PROJECT SUMMARY Configurations of the gut microbiome and specific bacterial species are associated with CRC, and such data provide fresh perspectives and insights into CRC susceptibility, response to therapy, and therapeutics. A central challenge of cancer gut microbiome studies is to define the bioactive features of micro-organisms that influence CRC susceptibility and progression. Fusobacterium nucleatum, a Gram-negative anaerobic bacterium, is a normal constituent of the human oral microbiome. However, numerous studies now demonstrate that F. nucleatum (Fn) is enriched in human colorectal adenomas and CRC compared to healthy colonic tissues, as well as in stool from individuals with CRC, and that tumoral Fn-enrichment correlates with worse overall-survival, progress-free survival and chemoresistance. My lab has been involved in studies detecting Fn in adenomas and CRC, correlating fusobacterial presence with various CRC risk factors, and determining how Fn may contribute to CRC using preclinical CRC models with a focus on its interactions with immune cells. Yet, much remains to discover about the mechanisms by which Fn contributes to CRC. To date, the bulk of studies on Fn virulence mechanisms in CRC have focused on its adhesins and its lipid polysaccharide. While such studies have revealed important biology about Fn, there are additional mechanisms that may underlie Fn's contribution to CRC. In this renewal proposal, we build upon our expertise with Fn, microbial metabolism, and immunology to further understanding of how Fn contributes to CRC. Specifically, we will investigate the effects of three bioactivities, Fn hydrogen sulfide production, short-chain fatty acids and Fic proteins, on the colonic epithelium and immune cells in the evolving tumor microenvironment. In considering broader mechanisms used by but not exclusive to Fn, we hope to extend the applicability of our findings to Fn-negative CRC and other malignancies.