Consecutive patients who underwent upper endoscopy due to dyspeptic symptoms at Southwest Hospital, Chongqing, China during January 2013 and December 2014, were recruited. One hundred and six (49 women and 57 men with age of 43 ± 20 years) patients were eligible for enrollment into the H. pylori positive group if they had a positive [¹³C] urea breath test, a positive rapid urease test, and H. pylori culture. Eleven (six women and five men with age of 35 ± 20 years) with normal gastric mucosa were eligible for enrollment into the H. pylori negative group, and the clinical characteristics are shown in Supplementary Table 2. The study was approved by the Institutional Review Board at Third Military Medical University, and all patients signed informed consent before participation. All experiments were performed in accordance with relevant guidelines and regulations.

H. pylori was successfully isolated from 106 patients, and genotyping for cagA and vacA was performed for 106 isolates. All H. pylori strains carry the vacA gene. To exclude the effect of VacA, the toxigenic vacA genotype (vacA^s1m1, 42 cases), expressing a functional VacA toxic, were excluded from the study. The rest of the cases include: normal control (11 cases), cagA⁻/vacA^s1m2 (7 cases), cagA⁺/vacA^s1m2 (57 cases). To ensure that approximately equal numbers of each group, 23 selective patients were chosen randomly for analyzing autophagy and inflammation, dividing into normal control (8 cases), cagA⁻/vacA^s1m2 (7 cases), cagA⁺/vacA^s1m2 (8 cases).

The selected gastric biopsy samples among the genotype subgroups were obtained to perform H&E staining. The intensity of inflammation was evaluated independently by two pathologists according to previously established criteria. The degree of neutrophil infiltration, mononuclear cell infiltration, atrophy, and metaplasia was assessed according to the updated Sydney classification as follows: 0, absent; 1, minimal; 2, mild; 3, moderate; 4, marked. So the biopsies were from different stages of gastritis (Dixon et al., 1996).

H. pylori infection status was detected by rapid urease test, bacterial culture, ¹³C-urea breath test, and histological examination (Vaira et al., 1999). In patients with positive culture, H. pylori isolates were subcultured for a maximum of five passages, and genomic DNA was extracted to genotype for the cagA and vacA genes, as previously described (Argent et al., 2008). The primers used for PCR amplification and nucleotide sequencing are listed in Supplementary Table 1.

AGS (a human gastric cancer cell line) purchased from the cell bank of Chinese Academy of Sciences, were cultured in F12 cell culture medium (Gibco, Grand Island, NY, USA, #11765-054) supplemented with 10% FBS (Gibco, #10099-141) in a humidified incubator (5% CO₂) at 37°C. The starvation condition was established by culturing the cells with serum-free medium for 4 h.

The wide-type cagA⁺/vacA⁺ H. pylori strain, NCTC11637 (Hp-WT, obtained from ATCC), cagA-knockout H. pylori with NCTC11637 background (Hp-ΔcagA, kindly provided by Dr. Sasakawa (Asahi et al., 2000; Suzuki et al., 2009) and H. pylori cagA-knockout complementation mutant (Hp-c-cagA, constructed by our group), were cultured on brain-heart infusion medium (10% rabbit blood) under microaerophilic conditions (5% O₂, 10% CO₂, and 85% N₂) at 37°C. Hp-c-cagA mutant was obtained by amplifying the cDNA fragments of cagA gene from the gene of NCTC11637 by polymerase chain reaction (PCR), and the primers of PCR is following: forward: 5′-GCGCTCGAGATGACTAACGAACC-3′; reverse: 5′-GCGCTGCAGTTAAGATTTTTGG-3′. The product of PCR was digested with XhoI and PstI, and then ligating the cDNA fragments of cagA gene between cagA-upstream and -downstream sequences cloned on the pHel3 shuttle vector. The pHel3 shuttle vector with cagA gene was electroporated into Hp-ΔcagA cells carrying the kanamycin resistance. Hp-c-cagA clones were cultured in brain-heart infusion medium as previous described.

AGS cells transfected with plasmids and/or siRNAs were infected with Hp-WT, Hp-ΔcagA, or Hp-c-cagA, respectively, with different multiplicity of infection (MOI = 10, 50, 100, 200), for 6 h. Cells without infection served as controls.

