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Oligosaccharides increase the genotoxic effect of colibactin produced by pks+ Escherichia coli strains

Oliero, Manon, Calvé, Annie, Fragoso, Gabriela, Cuisiniere, Thibault, Hajjar, Roy, Dobrindt, Ulrich, Santos, Manuela M.·2021·BMC Cancer
DOI 10.1186/s12885-021-07876-8PMC PMC7890614

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Abstract

Background: Colibactin is a genotoxin that induces DNA double-strand breaks that may lead to carcinogenesis and is produced by Escherichia coli strains harboring the pks island. Human and animal studies have shown that colibactin-producing gut bacteria promote carcinogenesis and enhance the progression of colorectal cancer through cellular senescence and chromosomal abnormalities. In this study, we investigated the impact of prebiotics on the genotoxicity of colibactin-producing E. coli strains Nissle 1917 and NC101. Methods: Bacteria were grown in medium supplemented with 20, 30 and 40 mg/mL of prebiotics inulin or galacto-oligosaccharide, and with or without 5 μM, 25 μM and 125 μM of ferrous sulfate. Colibactin expression was assessed by luciferase reporter assay for the clbA gene, essential for colibactin production, in E. coli Nissle 1917 and by RT-PCR in E. coli NC101. The human epithelial colorectal adenocarcinoma cell line, Caco-2, was used to assess colibactin-induced megalocytosis by methylene blue binding assay and genotoxicity by γ-H2AX immunofluorescence analysis. Results: Inulin and galacto-oligosaccharide enhanced the expression of clbA in pks+ E. coli . However, the addition of 125 μM of ferrous sulfate inhibited the expression of clbA triggered by oligosaccharides. In the presence of either oligosaccharide, E. coli NC101 increased dysplasia and DNA double-strand breaks in Caco-2 cells compared to untreated cells. Conclusion: Our results suggest that, in vitro, prebiotic oligosaccharides exacerbate DNA damage induced by colibactin-producing bacteria. Further studies are necessary to establish whether oligosaccharide supplementation may lead to increased colorectal tumorigenesis in animal models colonized with pks+ E. coli .

