Molecular screening of Mycobacterium vaccae for identification of novel anti-inflammatory compounds (IRM12P.651)

Abstract

Abstract Mycobacterium vaccae is a saprophytic, non-pathogenic bacterium. Administration of M. vaccae in activates regulatory T cells (Treg) and attenuates allergic responses. M. vaccae-induced activation of Treg may be initiated through interactions with sentinel immune cells, such as macrophages. We are interested in identifying the particular immunoregulatory molecules produced by M. vaccae that suppress inflammatory signaling in macrophages. Consequently, we developed a reiterative cell-based screen as an unbiased approach to search the M. vaccae chemical landscape for potential anti-inflammatory small molecules. M. vaccae was grown in 7H9 broth as a mixture of both smooth and rough variants.We prepared hexane, ethyl acetate, and aqueous extracts of live M. vaccae cultures; cell-based assays were used to guide successive steps of purification. For cell-based assays, 100 µg/ml of the extracts were incubated with freshly isolated murine peritoneal macrophages, and, after one hour, the cultures were stimulated with 1 µg/ml of lipopolysaccharide (LPS). Interleukin 6 (IL-6) concentrations in the supernatant 12h after stimulation were measured via ELISA as a determinant of inflammatory response. After 5 iterations of screening and evaluation of 80 fractions, we identified 3 fractions from the ethyl acetate extract with potent anti-inflammatory properties. From mass spectrometry, we expect the molecules to be lipid conjugates, and we are in preparation for a structural analysis.