Isolation and Proteomic Characterization of Outer Membrane Vesicles in Fusobacterium nucleatum

Fusobacterium nucleatum is an anaerobic Gram-negative bacterium associated with periodontal disease and colorectal cancer. Outer membrane vesicles (OMVs) produced by F. nucleatum are nanoscale lipid vesicles enriched with immunogenic and virulence-associated proteins, making them promising candidates for vaccine development. This protocol describes a comprehensive workflow for culturing F. nucleatum, isolating and purifying OMVs via ultracentrifugation and density gradient ultracentrifugation, and characterizing their biophysical properties using transmission electron microscopy (TEM) and dynamic light scattering (DLS). Proteomic profiling of purified OMVs was conducted using LC-MS/MD, and the resulting data were analyzed through bioinformatics tools. Protein function, subcellular localization, and immunogenic features were predicted using Vaxign, PRED-TMBB2, InterPro, and EMBOSS. Pathway and functional classification were performed using KEGG and EggNOG databases. Together, this methodology provides a reproducible pipeline for characterizing OMV-associated proteins and identifying potential antigens for F. nucleatum.