Rapamycin (Rapa, R8781), 3-methyladenine (3-MA, M9281), bafilomycin A1 (Baf-A1, B1793), antibodies against ATG12 (WH0009140M1), MAP1LC3B (L7543), and ATG5 (WH0009474M1) were purchased from Sigma-Aldrich (Shanghai, CHINA), and MK-2206 2HCL (S1078) purchased from Selleckchem (Houston, TX, USA). Antibodies against Akt (9272), mTOR (2972), AMPK (2532), phospho-Akt (Ser473) (4060), LAMP1 (9091), phospho-mTOR (Ser2448) (5536), phospho-S6 ribosomal protein (Ser235/236) (2211), ribosomal protein (2217), phospho-c-Met (Y1234/Y1235) (4033), and c-Met (4560) were obtained from Cell Signaling Technology (Beverly, MA, USA), whereas antibodies against β-actin (sc-10731), VacA (sc-25790), CagA (sc-17450), phospho-tyrosine (PY99) (sc-7020) and SQSTM1 (sc-28359), and siRNAs specific for SQSTM1 (human, sc-29679), ATG12 (human, sc-72578), c-Met (human, sc-29397), and ATG5 (human, sc-41445), along with a control siRNA (sc-44230) were obtained from Santa Cruz Biotechnology (CA, USA).

Gastric biopsy sections from patients infected with different genotypes of H. pylori were stained using SQSTM1 antibody (Enzo Life Sciences, BML-PW9860-0025) by the DAB reagent technique. SQSTM1 staining was assessed by three pathologists with no prior knowledge of the different groups, and scored as 0 (no staining), 1 (< 10% of SQSTM1 staining), 2 (10–50% of SQSTM1 staining), or 3 (>50% of SQSTM1 staining).

The protein level of SQSTM1, LAMP1, phospho-mTOR (Ser2448), MAP1LC3B, ATG12, ATG5, AMPK, phospho-Akt (Ser473), Akt, mTOR, ribosomal protein, phospho-c-Met (Y1234/Y1235), c-Met, phospho-S6 ribosomal protein (Ser235/236), VacA, CagA, and phospho-tyrosine (PY99) were performed to determine by Western blotting in gastric tissues or AGS cells as described previously (Tang et al., 2015).

Trizol reagent (Invitrogen, 15596-026) was used to extract total RNA from gastric biopsy sections. qRT-PCR analysis of gastric biopsy sections from patients for the mRNA of SQSTM1, BECN1, IL-8, TNF-α, β-actin, and IL-1β were performed by using PrimeScript RT-PCR kits (Takara, Tokyo, Japan, DRR037), and run on a Bio-Rad IQ5 thermocycler (Bio-Rad Laboratories, Inc., Hercules, USA), β-actin as an internal control. The conditions for PCR were as follows: 1 cycle of 95°C for 30 s, 40 cycles of 95°C for 6 s, 60°C for 6 s, and 72°C for 31 s, and the primer sequences used are shown in Supplementary Table 1.

AGS cells were cotransfected with Renilla control vector (pRL-TK, Promega, #2241) and luciferase reporter vector pNF-κB-TA-Luc (Clontech, #631904) with lipofectamine 2000 (the ratio of 20:1) for 1 day, followed with H. pylori infection. The Dual-Luciferase Reporter Assay System (Promega, E1910) detected firefly luciferase activity and Renilla luciferase activity according to the manufacture's protocol.

The supernatant of AGS cells with different treatment were detected by DuoSet ELISA Development System (IL-8, IL-1β, and TNF-α; R&D, Minneapolis, USA) as our previous study (Tang et al., 2016).

