Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Biological containment of genetically modified Lactococcus lactis for intestinal delivery of human interleukin 10


Genetically modified Lactococcus lactis secreting interleukin 10 provides a therapeutic approach for inflammatory bowel disease. However, the release of such genetically modified organisms through clinical use raises safety concerns. In an effort to address this problem, we replaced the thymidylate synthase gene thyA of L. lactis with a synthetic human IL10 gene. This thyAhIL10+ L. lactis strain produced human IL-10 (hIL-10), and when deprived of thymidine or thymine, its viability dropped by several orders of magnitude, essentially preventing its accumulation in the environment. The biological containment system and the bacterium's capacity to secrete hIL-10 were validated in vivo in pigs. Our approach is a promising one for transgene containment because, in the unlikely event that the engineered L. lactis strain acquired an intact thyA gene from a donor such as L. lactis subsp. cremoris, the transgene would be eliminated from the genome.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: Exchange between thyA and hIL10 genes.
Figure 2: hIL-10 production by Thy11–16.
Figure 3: Growth and survival of Thy12 and MG1363.

Accession codes




  1. Steidler, L. et al. Mucosal delivery of murine interleukin-2 (IL-2) and IL-6 by recombinant strains of Lactococcus lactis coexpressing antigen and cytokine. Infect. Immun. 66, 3183–3189 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. Steidler, L. et al. Treatment of murine colitis by Lactococcus lactis secreting interleukin-10. Science 289, 1352–1355 (2000).

    Article  CAS  Google Scholar 

  3. Beninati, C. et al. Therapy of mucosal candidiasis by expression of an anti-idiotype in human commensal bacteria. Nat. Biotechnol. 18, 1060–1064 (2000).

    Article  CAS  Google Scholar 

  4. Kruger, C. et al. In situ delivery of passive immunity by lactobacilli producing single- chain antibodies. Nat. Biotechnol. 20, 702–706 (2002).

    Article  Google Scholar 

  5. van Deventer, S.J., Elson, C.O. & Fedorak, R.N. Multiple doses of intravenous interleukin 10 in steroid-refractory Crohn's disease. Crohn's Disease Study Group. Gastroenterology 113, 383–389 (1997).

    Article  CAS  Google Scholar 

  6. Fedorak, R.N. et al. Recombinant human interleukin 10 in the treatment of patients with mild to moderately active Crohn's disease. The Interleukin 10 Inflammatory Bowel Disease Cooperative Study Group. Gastroenterology 119, 1473–1482 (2000).

    Article  CAS  Google Scholar 

  7. Schreiber, S. et al. Safety and efficacy of recombinant human interleukin 10 in chronic active Crohn's disease. Crohn's Disease IL-10 Cooperative Study Group. Gastroenterology 119, 1461–1472 (2000).

    Article  CAS  Google Scholar 

  8. Tilg, H., Ulmer, H., Kaser, A. & Weiss, G. Role of IL-10 for induction of anemia during inflammation. J. Immunol. 169, 2204–2209 (2002).

    Article  CAS  Google Scholar 

  9. Schotte, L., Steidler, L., Vandekerckhove, J. & Remaut, E. Secretion of biologically active murine interleukin-10 by Lactococcus lactis. Enzyme Microb. Technol. 27, 761–765 (2000).

    Article  CAS  Google Scholar 

  10. Masood, R. et al. Interleukin-10 is an autocrine growth factor for acquired immuno-deficiency syndrome-related B-cell lymphoma. Blood 85, 3423–3430 (1995).

    CAS  PubMed  Google Scholar 

  11. Ross, P., O'Gara, F. & Condon, S. Cloning and characterization of the thymidylate synthase gene from Lactococcus lactis subsp. lactis. Appl. Environ. Microbiol. 56, 2156–2163 (1990).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Biswas, I., Gruss, A., Ehrlich, S.D. & Maguin, E. High-efficiency gene inactivation and replacement system for gram-positive bacteria. J. Bacteriol. 175, 3628–3635 (1993).

    Article  CAS  PubMed Central  Google Scholar 

  13. Bolotin, A. et al. The complete genome sequence of the lactic acid bacterium Lactococcus lactis ssp. lactis IL1403. Genome Res. 11, 731–753 (2001).

    Article  CAS  PubMed Central  Google Scholar 

  14. Law, J. et al. A system to generate chromosomal mutations in Lactococcus lactis which allows fast analysis of targeted genes. J. Bacteriol. 177, 7011–7018 (1995).

    Article  CAS  PubMed Central  Google Scholar 

  15. Waterfield, N.R., Le Page, R.W., Wilson, P.W. & Wells, J.M. The isolation of lactococcal promoters and their use in investigating bacterial luciferase synthesis in Lactococcus lactis. Gene 165, 9–15 (1995).

    Article  CAS  Google Scholar 

  16. Stemmer, W.P., Crameri, A., Ha, K.D., Brennan, T.M. & Heyneker, H.L. Single-step assembly of a gene and entire plasmid from large numbers of oligodeoxyribonucleotides. Gene 164, 49–53 (1995).

    Article  CAS  Google Scholar 

  17. van Asseldonk, M. et al. Cloning of usp45, a gene encoding a secreted protein from Lactococcus lactis subsp. lactis MG1363. Gene 95, 155–160 (1990).

