Carcinogenic bacterial pathogen Helicobacter pylori triggers DNA double-strand breaks and a DNA damage response in its host cells

Abstract

The bacterial pathogen Helicobacter pylori chronically infects the human gastric mucosa and is the leading risk factor for the development of gastric cancer. The molecular mechanisms of H. pylori-associated gastric carcinogenesis remain ill defined. In this study, we examined the possibility that H. pylori directly compromises the genomic integrity of its host cells. We provide evidence that the infection introduces DNA double-strand breaks (DSBs) in primary and transformed murine and human epithelial and mesenchymal cells. The induction of DSBs depends on the direct contact of live bacteria with mammalian cells. The infection-associated DNA damage is evident upon separation of nuclear DNA by pulse field gel electrophoresis and by high-magnification microscopy of metaphase chromosomes. Bacterial adhesion (e.g., via blood group antigen-binding adhesin) is required to induce DSBs; in contrast, the H. pylori virulence factors vacuolating cytotoxin A, γ-glutamyl transpeptidase, and the cytotoxin-associated gene (Cag) pathogenicity island are dispensable for DSB induction. The DNA discontinuities trigger a damage-signaling and repair response involving the sequential ataxia telangiectasia mutated (ATM)-dependent recruitment of repair factors--p53-binding protein (53BP1) and mediator of DNA damage checkpoint protein 1 (MDC1)--and histone H2A variant X (H2AX) phosphorylation. Although most breaks are repaired efficiently upon termination of the infection, we observe that prolonged active infection leads to saturation of cellular repair capabilities. In summary, we conclude that DNA damage followed by potentially imprecise repair is consistent with the carcinogenic properties of H. pylori and with its mutagenic properties in vitro and in vivo and may contribute to the genetic instability and frequent chromosomal aberrations that are a hallmark of gastric cancer.

Fig. 1.

H. pylori infection of cultured cells induces DSBs and DNA damage-response pathways. (A) AGS cells were infected with H. pylori strain G27 for 6 h at MOIs of 10 and 200. Hydroxyurea (HU) treatment (5 mM) served as positive control. DNA integrity was assessed by PFGE. (B–D) AGS cells (B), primary murine gastric epithelial cells (GEC) (C), and U2OS cells (D) were infected with H. pylori strain G27 (MOI 200) for 6 h and immunostained for 53BP1 and stained with DAPI for nuclear DNA. Cells irradiated with 10 Gy served as positive control as indicated. (E) AGS cells were infected for 6 or 48 h with G27 and stained for 53BP1, γH2AX, and nuclear DNA. (F and G) U2OS cells were infected with H. pylori G27 (MOI 200) for 6 h and/or treated with the ATM inhibitor KU-55922 before staining for 53BP1 and γH2AX. (Scale bars: 10 μm.) In B, C, D, and G, cells with more than four foci per nucleus were scored as positive; 150 cells were scored per condition. SEM and P values (Student's two-tailed t test) were calculated from three independent experiments.

Fig. 2.

DSB induction is H. pylori specific and depends on the direct contact of live bacteria with their host cells. (A and B) U2OS cells were infected with H. pylori strain G27 (MOI 200) followed by visualization of chromatid breaks by chromosome spreading. The integrity of chromatids was assessed by visual inspection of the chromosome spreads of 153 control cells and 198 infected cells in three independent experiments. The number of chromatid breaks per cell and number of cells with chromatid breaks (%) is indicated in B; error bars indicate the variation within the three experiments. (C) AGS cells were cocultured with live H. pylori G27, ethanol-killed bacteria, or live E. coli DH10B at the indicated MOIs for 16 h. DNA fragmentation was assessed by PFGE. (D) AGS cells were infected with H. pylori G27 for 6 h at the indicated MOIs (lanes 1–3). In lanes 4 and 5, cells were treated with sterile-filtered cell-culture supernatants obtained from 16-h control- or H. pylori-infected (MOI 200) AGS cells. In lanes 6–8, Transwell filters were used to separate AGS cells physically from a concentrated overnight H. pylori culture supernatant (lane 6) or from living bacteria at MOIs of 10 (lane 7) or 200 (lane 8). DNA fragmentation was assessed by PFGE. (E) Kato III cells were infected with H. pylori J99 or the isogenic mutant ΔbabA for 6 h at the indicated MOIs. DNA integrity was assessed by PFGE. (F–H) AGS cells were infected with wild-type G27 or ΔPAI or ΔvacA isogenic mutants and assessed by PFGE (F) or staining for 53BP1 (G and H). In H, cells with more than four foci per nucleus were scored as positive; SEM and P values were calculated from three independent experiments.

Fig. 3.

H. pylori-associated DSBs occur independently of cell-cycle phase and oxidative DNA damage. (A) U2OS cells were arrested in G2/M by 12-h treatment with 250 ng/mL nocodazole, released for 2 h, and infected with H. pylori G27 for 6 h. DNA integrity was assessed by PFGE. (B) AGS cells were left untreated, were treated with 5 mM N-acetyl-cysteine (NA), and/or were infected with H. pylori G27 at MOIs of 10 and 200. Relative induction of ROS was assessed by oxidation of 2,7-dichlorofluorescein diacetate (DCF-DA) to fluorescent DCF. (C) AGS cells were infected with H. pylori G27 for 6 h at the indicated MOIs in 20% oxygen (normoxia) or 1% oxygen (hypoxia). (D) AGS cells were infected at the indicated MOIs for 6 h. N-acetyl-cysteine (NA) was added at 1 or 5 mM as indicated. H₂O₂ treatment for 1 h served as positive control. In C and D, DNA fragmentation was visualized by PFGE. (E and F) Primary gastric epithelial cells explanted from wild-type C57BL/6 or iNOS^−/−/NOX2^−/− mice were infected with H. pylori for 6 h and immunostained for 53BP1. Cells with more than four 53BP1 foci per nucleus were graded as positive; SEM and P values were calculated from three independent experiments.

Fig. 4.

H. pylori-induced breaks are repaired only after bacterial killing. (A–C) AGS cells were infected with H. pylori G27 at the indicated MOIs for 6 h before antibiotic killing of the bacteria (eradication therapy, ET). Cells then were grown in full medium containing penicillin/streptomycin for 48 h to allow DNA repair to occur, were harvested, and were subjected to PFGE (A) or to immunostaining for 53BP1 and MDC1 (B) and compared with control cells that had been infected either for 6 h or continuously for 54 h. In C, cells with more than four foci per nucleus were graded as positive; SEM and P values were calculated from three independent experiments. (D) Cells were infected with H. pylori G27 for 6 and 48 h, subjected to H. pylori eradication, and cultured for another 10 d. Proliferative capacity was assessed by clonogenic assay; the survival and clonogenic capacity of never-infected (ctrl) cells is set at 100%.