The GFP-MAP1LC3B plasmid and RFP-MAP1LC3B expression plasmid were kindly provided by Dr. Tamotsu Yoshimori (Department of Cell Biology, National Institute for Basic Biology, Presto, Japan) and Dr. Maria Colombo (Universidad Nacional de Cuyo, Mendoza, Argentina), respectively. The CagA expression plasmid, pEGFP-C1-CagA (GFP-CagA) (Asahi et al., 2000; Suzuki et al., 2009), was kindly provided by Dr. Chihiro Sasakawa. The cagA mutant plasmid, pEGFP-C1-CagA-Mut (GFP-CagA-Mut) was constructed by Life Technologies, Shanghai, China, and a series of CagA mutants with the Tyr residues of 899, 918, and 972 being substituted by Ala were generated from a plasmid-encoding fragment of cagA gene of H. pylori ATCC 26695 on pBluescript (Promega, Madison, USA) using a Gene Editor in vitro Site-Directed Mutagenesis System (Promega). These mutants were at the sites 2,695–2,697, 2,752–2,754, and 2,914–2,915 bp, respectively, started from the sequence ATG. Lipofectamine 2000 (Invitrogen, #11668019) was used to transfect plasmids and/or siRNAs into AGS cells. 3 × 10⁶ AGS cells were seeded into a 100-mm dish and incubated with transfection complexes containing 100 nM siRNA for 24 h.

AGS cells were harvested in RIPA buffer on crushed ice, and centrifuged at 6,000 rpm for 5 min, and commercial Lowry Assay (Bio-Rad DC) detected the concentration of protein. Five milligrams per milliliter of protein and 1 μg/μL anti-CagA (sc-17450, Santa Cruz Biotechnology) was incubated overnight at 4°C, then added 50 μL 50% protein A/G-sepharose bead suspension for 2 h, and washed three times with pre-cold RIPA buffer, and added 50 μL protein sample buffer to collect in each tube, and proteins detected by western blotting analysis.

AGS cells were transfected with GFP-MAP1LC3B or RFP-MAP1LC3B plasmid for 24 h, following H. pylori infection for another 24 h. Radiance 2000 laser scanning confocal microscope detected the images of the cells, and image analysis with LaserSharp 2000 software (Bio-Rad, San Francisco, CA) as our previous study (Tang et al., 2016). According to methods for monitoring GFP-LC3 and mRFP-GFP-LC3 puncta formation assays (Klionsky et al., 2007; Mizushima et al., 2010), the average number of MAP1LC3B puncta per cell in GFP-MAP1LC3B or RFP-MAP1LC3B-positive cells (200 cells per sample) was determined (Pattingre et al., 2005).

AGS cell viability was assessed using an MTT assay (Sigma) according to the manufacturer's instructions. Five milligrams per liter of MTT was added to each wells of AGS cells for 1–2 h, and dissolved in MTT solubilization solution. The absorbance at 590 nm (A590) was determined for each well using a microplate reader (Bio-Rad). After subtracting the background absorbance, the A590 value of the treated cells was divided by that of the untreated cells to determine the percentage of viable cells.

Monodansylcadaverine (MDC) and acridine orange (AO) staining was used to quantify the number of autolysosomes in AGS cells. Following treatment with H. pylori or transfection with plasmids/siRNAs, cells were stained with 10 mM MDC (sigma, 30432) and 1 mg/mL AO solution (sigma, A8097) at 37°C for 10 min, and fixed in 3% paraformaldehyde in PBS for 30 min. Photographs were obtained with a Radiance 2000 laser scanning confocal microscope (MDC, excitation wave length about 380 nm and emission filter 525 nm; AO, emission peak at 650 nm). The cells were then trypsinised and quantified by flow cytometry using a FACScan cytometer and CellQuest software (BD, New Jersey, USA). The percentage of cells with characteristic MDC or AO staining over the total cells was assessed.

AGS cells or gastric biopsy sections were collected and fixed in 2% paraformaldehyde, 0.1% glutaraldehyde and 0.1 M sodium cacodylate buffer (pH 7.4) for 2 h, then post-fixed in 1% OsO4, 0.5% potassium ferricyanide in cacodylate buffer for 1.5 h, then dehydrated with graded alcohol, and embedded in straight resin. Ultrathin sections were counterstained with 0.3% lead citrate and detected by Philips EM420 electron microscope. The method of counting autophagosomes' numbers was followed as described previously by Yla-Anttila et al. (2009). Data obtained by scoring for the presence of autophagic vacuoles (autophagosomes, autolysosomes) profiles per cell profile on the sections, and a total of 35 cells were recorded for triplicate samples per condition per experiment.

