Colorectal cancer (CRC) remains a major public health issue, with approximately 145,000 new cases and 51,000 deaths from this disease per year in the US alone. Alterations in the composition of the gut microbiota and elevated inflammation are established risk factors for CRC. We made the novel observation that the normal gut bacterium Parabacteroides distasonis (Pd) suppresses colorectal inflammation and tumor formation in mice. Importantly, we show that Pd suppresses the activation of the pro-inflammatory, pro-tumorigenic TLR4 signaling pathway. The objectives of this application are to determine whether blocking TLR4 is required for the anti-tumor effects of Pd, to identify the active factor of Pd and its host target and finally to define how Pd influences immune cell populations in the colon. Based on our robust data, we hypothesize that the chemopreventive effect of Pd is achieved by the suppression of pro-inflammatory TLR4 signaling and the recruitment of inflammation quelling T regulatory (Treg) cells to the colon. The specific aims of the study are to: 1) To identify anti-inflammatory factor of Pd; 1A) To test whether the anti-TLR4 function of Pd is required for its chemopreventive effect; 1B) To identify the molecular target of the Pd anti-inflammatory molecule and 2) To define how Pd influences immune cell populations in the colonic mucosa. The aims of this study will be addressed by comparing the ability of various closely-related bacterial species to inhibit TLR4 signaling in vitro followed by comparative genomics to identify genes common to inhibitory species but without homologs in non-inhibitory species. The function of candidates will be evaluated by expressing them in E.coli Nissle 1917 (EcN) and mutating them in Pd. To evaluate whether TLR4 inhibition is required for chemoprevention by Pd we will compare the ability of Pd and Pd lacking anti-TLR4 function to suppress colon tumorigenesis with EcN and EcN expressing Pd anti-TLR4 genes. Host target proteins will be identified by co-immunoprecipitation with the recombinant tagged Pd protein. Finally, the influence of Pd on colonic immune cells will be evaluated by comparing specific cancer and microbiota relevant immune cell populations between Pd fed and control mice over time. Understanding Pd’s mechanism of action and identifying its active and target molecules will pave the way for this bacterium, or its active component, to be utilized in the prevention of CRC. Our long-term goal is to identify bacteria with chemopreventive properties and to understand their mechanisms of action. Importantly, Pd appears to be compatible with other microbial therapies and does not disrupt the resident microbiota. These studies are highly worthwhile because although a few agents show promise as chemopreventive agents in pre-clinical studies, aspirin (a drug developed over 100 years ago) remains the only agent for which we have compelling evidence of efficacy and its detrimental effects may outweigh those benefits. These studies directly address the mission of the NCI to develop the knowledge base that will lessen the burden of cancer in the United States and around the world.