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Fig. 1 Inulin and GOS increase the clbA promoter activity. a-b Growth curves and luminescence measurement of EcN carrying the translational clbA-luciferase reporter fusion. The effect of 0, 20, 30 and 40 mg/mL of inulin (a) and GOS (b) on growth (OD600; filled symbols) and on clbA-lux relative luminescence (RLU 106; open symbols). c-d Area under the curve (AUC) of RLU/OD600 for inulin (c) and GOS (d). *P < 0.05, **P < 0.01, ***P < 0.001 compared to control (0 mg/mL oligosaccharides) and &P < 0.05; ANOVA
Fig. 1 Inulin and GOS increase the clbA promoter activity. a-b Growth curves and luminescence measurement of EcN carrying the translational clbA-luciferase reporter fusion. The effect of 0, 20, 30 and 40 mg/mL of inulin (a) and GOS (b) on growth (OD600; filled symbols) and on clbA-lux relative luminescence (RLU 106; open symbols). c-d Area under the curve (AUC) of RLU/OD600 for inulin (c) and GOS (d). *P < 0.05, **P < 0.01, ***P < 0.001 compared to control (0 mg/mL oligosaccharides) and &P < 0.05; ANOVACC BY · PubMed Central
Fig. 2 Iron decreases the transcription of clbA promoter activity promoted by oligosaccharides. a Growth curves and luminescence measurement of EcN the translational clbA luciferase reporter fusion and b area under the curve (AUC) of RLU/OD600, in media containing 5 μM, 25 μM and 125 μM ferrous sulfate without inulin and with c 20, d 30, e 40 mg/mL inulin and f-h corresponding area under the curve (AUC) of RLU/OD600. Growth curves and luminescence measurement of EcN in media containing 5 μM, 25 μM and 125 μM ferrous sulfate supplemented with i 20, j 30, k 40 mg/mL GOS and l-n corresponding area under the curve (AUC) of RLU/OD600. *P < 0.05, **P < 0.01, ***P < 0.001 compared to control (0 mg/mL oligosaccharides); &P < 0.05, &&P < 0.01, and &&&P < 0.001; ANOVA
Fig. 2 Iron decreases the transcription of clbA promoter activity promoted by oligosaccharides. a Growth curves and luminescence measurement of EcN the translational clbA luciferase reporter fusion and b area under the curve (AUC) of RLU/OD600, in media containing 5 μM, 25 μM and 125 μM ferrous sulfate without inulin and with c 20, d 30, e 40 mg/mL inulin and f-h corresponding area under the curve (AUC) of RLU/OD600. Growth curves and luminescence measurement of EcN in media containing 5 μM, 25 μM and 125 μM ferrous sulfate supplemented with i 20, j 30, k 40 mg/mL GOS and l-n corresponding area under the curve (AUC) of RLU/OD600. *P < 0.05, **P < 0.01, ***P < 0.001 compared to control (0 mg/mL oligosaccharides); &P < 0.05, &&P < 0.01, and &&&P < 0.001; ANOVACC BY · PubMed Central
Fig. 3 Inulin and GOS increase the expression of clbA in E. coli strain NC101. a-b Growth curves of E. coli NC101 grown in 100 μM of iron sulfate and 40 mg/mL of inulin (a) or 40 mg/mL of GOS (b). c Fold change of clbA/16S rRNA expression in minimal medium supplemented with inulin or GOS, with or without 100 μM iron sulfate. *P < 0.05, **P < 0.01, n.s. non-significant compared to control (0 mg/mL oligosaccharides) and &&&P < 0.001; ANOVA
Fig. 3 Inulin and GOS increase the expression of clbA in E. coli strain NC101. a-b Growth curves of E. coli NC101 grown in 100 μM of iron sulfate and 40 mg/mL of inulin (a) or 40 mg/mL of GOS (b). c Fold change of clbA/16S rRNA expression in minimal medium supplemented with inulin or GOS, with or without 100 μM iron sulfate. *P < 0.05, **P < 0.01, n.s. non-significant compared to control (0 mg/mL oligosaccharides) and &&&P < 0.001; ANOVACC BY · PubMed Central
Fig. 4 Inulin and GOS enhance DNA damage in Caco-2 cells infected with pks+ E. coli strain NC101. a Representative images of methylene blue staining for quantification of megalocytosis of Caco-2 cells 4 h post-infection (multiplicity of infection = 50 bacteria/cell; magnification 200x). E. coli K-12 was used as a pks- control strain. b-c Quantification of megalocytosis of Caco-2 cells with addition of inulin (b) and GOS (c) in medium. d Scan of In Cell Western image showing DNA DSBs with merged detection of total DNA (red, 680 nm) and γ-H2AX (green, 800 nm). e γ-H2AX fold induction in inulin and GOS (analysis were done on single colored picture). *P < 0.05, ***P < 0.001, compared to the control, pks- E. coli K-12 and &P < 0.05, &&P < 0.01, &&&P < 0.001; ANOVA
Fig. 4 Inulin and GOS enhance DNA damage in Caco-2 cells infected with pks+ E. coli strain NC101. a Representative images of methylene blue staining for quantification of megalocytosis of Caco-2 cells 4 h post-infection (multiplicity of infection = 50 bacteria/cell; magnification 200x). E. coli K-12 was used as a pks- control strain. b-c Quantification of megalocytosis of Caco-2 cells with addition of inulin (b) and GOS (c) in medium. d Scan of In Cell Western image showing DNA DSBs with merged detection of total DNA (red, 680 nm) and γ-H2AX (green, 800 nm). e γ-H2AX fold induction in inulin and GOS (analysis were done on single colored picture). *P < 0.05, ***P < 0.001, compared to the control, pks- E. coli K-12 and &P < 0.05, &&P < 0.01, &&&P < 0.001; ANOVACC BY · PubMed Central

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Metadata

Authors
Oliero, M., Calvé, A., Fragoso, G., Cuisiniere, T., Hajjar, R., Dobrindt, U., & Santos, M. M.
Venue
BMC Cancer
Publisher
Springer Science and Business Media LLC
Published
2021-02-17
Volume
21
Issue
1
ISSN
1471-2407
DOI
10.1186/s12885-021-07876-8
PMC
PMC7890614
Cited by
47
References
8
Language
en
Licence
Funders
  • Canadian Institutes of Health Research (PJT-159775)
  • Natural Sciences and Engineering Research Council of Canada (RGPIN-2018-06442)
  • Institut du Cancer de Montréal (scholarship)
  • Université de Montréal (scholarship)
  • Fonds de Recherche du Québec - Santé (MSSS Resident Physician Health Research Career Training Program)
  • Interdisciplinary Center for Clinical Research of the Medical Faculty Münster (Dob2/013/12)

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