The Student t-test was used to analyze between two groups, and one-way analysis of variance (ANOVA) was used to analyze among multiple group data, and expressed as mean ± standard error (SEM). GraphPad Prism software (GraphPad, San Diego, CA) was used for all statistical analyses. For all inferential statistics a P < 0.05 was considered significant.

The clinical characteristics of 117 patients with (106) and those without (11) H. pylori infection are shown in Supplementary Table 2. H. pylori was successfully isolated from 106 patients, and genotyping for cagA and vacA. All H. pylori strains carry the vacA gene. To exclude the effect of VacA, the toxigenic vacA genotype (vacA^s1m1), expressing a functional VacA toxic, were excluded from the study. In order to ensure that approximately equal numbers of each group, three equal groups were created for analyzing via random sampling methods, including normal control (8 cases), cagA⁻/vacA^s1m2 (7 cases), cagA⁺/vacA^s1m2 (8 cases).

To verify the effect of CagA in severe tissue inflammation, we evaluated the level of inflammation in gastric mucosa. Firstly, the degree of gastric inflammation was higher in patients infected with cagA⁺/vacA^s1m2 strains than in those infected with cagA⁻/vacA^s1m2 strains (Figure 1A). Notably, the mRNA levels of IL-8, TNF-α, and IL-1β in the gastric epithelial cells were significantly higher in patients infected with cagA⁺/vacA^s1m2 strains than in patients without H. pylori infection or those infected with cag⁻/vacA^s1m2 strains (Figure 1B).

Furthermore, we evaluate the autophagic activity in gastric mucosal tissues from patients infected with different genotypes H. pylori. The SQSTM1/p62 (sequestosome1) protein serves as a link between LC3 and ubiquitinated substrates (Wang et al., 2010). Dysfunctional autophagy could result in an accumulation of SQSTM1, which has been involved in promoting inflammation (Raju et al., 2012). Therefore, we detected the levels of SQSTM1 in human gastric biopsies. As shown in Figure 2A, accumulation of SQSTM1 in the gastric biospy with H. pylori infection was significantly higher than those uninfected normal control, and the accumulation of SQSTM1 was significantly higher in the gastric epithelium cells in patients infected with cagA⁺/vacA^s1m2 strains than in those infected with cagA⁻/vacA^s1m2 strains (P < 0.001) as determined by immunohistochemistry. Moreover, in patients infected with cagA⁻/vacA^s1m2 strains, the ratio of microtubule-associated protein 1 light chain 3 beta-II (MAP1LC3B-II) to β-actin and the lysosomal-associated membrane protein 1 (LAMP1, the late endosomal lysosomal marker) protein levels was higher than that in patients infected with cagA⁺/vacA^s1m2 strains, and the SQSTM1 protein levels increased in patients infected with CagA-positive H. pylori (Figure 2B). In addition, infection of cagA⁺/vacA^s1m2 strains was significantly associated with increased levels of mRNA expression of SQSTM1 (Supplementary Figure 1A), but not of BECN1 (Supplementary Figure 1B). Furthermore, it was also revealed an increase in the number of autophagosomes in patients infected with cagA⁻/vacA^s1m2 strains compared with that in cagA⁺/vacA^s1m2 group in TEM analysis (Figure 2C). Both cagA⁺/vacA^s1m2 and cagA⁻/vacA^s1m2 groups displayed high autophagy activity than the normal control group. These findings indicate that H. pylori infection could induce inflammation response and autophagy activity in the gastric epithelium cells in vivo, but CagA-positive H. pylori are associated with more severe inflammation, and down-regulates autophagic response in vivo.

