Helicobacter pylori inhibits autophagic flux and promotes its intracellular survival and colonization by down-regulating SIRT1

Abstract

Helicobacter pylori (H. pylori) is the strong risk factor for a series of gastric pathological changes. Persistent colonization of H. pylori leading to chronic infection is responsible for gastritis and malignancy. Autophagy is an evolutionary conserved process which can protect cells and organisms from bacterial infection. Here, we demonstrated that H. pylori infection induced autophagosome formation but inhibited autophagic flux. SIRT1, a class III histone deacetylase, was down-regulated at both mRNA and protein levels by H. pylori infection in gastric cells. Further investigation showed that the transcriptional factor RUNX3 accounted for down-regulation of SIRT1 in H. pylori-infected gastric cells. SIRT1 promoted autophagic flux in gastric cells and activation of SIRT1 restored the autophagic flux inhibited by H. pylori infection. Furthermore, SIRT1 exerted inhibitory effects on intracellular survival and colonization of H. pylori. And activation of autophagic flux in SIRT1-inhibited gastric cells could significantly reduce intracellular load of H. pylori. Moreover, the relationship between H. pylori infection and SIRT1 expression was identified in clinical specimen. Our findings define the importance of SIRT1 in compromised autophagy induced by H. pylori infection and bacterial intracellular colonization. These results provide evidence that SIRT1 can serve as a therapeutic target to eradicate H. pylori infection.

Keywords: Helicobacter pylori; SIRT1; autophagic flux; autophagosome; intracellular colonization; intracellular survival.

FIGURE 1

H. pylori infection induces autophagosome formation but inhibits autophagic flux leading to increased colonization in vitro. A, Western blot was performed to detect the protein levels of LC3BI/II and SQSTM1/p62 in cells infected with Hp26695 at an MOI of 100 for indicated time. Data from 3 independent experiments are presented as mean ± SD. B and C, The mCherry‐EGFP‐LC3B fluorescence microscopy assays were performed in cells treated with EBSS (3 h, to induce autophagic flux) or infected with Hp26695 (3 h). Data from 5 independent experiments are presented as mean ± SD. Scale bars: 10 µm. D–G, Colony formation assays (D and F) and bacterial DNA quantitation (E and G) were performed after treating Hp26695‐infected cells with EBSS (12 h) or Baf A1 (10 nM, 1 h, an inhibitor of autophagic flux). Data from 3 independent experiments are presented as mean ± SD. (H and I) Immunofluorescence staining of H. pylori after treating Hp26695‐infected cells with EBSS (12 hr) or Baf A1 (10 nM, 1 hr). Data from 3 independent experiments are presented as mean ± SD. Scale bars: 10 µm. *represents P < 0.05, **represents P < 0.01, *** represents P < 0.001 and **** represents P < 0.0001

FIGURE 2

H. pylori infection inhibits expression levels of SIRT1 in gastric cells. A, The qRT‐PCR analysis of SIRT1 mRNA levels in cells infected with Hp26695. B and C, Western blot analysis of SIRT1 protein levels in cells infected with Hp26695. D, The qRT‐PCR analysis of SIRT1 mRNA levels in cells infected with Hp11637. E and F, Western blot analysis of SIRT1 protein levels in cells infected with Hp11637. Data from 3 independent experiments are presented as mean ± SD. ***represents P < 0.001 and **** represents P < 0.0001

FIGURE 3

Regulation of SIRT1 expression by RUNX3 in gastric cells. A and B, The qRT‐PCR analysis of RUNX3 (A) and SIRT1 (B) mRNA levels in cells transfected with siRNAs targeting RUNX3. Data from 3 independent experiments are presented as mean ± SD. C, Western blot analysis of RUNX3 and SIRT1 protein levels in cells transfected with siRNAs targeting RUNX3. The mean values are indicated (n = 3). D and E, The qRT‐PCR analysis of RUNX3 (D) and SIRT1 (E) mRNA levels in cells transfected with RUNX3‐expressing vector. Data from 3 independent experiments are presented as mean ± SD. F, Western blot analysis of RUNX3 and SIRT1 protein levels in cells transfected with RUNX3‐expressing vector. The mean values are indicated (n = 3). G and H, The qRT‐PCR analysis of RUNX3 (G) and SIRT1 (H) mRNA levels in cells transfected with Runt domain mutant RUNX3‐expressing vector. Data from 3 independent experiments are presented as mean ± SD. I, Western blot analysis of RUNX3 and SIRT1 protein levels in cells transfected with Runt domain mutant RUNX3‐expressing vector. The mean values are indicated (n = 3). ** represents P < 0.01, *** represents P < 0.001 and **** represents P < 0.0001

