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Bacteriophage-Mediated Restoration of Epithelial Integrity, DNA Damage Response, and Transcriptomic Homeostasis in Colon Organoids Exposed to ETBF and pks+ E. coli

ABSTRACT: Colorectal cancer (CRC)–associated pathobionts such as Enterotoxigenic Bacteroides fragilis (ETBF) and colibactin-producing (pks⁺) Escherichia coli disrupt epithelial barrier function, promote chronic inflammation, and induce genotoxic stress. To investigate how these pathogenic interactions with the host epithelium can be modulated, we employed patient-derived normal human colon organoids as an ex vivo model. Two lytic bacteriophages, Bac-FRP-3 (targeting ETBF) and Esc-COP-23 (targeting pks⁺ E. coli), were applied to bacterial cultures, and the resulting phage-conditioned supernatants were used to challenge organoids. Exposure to ETBF and pks⁺ E. coli induced strong pro-inflammatory transcriptional responses, DNA damage signaling, and epithelial disruption, whereas phage treatment attenuated these effects. Transcriptomic profiling revealed restoration of inflammatory and DNA damage–related gene expression, while confocal imaging confirmed recovery of epithelial junctional integrity and reduced DNA double-strand breaks. These findings highlight phage-mediated modulation of host–pathobiont interactions and provide mechanistic insight into how targeted viral predation can reshape pathogenic impacts within the human intestinal environment.

ORGANISM(S): Homo sapiens

PROVIDER: GSE312429 | GEO | 2026/08/26

REPOSITORIES: GEO

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Project description:The complex reservoir of metabolite-producing bacteria in the gastrointestinal tract contributes tremendously to human health and disease. Bacterial composition, and by extension gut metabolomic composition, is undoubtably influenced by the use of modern antibiotics. Herein, we demonstrate that polymyxin B, a last resort antibiotic used for chronic multidrug resistant infections infections, influences the production of the genotoxic metabolite colibactin from adherent-invasive Escherichia coli (AIEC) NC101. Colibactin can augment colorectal cancer (CRC) through DNA double stranded breaks and interstrand crosslinks. While the structure and biosynthesis of colibactin has been elucidated, chemical-induced regulation of its biosynthetic gene cluster and subsequent production of the genotoxin by pathogenic E. coli are largely unexplored. This research highlights the regulation of the colibactin-producing biosynthetic gene cluster under polymyxin stress. Using a multi-omic approach, we have identified that polymyxin stress enhances the abundance of colibactin biosynthesis proteins (Clb’s) in multiple pks+ E. coli strains, including pro-carcinogenic AIEC: NC101, the probiotic strain: E. coli Nissle 1917, and the antibiotic testing strain: E. coli ATCC 25922. Expression analysis via qPCR revealed that increased transcription of clb genes likely contributes to elevated Clb protein levels in NC101. Enhanced production of Clb’s by NC101 under polymyxin stress matched an increased production of the colibactin prodrug motif, a proxy for the mature genotoxic metabolite. Furthermore, E. coli with heightened tolerance for polymyxin antibiotics induced greater DNA damage, assessed by quantification of γH2AX staining in cultured intestinal epithelial cells. This study establishes a key link between the polymyxin B stress response and colibactin production in pks+ E. coli. Ultimately, our findings will inform future studies investigating colibactin regulation, the microbial response to antibiotics in the gut, and the ability of seemingly innocuous commensal microbes to induce host disease.

Project description:The complex reservoir of metabolite-producing bacteria in the gastrointestinal tract contributes tremendously to human health and disease. Bacterial composition, and by extension gut metabolomic composition, is undoubtably influenced by the use of modern antibiotics. Herein, we demonstrate that polymyxin B, a last resort antibiotic used for chronic multidrug resistant infections infections, influences the production of the genotoxic metabolite colibactin from adherent-invasive Escherichia coli (AIEC) NC101. Colibactin can augment colorectal cancer (CRC) through DNA double stranded breaks and interstrand crosslinks. While the structure and biosynthesis of colibactin has been elucidated, chemical-induced regulation of its biosynthetic gene cluster and subsequent production of the genotoxin by pathogenic E. coli are largely unexplored. This research highlights the regulation of the colibactin-producing biosynthetic gene cluster under polymyxin stress. Using a multi-omic approach, we have identified that polymyxin stress enhances the abundance of colibactin biosynthesis proteins (Clbs) in multiple pks+ E. coli strains, including pro-carcinogenic AIEC: NC101, the probiotic strain: E. coli Nissle 1917, and the antibiotic testing strain: E. coli ATCC 25922. Expression analysis via qPCR revealed that increased transcription of clb genes likely contributes to elevated Clb protein levels in NC101. Enhanced production of Clbs by NC101 under polymyxin stress matched an increased production of the colibactin prodrug motif, a proxy for the mature genotoxic metabolite. Furthermore, E. coli with heightened tolerance for polymyxin antibiotics induced greater DNA damage, assessed by quantification of yH2AX staining in cultured intestinal epithelial cells. This study establishes a key link between the polymyxin B stress response and colibactin production in pks+ E. coli. Ultimately, our findings will inform future studies investigating colibactin regulation, the microbial response to antibiotics in the gut, and the ability of seemingly innocuous commensal microbes to induce host disease.

Dataset Information

Bacteriophage-Mediated Restoration of Epithelial Integrity, DNA Damage Response, and Transcriptomic Homeostasis in Colon Organoids Exposed to ETBF and pks+ E. coli

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