To further validate the role of CagA in autophagy regulation, we next infected AGS cells with the H. pylori wide-type (Hp-WT), H. pylori cagA-knockout mutant (Hp-ΔcagA) or H. pylori cagA-knockout complementation (Hp-c-cagA) (MOI = 100:1), which strains the expression of VacA is similar during infection (Supplementary Figure 2A), and evaluated the kinetics of autophagosome formation by a GFP-MAP1LC3B puncta formation assay. Formation of MAP1LC3B puncta, peaked at 12 h and decreased at 24 h (Figure 3A and Supplementary Figure 2B). And compared with cells infected with Hp-WT or Hp-c-cagA, there was a significantly increased percentage of cells with formation of MAP1LC3B puncta for cells infected with Hp-ΔcagA (Figure 3A). TEM revealed an increase in the number of autophagic vacuoles (autophagosomes and autolysosomes) in AGS cells infected with Hp-ΔcagA-infected cells, compared with cells infected with Hp-WT or Hp-c-cagA (Figure 3B). Similar results were obtained in MDC (Figure 3C and Supplementary Figure 2D) and AO (Figure 3D and Supplementary Figure 2D) staining. Additionally, Hp-ΔcagA induced MAP1LC3B-II formation, and decreased SQSTM1 protein expression at a higher level, compared with Hp-WT or Hp-c-cagA, at 6, 12, and 24 h (Figure 3E and Supplementary Figure 2C). Furthermore, inhibition of autophagy by Baf-A1 challenge resulted in further accumulation of both MAP1LC3B-II and SQSTM1 in AGS cells after 6 h of Hp-WT or Hp-ΔcagA infection (Figure 3F), suggesting that H. pylori CagA did not inhibit the fusion of autophagosomes with lysosomes. Furthermore, under Hp-WT or Hp-ΔcagA infection, the levels of MAP1LC3B-II in AGS cells transfected with the CagA expression plasmid (GFP-CagA) were decreased in comparison to that in transfected-control cells (Figure 3G), suggesting that over-expression of CagA lead to further reduction of autophagic flux. Collectively, these data suggest that H. pylori CagA may inhibit the generation of autophagosomes in AGS cells.

In order to eliminate the influence of H. pylori itself on autophagy, starvation-triggered autophagy was performed in AGS cells after transfecting the CagA expression plasmid (GFP-CagA) or tyrosine phosphorylation point mutant of CagA plasmid (GFP-CagA-Mut). At least 50% transfection efficiency was achieved for transfection of GFP-CagA and GFP-CagA-Mut in AGS (Supplementary Figure 3A). Although cell viability was influenced by starvation to a certain extent during the first 4 h, it appears not to be significantly influenced afterwards (Supplementary Figure 3B). During nutrient starvation, in the AGS cells transfected with GFP-CagA or GFP-CagA-Mut, there was a significant decrease in the number of autophagosomes as determined by TEM, compared with cells transfected with control plasmid (P < 0.05, Figure 4A). The ratio of MAP1LC3B-II to β-actin was also significantly decreased in cells transfected with GFP-CagA or GFP-CagA-Mut following starvation treatment (P < 0.05, Figure 4B). Similarly, SQSTM1 expression was increased in cells transfected with GFP-CagA or GFP-CagA-Mut following starvation treatment (P < 0.05, Figure 4B). Interestingly, we observed that transfection with GFP-CagA or GFP-CagA-Mut had no effect on the expression of the AMP activated protein kinase (AMPK, an energy sensor; Figure 4B), indicating that CagA suppressed starvation-induced autophagy may not via the AMPK signal pathway. Furthermore, as shown in Figure 4C, the number of RFP-MAP1LC3B puncta in AGS cells co-transfected with RFP-MAP1LC3B and GFP-CagA was decreased after starvation treatment (P < 0.05). Taken together, these results suggest that CagA suppressed starvation-induced autophagy, which may not be dependent on tyrosine phosphorylation of CagA.

To clarify the role of CagA in the inflammation, the expression of proinflammatory cytokines (IL-8, TNF-α, and IL-1β), which are involved in gastritis during H. pylori infection (Nakachi et al., 2000), was examined by ELISA assay. These cytokines were significantly higher in AGS cells infected with Hp-WT or Hp-c-cagA than in those infected with Hp-ΔcagA at different time points (Figure 5A). Moreover, These cytokines in AGS cells infected with Hp-WT and Hp-ΔcagA was increased following the increase of the bacterial load. AGS cells infected with Hp-WT produced greater amounts of the cytokines than cells infected with Hp-ΔcagA (Figure 5B).

