The Colorectal Cancer Associated Microbe Odoribacter Splanchnicus Produces Genotoxic Hydrogen Sulfide Via Cysteine Metabolism

The proinflammatory and genotoxic properties of hydrogen sulfide (H2S) have been implicated as an environmental trigger of colonic disease. Recent evidence links sulfidogenic bacteria capable of metabolizing organic sulfur substrates, like dietary sulfur amino acids and taurine conjugated bile acids, with increased colorectal cancer (CRC) and colitis risk as a consequence of a “western” type diet. Our previous work demonstrates that the sulfidogenic bacterium, Odoribacter splanchnicus, is a significant indicator of CRC in African Americans who are at higher risk of CRC incidence and death than other races and ethnicities in the United States. This observation has been corroborated by several recent studies that observed O. splanchnicus to be associated with CRC and colitis-induced CRC. While previous work has reported that O. splanchnicus may be sulfidogenic, its method of H2S production has yet to be characterized. Production of H2S by O. splanchnicus was confirmed in vitro using sulfide indole and motility media, and varying sulfur substrates. A predictive metabolic analysis of 74 O. splanchnicus metagenomes taken from publicly available CRC cohorts did not reveal complete pathways commonly associated with H2S production, however genes for a polar amino acid transporter were revealed. Therefore, a genomic analysis was performed revealing that O. splanchnicus harbors the sulfidogenic genes tryptophanase (tnaA) and cystathionine-β-synthase. The enzyme TnaA has been previously shown in Eschericia coli to produce indole or H2S from tryptophan or cysteine, respectively. However, the E. coli amino acid sequence shares only 44.85% identity with that from O. splanchnicus, thus heterologous protein expression of recombinant tnaA was performed to determine enzyme functionality. Enzyme kinetics and pH optimum were obtained using the spectrophotometric BiCl3 assay, which turns black in presence of H2S. Functional analysis of recombinant purified TnaA revealed bifunctionality of the enzyme with positive tryptophanase and cysteine desulfhydrase activity with differing substrates. Cysteine desulfhydrase activity resulted in consumption of L-cysteine with subsequent production of H2S and pyruvate. Thus, TnaA is a functional enzyme that warrants further evaluation as possibly relating to sulfidogenic O. splanchnicus being linked to CRC and colitis-associated CRC. Citation Format: Patricia G. Wolf, Vladimir Kolosslov, Zhichao Zhou, Lindsey Ly, Heidi Doden, Saravanan Devendran, Adam M. Breister, Luke Lucio, Paige Polak, Sarah Matatov, Karthik Anantharaman, Jason M. Ridlon, Rex Gaskins. The colorectal cancer associated microbe Odoribacter splanchnicus produces genotoxic hydrogen sulfide via cysteine metabolism [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3342.