Quorum sensing as a tool to detect and target pro-oncogenic microbial biofilms associated with right-sided colorectal cancer

Project Information

BIFCOLC-QS

Grant agreement ID: 101109251

DOI

10.3030/101109251

Project closed

EC signature date 3 April 2023

Start date 15 June 2023

End date 14 June 2025

Funded under

• Marie Skłodowska-Curie Actions (MSCA)

Total cost

No data

EU contribution

€ 173 847,36

Coordinated by

EUROPEAN MOLECULAR BIOLOGY LABORATORY
Germany

Periodic Reporting for period 1 - BIFCOLC-QS (Quorum sensing as a tool to detect and target pro-oncogenic microbial biofilms associated with right-sided colorectal cancer)

Reporting period: 2023-06-15 to 2025-06-14

Summary of the context and overall objectives of the project

Colorectal cancer (CRC) is the second leading cause of cancer-related deaths worldwide, responsible for approximately 0.90 million deaths in 2022. CRC can be categorized into two major types based on the anatomical location of the tumors. The proximal (right-sided) and distal (left-sided) colons possess anatomical, developmental, and physiological distinctions, including differences in embryonic origin, vascularization, neuronal supply, and microbial composition. These fundamental differences influence disease onset, progression, pathological features, and clinical outcomes. Right-sided CRC is consistently associated with poorer prognosis, greater resistance to treatment, lower survival rates, and a higher risk of recurrence compared to left-sided CRC.
Being a multifactorial disease, CRC is also linked to gut microbiome dysbiosis and is closely associated with pro-oncogenic bacterial strains, including Fusobacterium nucleatum, pks+ Escherichia coli, and enterotoxigenic Bacteroides fragilis. These bacteria contribute to tumorigenesis by producing toxins that damage epithelial integrity, promote inflammation, and induce DNA mutations. They often form invasive polymicrobial biofilms that enhance tumor progression, particularly in right-sided CRC, where such biofilms are more prevalent. Biofilm development is regulated by quorum sensing (QS), a bacterial communication system in which small signaling molecules coordinate the expression of virulence genes and interactions between species
The current project has focused on elucidating the role of QS in regulating polymicrobial biofilm formation by CRC-associated bacteria and on developing a probiotic strain capable of sensing these QS signals to identify and target pro-oncogenic biofilms. This approach is being explored as a potential alternative to antibiotics and natural probiotics, which show limited efficacy against biofilms. Targeting CRC-associated microbial biofilms may help limit biofilm-associated disease progression in CRC and reduce disease severity. Furthermore, if administered in the early stages of CRC, it could slow disease progression and prevent recurrence following treatment.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

• Developed Phoenix, an in-house Nextflow-based pipeline for genome assembly, polishing, and annotation to identify quorum sensing (QS) genes in CRC-associated bacteria using sequencing data.
• Established LC-MS/QqQ-MS methods for the identification and quantification of quorum sensing molecules (autoinducers).
• Developed a multiplexed FISH method to visualize polymicrobial biofilms using confocal microscopy.
• Assessed the biological activity of identified autoinducers and developed protocols for an autoinducer-bioluminescence reporter assay.
• Designed and performed quorum quenching biofilm assays, demonstrating that disrupting QS pathways significantly reduced in vitro biofilm formation.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

Our findings reveal a diverse distribution of quorum sensing (QS) systems among CRC-associated bacterial isolates, with AI-2 being the most broadly conserved. The presence of synthase and receptor genes indicates potential differences in signal production and recognition, suggesting complex inter- and intra-species communication networks within these microbial communities. The biological activity of autoinducers was confirmed, as these molecules present in culture supernatants from these bacteria were able to activate QS pathways in reporter strains. Biofilm assays under anaerobic conditions showed both synergistic and inhibitory effects on biofilm formation in co-culture conditions. Quorum quenching (QQ) assays demonstrated that disrupting QS pathways inhibited biofilm development in these bacteria. In vivo colonization studies confirmed that Bacteroides fragilis, Fusobacterium nucleatum, and pks⁺ Escherichia coli could stably colonize the mouse gut, demonstrating their persistence and compatibility within the host environment. This project investigates the potential of a non-invasive, probiotic-based approach to target CRC-associated bacterial biofilms. We demonstrated that CRC-associated bacteria can communicate through QS and that these signalling molecules could be harnessed as cues to sense and target these bacteria within tumor tissue.

Schematic representation of engineered probiotic bacteria to target CRC-associated biofilms.