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.


| INTRODUC TI ON
Helicobacter pylori (H. pylori) is one of the most common human pathogens that has colonizes more than 50% of the world's population.
Chronic infection of H. pylori gives rise to a series of well-defined histological stages processing through gastritis, metaplasia, dysplasia and eventually the malignancy, gastric cancer. 1 Although H. pylori was previously regarded as an extracellular bacterium, a growing evidence has reported the facultative intracellular nature of H. pylori. 2,3 This bacterium was found to survive and even multiply inside gastric epithelial cells. 3,4 Presence of intracellular H. pylori in progenitor or stem cells serves as a strategy for this invasive pathogen to persist in human stomach. 5,6 Hiding inside the host cells enables H. pylori to escape from the innate immune response. 7 Besides, intracellular H.
pylori are more resistant to antibiotic treatment and account for repopulation following eradication therapy. 2,8,9 However, the host factors that affect the intracellular survival and colonization of H. pylori remain poorly understood.
Autophagy is an evolutionary conserved process which may be divided into two steps. First, double-membrane autophagosomes formed sequestrating intracellular components. Second, autophagosome-lysosome fused and eventually degraded the contents. The second step is also named as autophagic flux. to inhibit autophagosome maturation or escape from the host cell autophagosomes. 12,13 It has been reported that H. pylori can induce autophagosomes formation, survive and even multiply in autophagosomes before autophagic flux occurs. 14,15 When autophagosomes fused with lysosome, which is referred to autophagic flux, clearance of H. pylori in a lysosome-dependent manner occurred.
Therefore, inhibiting autophagic flux may increase intracellular survival and colonization of H. pylori. 15 Nevertheless, the mechanism by which H. pylori antagonizes degradation by autophagic flux remains to be elucidated.
Sirtuin 1 (SIRT1) belongs to class III histone deacetylases and is the mammalian ortholog of yeast silent information regulator (Sir 2).
As Sir 2 was proved to extend lifespan in yeast, research on SIRT1 first concentrated on anti-ageing and then expanded to stress response and metabolism. [16][17][18] By interacting with the energy sensor, AMP-activated protein kinase, SIRT1 was shown to inhibit mammalian target of rapamycin (mTOR) signalling and thus promote formation of autophagosomes. 19 SIRT1 can also directly deacetylate ATGs and BECLIN1 and stimulates the initial process of autophagy. 20,21 Moreover, SIRT1 deacetylates and activates transcription factor FOXO1 and its target Rab7, which was both necessary and sufficient for increasing autophagic flux. 22 Moreover, SIRT1-dependent autophagy activation was found to reduce bacterial growth and anti-M. tuberculosis responses. 21,23 However, whether SIRT1 and SIRT1induced autophagy play a role in intracellular survival and colonization of H. pylori has not been elucidated.
In this study, we investigated regulation of SIRT1 expression by H. pylori infection and the underlying mechanism was explored in detail. Then the effects of SIRT1 and SIRT1-induced autophagic flux in intracellular survival and colonization of H. pylori were also explored. with 10% foetal bovine serum (Biological Industries, Israel), 100 units/ ml penicillin and 100 µg/ml streptomycin. All cells were incubated at 37°C with 5% CO 2 . Mycoplasma PCR (GeneCopoeia) testing of these cells was performed every month following the protocol of the manufacturer. Reagents used in this study were: Earle's Balanced Salt Solution (EBSS, HyClone) used for serum starvation to induce autophagic flux, bafilomycin A 1 (Baf A1, S1413, Selleckchem) used as inhibitor of autophagic flux, SRT1720 (S1129, Selleckchem) used as activator of SIRT1, and EX 527 (S1541, Selleckchem) used as inhibitor of SIRT1.
The luciferase reporter plasmids containing the promoter sequence of SIRT1 assumed RUNX3 binding sits deleted promoter sequences of SIRT1 were constructed by BioSune Biotechnology (Shanghai, China). The plasmids were delivered into cells by X-tremeGENE HP Transfection Reagent (Roche Applied Science, Basel, Switzerland).
The small interfering RNA (siRNA) targeting RUNX3 and FOXO3a, and the negative control siRNA were synthesized by GenePharma (Shanghai, China) and delivered into cells by Lipofectamine 2000 (Invitrogen). The sequences of siRNAs were summarized in Table S1.