We also examined the production of the cytokines with the autophagy enhancer (Rapa, Rapamycin) or inhibitors (3-MA or Baf-A1) treatment during Hp-WT and Hp-ΔcagA infection. The effects of two autophagy inhibitors, and one enhancer, are shown in Supplementary Figures 4A–C. Autophagy inhibitors significantly increased the cytokines and activated NF-κB, and enhancer Rapa decreased the ones in AGS cells infected Hp-ΔcagA infection (Figure 5C and Supplementary Figure 4E). After 24 h infection, the three proinflammatory cytokines were increased with the inhibitors in cells infected with Hp-WT and Hp-ΔcagA (Supplementary Figure 4F). Moreover, the production of proinflammatory cytokines and activity of NF-κB were significantly increased in AGS cells transfected with siRNAs for ATG5 or ATG12 upon H. pylori infection (Figure 5D and Supplementary Figure 4G). These data suggested that autophagy plays a critical role in the inflammation induced by H. pylori.

The previous study reported that CagA has been known to activate c-Met and the PI3K/AKT pathway (Churin et al., 2003). However, it is not clear whether c-Met could regulate autophagy. The wild type H. pylori infection activated c-Met in AGS cells (Figure 6A). CagA was coimmunoprecipitated with c-Met in AGS cells infection with Hp-WT (Figure 6B). This result was consistent with previous study (Oliveira et al., 2009). The effects of c-Met depletion through siRNA interference are shown in Supplementary Figure 4D. The number of GFP-MAP1LC3B puncta in c-Met siRNA group was higher than that of control group upon infection with the Hp-WT (P < 0.05, Figure 6C). It was a significant increase in the ratio of MAP1LC3B-II to β-actin in c-Met siRNA group than in the control siRNA upon infection with the Hp-WT (P < 0.05, Figure 6D). Furthermore, MDC and AO staining showed that c-Met siRNA group induced the formation of autophagolysosomes in AGS cells at a significantly higher level, compared with control siRNA group in AGS cells infected with Hp-WT (P = 0.008 and 0.018, respectively, Figures 6E,F and Supplementary Figure 5A). Moreover, in CagA-expressing AGS cells, the ratio of MAP1LC3B-II to β-actin significantly increased by c-Met siRNA regardless of infection status (Figure 6G). These results demonstrate that c-Met may be an important adaptor in CagA-mediated autophagy pathway.

Given that c-Met could activate PI3K/AKT/mTOR pathway (Lim and Walikonis, 2008; Tang et al., 2015), we hypothesized that PI3K/AKT/mTOR pathway might play an important role in the process of CagA-mediated autophagy. We analyzed the activation status of the key members of autophagy-related PI3K/Akt/mTOR pathways. As shown in Figure 7A, Hp-WT activated Akt kinase at Ser-473 site at a significantly higher level than did Hp-ΔcagA (P = 0.018), which was consistent with a previous report (Tabassam et al., 2009). Both Hp-WT and Hp-ΔcagA increased MAP1LC3B-II expression, but Hp-ΔcagA did at a significantly higher level than did Hp-WT (Figure 7A). There was a significant increase in the levels of phosphorylated mTOR (p-mTOR) and phosphorylated S6 ribosomal protein (p-S6) upon Hp-WT vs. Hp-ΔcagA (Figure 7A). Tyrosine phosphorylation of CagA did not affect the expression levels of proteins related to PI3K/Akt/mTOR pathway and autophagy in AGS cells (Figure 7B), whereas c-Met siRNA significantly decreased the level of p-Akt, p-mTOR, and p-S6, and increased MAP1LC3B-II levels (Figure 7C). Moreover, treatment of MK-2206, a specific inhibitor of Akt, abrogated Akt activation, and reversed the ratio of MAP1LC3B-II/β-action, and decreased the level of p-Akt, p-mTOR, and p-S6 (Figure 7D). Then, to investigate whether c-Met siRNA or MK-2206 reverse inflammatory response during H. pylori infection, we detected the expression of inflammatory cytokines. As shown in Figure 7E, there was a significant decrease in the production of proinflammatory cytokines in cells transfected with siRNA specific for c-Met upon infection. Similarly, the expression of inflammatory cytokines significantly decreased in AGS cells treated with MK2206 during infection (Figure 7F). Together, we concluded that the CagA-mediated autophagy pathway may be dependent on the c-Met/Akt signaling pathway, which could regulate the expression of inflammatory cytokines.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.