Engineering of the gut commensal bacterium Bacteroides ovatus to produce and secrete biologically active murine interleukin-2 in response to xylan M.D. Farrar 1 , T.R. Whitehead 2 , J. Lan 1 , P. Dilger 3 , R. Thorpe 3 , K.T. Holland 1 and S.R. Carding 1 1 School of Biochemistry and Microbiology, University of Leeds, Leeds, UK, 2 Fermentation Biotechnology Research, National Centre for Agricultural Utilization Research, Peoria, IL, USA, and 3 Division of Immunobiology, National Institute for Biological Standards and Control, Potters Bar, UK 2004/0920: received 18 June 2004, revised 30 November 2004 and accepted 30 November 2004 ABSTRACT M.D. FARRAR, T.R. WHITEHEAD, J. LAN, P. DILGER, R. THORPE, K.T. HOLLAND AND S.R. CARDING. 2005. Aims: 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. Methods and Results: Bacteroides 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 Bacteroides fragilis enterotoxin secretion signal sequence to the protein. The recombinant strains produced MuIL2 only in the presence of xylan as determined by ELISA 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. Conclusions: Bacteroides ovatus was successfully engineered to produce and secrete biologically active MuIL2 in a xylan-inducible manner. Significance and Impact of the Study: 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. Keywords: Bacteroides ovatus, biologically active, gut commensal, immunotherapy, interleukin-2 production, xylan-inducible. INTRODUCTION The mucosal immune response can be influenced by manipulation of the normal resident bacterial flora. This flora possesses a large variety of biological and immuno- modulatory properties that can, directly or indirectly, influence the development and function of the mucosal immune system (Savage 1999). Chronic disorders of the gut, 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 (Steidler et al. 2001). Animal models of mucosal inflammation have been used to try and determine the immune mechanisms involved in the pathogenesis of these diseases (Blumberg et al. 1999; Arseneau et al. 2000). Chronic colitis develops spontaneously in interleukin (IL) 2 )/) and IL10 )/) mice (Ku ¨hn et al. 1993; Sadlack et al. 1993), both of which are used as models of IBD. Many other mouse models of IBD have also been described, most of which have targeted deletions of immune response genes (Kulkarni et al. 1993; Mombaerts et al. 1993). Current treatment of IBD is restricted to anti-inflammatory and immunosuppressive drug therapies including recombinant Correspondence to: S.R. Carding, School of Biochemistry and Microbiology, University of Leeds, Leeds, LS2 9JT, UK (e-mail: s.r.carding@leeds.ac.uk). ª 2005 The Society for Applied Microbiology Journal of Applied Microbiology 2005, 98, 1191–1197 doi:10.1111/j.1365-2672.2005.02565.x