Submitted to: Journal of Applied Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/30/2004
Publication Date: 3/1/2005
Citation: Farrar, M.D., Whitehead, T.R., Lan, J., Dilger, P., Thorpe, R., Holland, K.T., Carding, S.R. 2005. Engineering of the gut commensal bacterium Bacteroides ovatus to produce and secrete biologically active murine interleukin-2 in response to xylan. Journal of Applied Microbiology. 98:1191-1197. Interpretive Summary: Chronic disorders of the human gastrointestinal (GI) tract, for example inflammatory bowel disease (IBD) which includes the disorders Crohn's disease and ulcerative colitis, affect a significant proportion of the population in developed countries such as the United States. Present therapies are not curative and may cause adverse side effects during treatment. Therefore, there is a need for more targeted and controlled forms of immunotherapy. Previous work has indicated that commensal bacteria that normally inhabit the GI tract might prove useful for treating certain forms of bowel disease. Therefore, a human isolate of the commensal bacterium, Bacteroides ovatus, was engineered as a probiotic for production and secretion of murein Interleukin-2, a compound important for the treatment of IBD. The production and secretion of a biologically active mammalian protein by a member of the GI microflora could lead to the development of new long-term immunotherapies for inflammatory gut diseases.
Technical Abstract: The aim of this work was to engineer a gut commensal bacterium, Bacteroides ovatus, to produce and secrete a biologically active cytokine in a regulated manner as a basis for novel immunotherapies for chronic gut disorders. B. ovatus was engineered to produce murine interleukin-2 (MuIL2) intracellularly in response to xylan in culture media by inserting the MuIL2 gene into the xylanase operon of the organism. A second strain was engineered to secrete MuIL2 by adding the B. fragilis enterotoxin secretion signal sequence to the protein. The recombinant strains produced MuIL2 only in the presence of xylan as determined by enzyme-linked immunosorbent assay of cell lysates and culture supernatants. The IL2-dependent cell line CTLL-2 was used to demonstrate that MuIL2 produced by both B. ovatus strains was biologically active. This activity could be blocked by an anti-IL2 neutralizing antibody. The xylan-inducible nature of this system was demonstrated by RT-PCR. B. ovatus was successfully engineered to produce and secrete biologically active MuIL2 in a xylan-inducible manner. The production and secretion of a biologically active mammalian protein by a member of the gut microflora could lead to the development of new long-term immunotherapies for inflammatory gut diseases.