| RNA extraction and quantitative real-time PCR (qRT-PCR)
Total RNA was extracted with Trizol, converted into cDNA and amplified by qRT-PCR as previously described. 24 Primers were synthesized by BioSune Biotechnology, and the primer sequences were listed in Table S1.
The protein concentrations were measured using a BCA reagent kit (Beyotime). The membranes were probed, and the protein bands were visualized as previously described. 24 The primary antibod-

| Luciferase assay
For luciferase assay, cells were seeded in 24-well plates (5 × 10 4 cells per well). After transfection with plasmids or siRNAs, appropriate reporter plasmid was transfected into cells. Then, the relative luciferase activities were measured and calculated using the Dual-Luciferase Reporter Assay System (Promega). 24

| Chromatin immunoprecipitation (ChIP)
With the SimpleChIP ® Enzymatic Chromatin IP Kit (Cell Signaling), ChIP assays were performed with anti-RUNX3 (ab11905, Abcam), and IgG (negative control) in AGS cells transfected with pcDNA3.1-RUNX3 plasmid. The purified DNA served as the template to amplify SIRT1 promoter. Agarose gel electrophoresis was performed to visualize the PCR products. The primers were listed in Table S1.

| The mCherry-EGFP-LC3B fluorescence microscopy assay
Cells were seeded on glass coverslips in 24-well plates (5 × 10 4 cells per well) and incubated overnight. Then cells were transfected with AdM-CMV-mCherry-EGFP-LC3B adenovirus (MOI = 100, Vigene, Jinan, China) for 24 h. After cells were treated with chemicals or infected with H. pylori, cells were fixed with 4% fixative solution and the coverslips were taken out of the plates for observation. Over 20 cells in one field were randomly selected to examine the numbers of autophagosomes (yellow puncta) and autolysosomes (red puncta) using an Olympus microscope (Tokyo, Japan). Five fields were randomly selected from each group, and the EGFP/mCherry co-localization efficiency was calculated. The primer sequences were listed in Table S1. The levels of 16S rDNA in gastric cells were measured and normalized to β-actin based on 2 -ΔΔCt method.

| Immunofluorescence stain
Cells (2.5 × 10 4 ) were seeded onto coverslips in 24-well plates and incubated overnight. After infected with Hp26695 for 10 h, extracellular H. pylori were killed using 100 µg/ml gentamicin for 1 hour at 37°C and then washed with PBS two times. Next, cells were fixed with 4% fixative solution and permeabilized with 0.2% Triton X-100.
Then, after blocking, cells were incubated with anti-H. pylori antibody (ab7788, 1: 100, Abcam) and the fluorescent secondary antibody. Nuclei were stained with 4', 6-diamidino-2-phenylindole (DAPI, Beyotime). Slides were observed under microscope (Olympus), and photographs were taken using CellSens Dimension software. Red fluorescence intensities for the H. pylori-infected group were calculated by randomly selecting over 30 cells in one field, and three fields were detected for each group (Image J).

F I G U R E 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

| Immunohistochemistry (IHC) stain
We obtained paraffinized gastric tissue sections from Jinan Central Hospital. H. pylori infection was examined by rapid urease test and pathological examination using methylene blue staining. These paraffinized gastric tissue sections were divided into two groups: H. pylori-positive (n = 15) and H. pylori-negative (n = 15). For each group, there were three types of pathological changes: superficial gastritis (n = 5), atrophic gastritis (n = 5) and dysplasia (n = 5). To investigate SIRT1 expression levels in these sections, IHC analysis was performed. Paraffin-embedded sections were deparaffinized, rehydrated and antigen-retrieved. The remaining steps were performed as previously described. 24 The primary antibody anti-SIRT1 (ab32441, 1: 100, Abcam) was used. The images were obtained under microscope (Olympus) using CellSens Dimension software.
The intensity of SIRT1 staining was scored as follows: 0, no staining; 1, light brown; 2, medium brown; and 3, dark brown. The expression score was calculated by multiplying the staining intensity score and the positive percentages.

| Statistical analysis
All experimental data were analysed with GraphPad Prism 8.0 software. Data were presented as mean ± SD. Comparisons between different groups were determined by Student's t test or one-way ANOVA. A value of P < 0.05 was considered statistically significant.

| H. pylori infection induces autophagosome formation but inhibits autophagic flux leading to increased colonization
We first verified increased autophagosome formation induced by H. pylori infection (Hp26695 and Hp11637). As revealed in Figure 1A and Figure S1A

| H. pylori infection inhibits expression levels of SIRT1 in gastric cells
Since SIRT1 can activate autophagy to eliminate bacterial load, 21,23 we have been suggested that SIRT1 might be the downstream tar-  Figure S2).

