close
Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2013 Oct 8;8(10):e77157.
doi: 10.1371/journal.pone.0077157. eCollection 2013.

Escherichia coli producing colibactin triggers premature and transmissible senescence in mammalian cells

Thomas Secher  1 , Ascel Samba-Louaka, Eric Oswald, Jean-Philippe Nougayrède
Affiliations

Escherichia coli producing colibactin triggers premature and transmissible senescence in mammalian cells

Thomas Secher et al. PLoS One. .

Abstract

Cellular senescence is an irreversible state of proliferation arrest evoked by a myriad of stresses including oncogene activation, telomere shortening/dysfunction and genotoxic insults. It has been associated with tumor activation, immune suppression and aging, owing to the secretion of proinflammatory mediators. The bacterial genotoxin colibactin, encoded by the pks genomic island is frequently harboured by Escherichia coli strains of the B2 phylogenetic group. Mammalian cells exposed to live pks+ bacteria exhibit DNA-double strand breaks (DSB) and undergo cell-cycle arrest and death. Here we show that cells that survive the acute bacterial infection with pks+ E. coli display hallmarks of cellular senescence: chronic DSB, prolonged cell-cycle arrest, enhanced senescence-associated β-galactosidase (SA-β-Gal) activity, expansion of promyelocytic leukemia nuclear foci and senescence-associated heterochromatin foci. This was accompanied by reactive oxygen species production and pro-inflammatory cytokines, chemokines and proteases secretion. These mediators were able to trigger DSB and enhanced SA-β-Gal activity in bystander recipient cells treated with conditioned medium from senescent cells. Furthermore, these senescent cells promoted the growth of human tumor cells. In conclusion, the present data demonstrated that the E. coli genotoxin colibactin induces cellular senescence and subsequently propel bystander genotoxic and oncogenic effects.

