysfunction, dynamic imbalance, mitophagy, and autophagy flux blockage primarily through its CagA. Mitophagy induction regulates the activation of the NLRP3 inflammasome, which may be one of the strategies of H. pylori for evading the immune response and enhancing the survival and viability of infected cells, potentially enhancing pathogen intracellular survival and causing persistent infections(Figure 10). The blockage of autophagy flux can cause the accumulation of dysfunctional mitochondria, which may play a role in tumorigenesis caused by H. pylori infection. Our findings improve our understanding of the pathogenesis of H. pylori-induced gastric cancer, and mitophagy inhibition can be one of the novel strategies for the prevention and treatment of H. pylori-induced gastric cancer.

Ethics approval and consent to participate

All animal study was approved by the Animal Care Welfare Committee of Guizhou Medical University (Guiyang, China; approval no. 1900801).

Author contributions

Yuan Xie conceived and designed the experiments, Dingyu Chen, Lixia Wu, Xi Liu and Qinrong Wang conducted most of the experiments and analyses. Shuqin Gui, Liya Bao, Zhengrong Wang and Xiaofeng He confirm the authenticity of all the raw data. Yuan Xie and Dingyu Chen drafted the manuscript. Yuan Xie , Dingyu Chen, Lixia Wu, Xi Liu ,Qinrong Wang, Liya Bao, Zhengrong Wang ,Yan Zhao and Jianjiang Zhou were responsible for writing and revising the manuscript, and contributed significantly to the study design, data collection, data analysis and interpretation of data. All authors read and approved the final version of the manuscript.

Funding information

The work was supported by National Natural Science Foundation of China (31960028, 82260405，32160166), The Guizhou Provincial Science and Technology Projects (QianKeHe JC [2020]1Z010, [2020]1Y333).

Competing Interests

The authors declare there are no competing interests.

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No competing interests reported.

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

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Abstract

Figures

Introduction

Materials and methods

Cultural conditions and cell lines

H. pylori culture and infection

Measurement of Adenosine Triphosphate (ATP)

Mitochondrial Membrane Potential (MMP) determination

Measurement of cell apoptosis

Transmission Electron Microscopy (TEM)

Generation of a c57bl/6 model

Immunochemistry

LC3 Immunofluorescence assay

Observation of the autophagic flux

Western blotting analyses

Cell Counting Kit (CCK)-8 assay

Statistical analysis

Results

H. pylori CagA induces mitochondrial dysfunction and apoptosis of infected cells

H. pylori CagA induces a mitochondrial dynamic imbalance in infected cells

H. pylori CagA mediates mitophagy in infected cells

Mitophagy-related proteins expression was increased in the gastric tissues of H. pylori-infected mice

Autophagic Flux blocked during H. pylori infection

H. pylori CagA induced mitophagy to attenuate NLRP3 inflammasome activation

Inhibition of mitophagy can increase apoptosis and decrease the viability of H. pylori-infected cells

Discussion

Declarations

References

Additional Declarations

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