| RUNX3 can regulate expression of SIRT1 in gastric cells
Next, we screened for transcriptional factors responsible for regu-  Figure 4B). Additionally, luciferase assays were used to verify the functional significance of this binding. We constructed luciferase reporter plasmids containing SIRT1 intact promoter (Pro) and mutant SIRT1 promoter (Mut). Overexpression of RUNX3 significantly increased promoter activities, while inhibition of RUNX3 exhibited the opposite effects ( Figure 4C,D). Nevertheless, mutation of RUNX-binding site abolished the changes in promoter activities ( Figure 4C,D). Moreover, expression of mutant RUNX3 did not regulate SIRT1 promoter activities ( Figure 4E). Therefore, in gastric cells SIRT1 was a direct target gene of RUNX3.

| RUNX3 accounts for down-regulation of SIRT1 in H. pylori-infected gastric cells
Then, we assessed whether RUNX3 was involved in SIRT1 expression inhibited by H. pylori infection. As shown in Figure 4F

| Activation of SIRT1 rescues autophagic flux inhibited by H. pylori infection
Next, we tested whether increasing SIRT1 activity could restore the autophagic flux inhibited by H. pylori infection. To this end, we activated SIRT1 using SRT1720 (a small molecule compound specific to activate SIRT1) and assessed autophagic flux by detecting levels of autophagy marker and the mCherry-EGFP-LC3B fluorescence microscopy assays. We showed that upon SIRT1 activation, protein levels of LC3B-II increased but SQSTM1/p62 decreased over time indicating induction of autophagic flux ( Figure 5A,B). In addition, cells with SIRT1 activation showed that most of the LC3B-positive autophagosomes lost EGFP signal but retained mCherry signal ( Figure 5C,D). This is consistent with the results from serum-starved cells shown in Figure 1B and confirmed that activation of SIRT1 leads to increased autophagic flux. Then, we investigated the effects of SIRT1 activator on autophagic flux in H. pylori-infected cells.
Therefore, these data indicated that activation of SIRT1 rescued autophagic flux that was inhibited by H. pylori infection.

F I G U R E 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

| SIRT1 inhibits intracellular colonization of H. pylori by activating autophagic flux
Then we investigated the effects of SIRT1 on intracellular survival and colonization of H. pylori. We pretreated H. pylori-infected cells with SIRT1 activator (SRT1720) and then determined intracellular survival and colonization of H. pylori by bacterial DNA measurement, colony formation assays and immunofluorescence staining.
As shown in Figure 6A

| Validation of relationship between SIRT1 expression levels and H. pylori infection in clinical specimens
To determine the relationship between SIRT1 expression levels and H. pylori infection, we performed IHC staining using clinical specimens. We collected gastric tissues diagnosed as superficial gastritis, atrophic gastritis and dysplasia. For each pathogenic stage, both H. pylori-positive and H. pylori-negative specimens were included.
For each histologic stage (superficial gastritis, atrophic gastritis or dysplasia), expression levels of SIRT1 in H. pylori-positive patients were significantly lower than those in patients H. pylori-negative ( Figure 7A,B). These results confirmed the relationship between SIRT1 expression and H. pylori infection in clinical specimen. On the other hand, intracellular bacteria develop diverse strategies to reprogram or stall autophagy process and thus escape from autophagic degradation. [29][30][31][32][33] H. pylori, which was previously thought only survive F I G U R E 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 F I G U R E 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 on the surface of gastric mucosa, have recently been found to invade epithelial cells and colonize intracellularly. Although the intracellular part represents a small percentage of overall bacteria, this proportion is strongly resistant to antibiotic treatment and play a critical role in infection recrudescence post-therapy. 3,8,9 Here, we show that in gastric cells, 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 compelling evidence suggests that intracellular pathogens may trigger nutritional deprivation and energy stress both of which were generally regarded as initial factors of autophagy. 29,33-35 SIRT1, a NAD + -dependent deacetylase, acts as an energy and nutrition sensor. It has been shown that SIRT1 plays an essential role in calorie restriction-induced longevity and starvation-activated autophagy. [36][37][38] By treating gastric cells with SIRT1 activator or inhibitor, we showed that SIRT1 can induce not only autophagosomes formation but also autophagic flux occurrence. The following rescue experiment indi- Our results provide evidence that SIRT1 can serve as a therapeutic target to eradicate H. pylori infection ( Figure 7C). All the authors had final approval of the submitted version.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.