PubMed Disclaimer

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Persistent γH2AX foci in IMR-90 cells infected with pks+ E. coli.
Non-transformed human IMR-90 cells were infected for 4h with live pks+ or pks- E. coli with a multiplicity of infection (MOI) of 20, 60 and 180 bacteria per cell or left uninfected. At the end of the infection, the cells were washed and further grown in medium supplemented with gentamicin. (A) Cells were examined for DNA (blue) and γH2AX (green) 3, 6 or 9 days after infection. Pictures of uninfected and MOI 180 -infected cells are shown, scale bars = 10µm. (B) The numbers of γH2AX foci per cell were quantified by a blinded observer in 30-100 nuclei for each condition in two independent experiments. Results represent the mean and standard error of the mean (SEM). Statistical significance was examined by one-way ANOVA with Bonferroni’s multiple comparison test; *P<0.05, **P<0.01, ***P<0.001 comparing infected and uninfected groups; #P<0.05, # #P<0.01, # # #P<0.001 comparing pks+ and pks- groups.
Figure 2
Figure 2. Permanent cell-cycle arrest and increased cell-cycle inhibitors expression in IMR-90 cells infected with pks+ E. coli.
(A) Cell-cycle distribution was analyzed by flow cytometry 3 days after infection. (B) 3 days after the infection the cells were incubated with EdU for 2 hours and its incorporation in S-phase cells was analyzed by flow cytometry. (C) Western blot analysis of p21 and p16 protein content in IMR-90 cells 4h, 1 and 6 days after infection. Actin was probed as a protein loading control. Results represent the mean and SEM of two independent experiments, one-way ANOVA with Bonferroni’s multiple comparison test; *P<0.05, ***P<0.001 comparing infected and uninfected groups; # #P<0.01, # # #P<0.001 comparing pks+ and pks- groups.
Figure 3
Figure 3. Increased senescence associated (SA)-β-galactosidase activity in IMR-90 cells infected with pks+ E. coli.
3, 6 or 9 days after infection, IMR90 cells were fixed with formaldehyde 4% and then stained with the galactosidase chromogenic substrate X-gal at pH 6 for 24h. Cells were examined for perinuclear blue staining by light microscopy at x20 magnification (A), and these SA-β-Gal positive cells (100-200 cells per condition) were quantified by a blinded observer (B). Results represent the mean and SEM of three independent experiments, one-way ANOVA with Bonferroni’s multiple comparison test; *P<0.05, **P<0.01 comparing infected and uninfected groups; #P<0.05, # #P<0.01, # # #P<0.001 comparing pks+ and pks- groups.
Figure 4
Figure 4. Accumulation of heterochromatin foci in IMR-90 cells infected with pks+ E. coli.
(A) Cells were stained with DAPI 6, 9 or 14 days after infection and examined for formation of heterochromatin foci (arrows) by confocal microscopy. Pictures of uninfected and MOI 180 –infected cells are shown, scale bars = 10µm. (B) Cells were stained with DAPI (blue) and with antibodies against H3K9me3 (green) 14 days after infection and examined by confocal microscopy, scale bar = 50µm. (C) Heterochromatin foci-positive cells (60-100 nuclei for each condition) were counted by a blinded observer. Results represent the mean and SEM of three independent experiments, one-way ANOVA with Bonferroni’s multiple comparison test; *P<0.05, **P<0.01 comparing infected and uninfected groups; #P<0.05 comparing pks+ and pks- groups.
Figure 5
Figure 5. Increased PML nuclear bodies in IMR-90 cells infected with pks+ E. coli.
(A) Cells were examined for DNA (blue) and PML protein (red) 3, 6 or 9 days after infection. Pictures of uninfected and MOI 180-infected cells are shown, scale bars = 10µm. (B) PML foci in 30-100 nuclei for each condition were counted by a blinded observer. Results represent the mean and SEM of three independent experiments, one-way ANOVA with Bonferroni’s multiple comparison test; *P<0.05, **P<0.01, ***P<0.001 comparing infected and uninfected groups; #P<0.05, # #P<0.01, # # #P<0.001 comparing pks+ and pks- groups.
Figure 6
Figure 6. Increased intracellular and mitochondrial ROS production in IMR-90 cells infected with pks+ E. coli.
(A) Cells were examined for intracellular ROS with the ROS sensor dihydro-dichloro-fluorescein diacetate (H2-DCFDA) (green). Pictures of uninfected and MOI 180-infected cells (at 3 days) are shown, scale bars = 10µm. (B) Mean H2-DCFDA fluorescence intensity was quantified by flow cytometry in 2x104 cells, 3 and 6 days after infection. (C) Cells were examined for mitochondrial superoxide production with the mitochondrial superoxide probe MitoSOX (orange) and for DNA (blue). Pictures from uninfected and MOI 180-infected cells (at 3 days) are shown, scale bars = 10µm. (D) Mean MitoSOX fluorescence intensity was quantified by flow cytometry 3 or 6 days after infection. Results in panels B and D represent the mean and SEM of three independent experiments, one-way ANOVA with Bonferroni’s multiple comparison test; **P<0.01, ***P<0.001 comparing infected and uninfected groups; # #P<0.01, # # #P<0.001 comparing pks+ and pks- groups.
Figure 7
Figure 7. Induction of a senescence-associated secretory phenotype (SASP) in IMR-90 cells following infection with pks+ E. coli.
IMR-90 cells were infected as before and incubated for 1-14 days. Conditioned cell culture media were prepared by incubating each culture in serum-free medium for 24 h. MMP-3, IL-6, IL-8 and MCP-1 secretion was quantified by Bio-Plex in one experiment (A) and by ELISA in two independent experiments (B) ; Results represent the mean and SEM, one-way ANOVA with Bonferroni’s multiple comparison test; **P<0.01, ***P<0.001 comparing infected and uninfected groups; # # #P<0.001 comparing pks+ and pks- groups.
Figure 8
Figure 8. Pks+ E. coli infected IMR-90 cells induce bystander γH2AX foci formation and SA-β-Gal expression in uninfected IMR-90 cells.
Naïve IMR-90 cells were treated for 1 day (γH2AX) or 3 days (SA-β-Gal) with conditioned media (CM) prepared 3-6-10 days after infection with pks+ or pks- E. coli with a MOI 180 (A, left panel). Cells were treated for 24 h with a 6 days-old CM and examined for DNA (Blue) and γH2AX (green), scale bars = 10µm (A, right panel). Cells were treated for 3 days with a 6 days old CM and examined for SA-β-Gal staining (Blue) (x20 magnification) (B) Numbers of γH2AX foci per cell (B) and percentages of SA-β-Gal positive cells (C) were quantified as before, 30-100 nuclei or 100-200 cells were evaluated for each condition. Results represent the mean and SEM of two independent experiments, one-way ANOVA with Bonferroni’s multiple comparison test; *P<0.05, ***P<0.001 comparing infected and uninfected groups; #P<0.05, # # #P<0.001 comparing pks+ and pks- groups. (D) IMR-90 grown on Transwells, were infected for 4h with live pks+ or pks- E. coli, washed and grown with gentamicin for 3 days. The Transwells were then transferred on top of naive IEC-6 cells and incubated for 24 hours with or without n-acetylcysteine (1mM). The IEC-6 cells were fixed and γH2AX was quantified by In-Cell Western. Results represent the mean and SEM of one experiments including triplicates for each conditions, two-way ANOVA with Bonferroni’s multiple comparison test; **P<0.01, ***P<0.001 comparing vehicle and n-acetylcysteine groups.
Figure 9
Figure 9. Senescent pks+ E. coli infected IMR-90 cells promote the growth of bystander tumour cells.
IMR-90 cells were infected for 4h with live pks+ or pks- E. coli with an MOI of 60 or 180 or left uninfected. At the end of the infection, the cells were washed and grown with gentamicin for 3 days. Then the IMR-90 cells were layered with 0.5% agar and 5000 HCT116 p53-/- cells embedded in 0.35% agar were added on top of the first layer and co-cultured for 10 days. (A) Representative photomicrographs of HCT116 p53-/- cell colonies, scale bars = 0.7 mm. (B) Large (>225 µm) microcolonies per microscopy field were counted by a blinded observer. The upper HCT116 p53-/- cell layer was harvested and stained with MTT for 24h. (C) Representative photomicrograph of experimental 6-well culture plates with viable cancer cell colonies coloured by MTT, scale bars = 3 mm. (D) The number of microcolonies was quantified using Image-J. (E) Total cell proliferation was assessed using MTT. Results represent the mean and SEM of two independent experiments, one-way ANOVA with Bonferroni’s multiple comparison test; *P<0.05 comparing infected and uninfected groups; #P<0.05 comparing pks+ and pks- groups.
See this image and copyright information in PMC

