Characterization of outer membrane vesicles (OMVs) from pks+ NC101 and pks- DH10B E. coli strands

Background & Objectives: Colibactin is a genotoxin encoded by the pks genomic island in pathogenic Gram-negative bacteria and has been implicated in colorectal cancer. Colibactin synthesis and activation occur in a two-step process in which pre-colibactin is produced in the bacterial cytoplasm and subsequently activated into colibactin within the periplasmic space. However, the mechanism by which pks⁺ bacteria export colibactin to host cells remains unclear. We hypothesize that outer membrane vesicles (OMVs), nanosized proteoliposomes released by Gram-negative bacteria capable of transporting diverse biomolecules, serve as a delivery system for this toxin to host cells. Methods: To investigate this, OMVs were isolated from pks⁺ NC101 and pks- DH10B E. coli strains using the exoEasy Maxi Kit and quantified with a BSA-based protein assay on a Synergy H1 microplate reader. Results: Yielding concentrations consistent with typical OMV preparations. The stability and morphological characteristics of the extracted OMVs will be evaluated using dynamic light scattering (DLS). Metabolites extracted from OMVs will be examined via mass spectrometry to confirm the presence of known colibactin-related intermediates. Additionally, OMVs will undergo proteomic analysis to identify proteins involved in OMV-host interaction mechanisms. Finally, we aim to assess the effects of OMV exposure on large intestinal epithelial cells to better understand their contribution to colibactin-associated toxicity and colorectal cancer development. Conclusion: Ultimately, this research seeks to clarify how OMVs mediate colibactin transport and cytotoxicity, providing new insights into bacterial factors that promote colorectal carcinogenesis. Acknowledgements: This research was funded by the National Institute of Allergy and Infectious Diseases (NIAID) Grant #5R25AI183304-02.