Proteomic identification of secreted protein candidates mediating anti-colorectal cancer activity in a human-derived non-enterotoxigenic Bacteroides fragilis

The contribution of gut microbiota-derived factors to colorectal cancer (CRC) progression has attracted increasing attention; however, the specific bioactive components and their underlying mechanisms remain poorly defined. In this study, an activity-guided fractionation approach combined with proteomic analysis was employed to identify NTBF-derived components associated with anti-proliferative activity in colorectal cancer cells. Bacterial conditioned supernatant was sequentially fractionated by ultrafiltration, solvent extraction, and chromatography, and the antiproliferative activity of each fraction was evaluated in HT-29 cells. Active fractions were subsequently subjected to LC–MS/MS analysis and bioinformatic characterization. Comparative proteomic analysis identified 41 candidate proteins associated with bioactive fractions. Functional annotation revealed that these proteins were primarily involved in iron acquisition, ATP-binding cassette (ABC) transport systems, and protein secretion pathways. Notably, a substantial proportion of these proteins were predicted to be localized to the outer membrane or to contain signal peptides, indicating potential roles in host–microbe interactions and possible association with extracellular structures such as outer membrane vesicles. These proteins should be regarded as candidate bioactive molecules associated with the anti-proliferative fractions rather than confirmed functional effectors. The observed enrichment patterns further suggest that multiple molecular components may contribute to the bioactivity, although the individual and combined functional roles of these candidates require direct experimental validation. Collectively, these findings provide a systematic framework for identifying microbiota-derived candidate molecules associated with anti-proliferative activity in CRC.