References

    1. Hayflick L (1965) The Limited in Vitro Lifetime of Human Diploid Cell Strains. Exp Cell Res 37: 614-636. doi: 10.1016/0014-4827(65)90211-9. PubMed: 14315085. - DOI - PubMed
    1. d’Adda di Fagagna F, Reaper PM, Clay-Farrace L, Fiegler H, Carr P et al. (2003) A DNA damage checkpoint response in telomere-initiated senescence. Nature 426: 194-198. doi: 10.1038/nature02118. PubMed: 14608368. - DOI - PubMed
    1. Kuilman T, Michaloglou C, Mooi WJ, Peeper DS (2010) The essence of senescence. Genes Dev 24: 2463-2479. doi: 10.1101/gad.1971610. PubMed: 21078816. - DOI - PMC - PubMed
    1. Rodier F, Campisi J (2011) Four faces of cellular senescence. J Cell Biol 192: 547-556. doi: 10.1083/jcb.201009094. PubMed: 21321098. - DOI - PMC - PubMed
    1. Sikora E, Arendt T, Bennett M, Narita M (2011) Impact of cellular senescence signature on ageing research. Ageing Res Rev 10: 146-152. doi: 10.1016/j.arr.2010.10.002. PubMed: 20946972. - DOI - PubMed

Publication types

MeSH terms