FIGURE 4

RUNX3 accounts for down‐regulation of SIRT1 in H. pylori‐infected gastric cells. A, The scheme of the putative RUNX3‐binding site in the SIRT1 promoter region. B, ChIP assay of RUNX3 directly binding to the promoter of SIRT1 (n = 3). C–E, Luciferase activities of different SIRT1 promoter constructs in cells transfected with RUNX3‐expressing vector (C), siRNAs targeting RUNX3 (D) or Runt domain mutant RUNX3‐expressing vector (E). Data from 3 independent experiments are presented as mean ± SD. F and G, The qRT‐PCR analysis of RUNX3 (F) and SIRT1 (G) mRNA levels in gastric cells transfected with RUNX3‐expressing vector and infected with Hp26695 for 3 h. Data from 3 independent experiments are presented as mean ± SD. H, Western blot analysis of RUNX3 and SIRT1 protein levels in gastric cells transfected with RUNX3‐expressing vector and infected with Hp26695 for 3 h. The mean values are indicated (n = 3). * represents P < 0.05 and **** represents P < 0.0001

FIGURE 5

Activation of SIRT1 rescues autophagic flux inhibited by H. pylori infection. A and B, Western blot was performed to detect the protein levels of LC3BI/II and SQSTM1/p62 in cells treated with SIRT1 activator, SRT1720 (5 µM) for indicated time. Data from 3 independent experiments are presented as mean ± SD. C and D, The mCherry‐EGFP‐LC3B fluorescence microscopy assay. Cells were infected with Hp26695 (3 h), or treated with SRT1720 (5 µM, 3 h) or pretreated with SRT1720 (5 µM, 1 h) and then infected with Hp26695 (3 h). Data from 5 independent experiments are presented as mean ± SD. Scale bars: 10 µm. E and F, Western blot was performed to detect the protein levels of LC3BI/II and SQSTM1/p62 in cells infected with Hp26695 for indicated time. Before Hp26695 infection, cells were pretreated with or without SRT1720 (5 µM, 1 h). Data from 3 independent experiments are presented as mean ± SD. ** represents P < 0.01 and **** represents P < 0.0001

FIGURE 6

SIRT1 inhibits intracellular colonization of H. pylori by activating autophagic flux. A‐C, Detecting 16S rDNA and colony formation assays for bacterial quantitation. After pretreatment with SRT1720 (SIRT1 activator, 5 µM, 1 h) (A) or EX 527 (SIRT1 inhibitor, 5 µM, 1 h) (B), cells were infected with Hp26695 and then used for further analysis. For rescuing experiments, EBSS (12 h) was used to induce autophagic flux in cells (C). Data from 3 independent experiments are presented as mean ± SD. D and E, Immunofluorescence staining of H. pylori. Cells were treated with SRT1720, or EX 527 or EBSS together with EX 527 as described in (A‐C). Data from 3 independent experiments are presented as mean ± SD. * represents P < 0.05, **represents P < 0.01, *** represents P < 0.001 and **** represents P < 0.0001

FIGURE 7

Validation of relationship between SIRT1 expression levels and H. pylori infection in clinical specimen. A, Representative images of IHC staining of SIRT1 in human superficial gastritis (SG), atrophic gastritis (AG) and dysplasia (DYS) samples. Scale bars: 200 µm for the left panel and 50 µm for the right panel. B, Statistics of IHC score of SIRT1 in clinical specimen. Comparison of SIRT1 expression score according to status of H. pylori infection. Data are presented as mean ± SD, n = 5. ** represents P < 0.01 and *** represents P < 0.001. C, Schematic model of study. Autophagy is a conserved protective process which can degrade intracellular pathogens such as H. pylori. Nevertheless, infection of H. pylori down‐regulated SIRT1 expression, inhibited autophagic flux in gastric cells and thus promoted its intracellular survival and colonization. Moreover, H. pylori infection inhibited expression of SIRT1 in a RUNX3‐dependent manner