    Article  CAS  Google Scholar 

  18. Barthelemy, I. et al. Production and secretion of human interleukin 6 into the periplasm of Escherichia coli: efficient processing of N-terminal variants of hIL6 by the E. coli signal peptidase. J. Biotechnol. 27, 307–316 (1993).

    Article  CAS  Google Scholar 

  19. Thompson-Snipes, L. et al. Interleukin 10: a novel stimulatory factor for mast cells and their progenitors. J. Exp. Med. 173, 507–510 (1991).

    Article  CAS  Google Scholar 

  20. Giard, J.C., Verneuil, N., Auffray, Y. & Hartke, A. Characterization of genes homologous to the general stress-inducible gene gls24 in Enterococcus faecalis and Lactococcus lactis. FEMS Microbiol. Lett. 206, 235–239 (2002).

    Article  CAS  Google Scholar 

  21. Decuypere, J.A., Vervaeke, I.J., Henderickx, H.K. & Dierick, N.A. Gastro-intestinal cannulation in pigs: a simple technique allowing multiple replacements. J. Anim. Sci. 45, 463–468 (1977).

    Article  CAS  Google Scholar 

  22. Molin, S. et al. Suicidal genetic elements and their use in biological containment of bacteria. Annu. Rev. Microbiol. 47, 139–166 (1993).

    Article  CAS  Google Scholar 

  23. Molina, L., Ramos, C., Ronchel, M.C., Molin, S. & Ramos, J.L. Construction of an efficient biologically contained Pseudomonas putida strain and its survival in outdoor assays. Appl. Environ. Microbiol. 64, 2072–2078 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Knudsen, S. et al. Development and testing of improved suicide functions for biological containment of bacteria. Appl. Environ. Microbiol. 61, 985–991 (1995).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Schweder, T., Hofmann, K. & Hecker, M. Escherichia coli K12 relA strains as safe hosts for expression of recombinant DNA. Appl. Microbiol. Biotechnol 42, 718–723 (1995).

    Article  CAS  Google Scholar 

  26. Torres, B., Jaenecke, S., Timmis, K.N., Garcia, J.L. & Diaz, E. A gene containment strategy based on a restriction-modification system. Environ. Microbiol. 2, 555–563 (2000).

    Article  CAS  Google Scholar 

  27. Diaz, E., Munthali, M., de Lorenzo, V. & Timmis, K.N. Universal barrier to lateral spread of specific genes among microorganisms. Mol. Microbiol. 13, 855–861 (1994).

    Article  CAS  Google Scholar 

  28. Bron, P.A. et al. Use of the alr gene as a food-grade selection marker in lactic acid bacteria. Appl. Environ. Microbiol. 68, 5663–5670 (2002).

    Article  CAS  PubMed Central  Google Scholar 

  29. MacCormick, C.A., Griffin, H.G. & Gasson, M.J. Construction of a food-grade host/vector system for Lactococcus lactis based on the lactose operon. FEMS Microbiol. Lett. 127, 105–109 (1995).

    Article  CAS  Google Scholar 

  30. Sorensen, K.I., Larsen, R., Kibenich, A., Junge, M.P. & Johansen, E. A food-grade cloning system for industrial strains of Lactococcus lactis. Appl. Environ. Microbiol. 66, 1253–1258 (2000).

    Article  CAS  PubMed Central  Google Scholar 

  31. Ahmad, S.I., Kirk, S.H. & Eisenstark, A. Thymine metabolism and thymineless death in prokaryotes and eukaryotes. Ann. Rev. Microbiol. 52, 591–625 (1998).

    Article  CAS  Google Scholar 

  32. Cohen, S.S. & Barner, H.D. Studies on unbalanced growth in Escherichia coli. Proc. Natl. Acad. Sci. USA 40, 885–893 (1954).

    Article  CAS  Google Scholar 

  33. Fu, X. & Xu, J.G. Development of a chromosome-plasmid balanced lethal system for Lactobacillus acidophilus with thyA gene as selective marker. Microbiol. Immunol. 44, 551–556 (2000).

    Article  CAS  Google Scholar 

  34. Pedersen, M.B., Jensen, P.R., Janzen, T. & Nilsson, D. Bacteriophage resistance of a ΔthyA mutant of Lactococcus lactis blocked in DNA replication. Appl. Environ. Microbiol. 68, 3010–3023 (2002).

    Article  CAS  PubMed Central  Google Scholar 

  35. Bringel, F., Van Alstine, G.L. & Scott, J.R. A host factor absent from Lactococcus lactis subspecies lactis MG1363 is required for conjugative transposition. Mol. Microbiol. 5, 2983–2993 (1991).

    Article  CAS  Google Scholar 

  36. Moon, H.W., Sorensen, D.K. & Sautter, J.H. Escherichia coli infection of the ligated intestinal loop of the newborn pig. Am. J. Vet. Res. 27, 11317–11325 (1966).

    Google Scholar 

Download references


The authors thank Inge Bruggeman, Hilde Devlies Koen Van Laer and Elena Yancheva for expert technical assistance, Karen Madsen, Claude Cuvelier and Frans Van Roy for critically reviewing the manuscript, and Jan Kok and Emmanuelle Maguin for providing genetic tools. This study was financed by the Vlaams Interuniversitair instituut voor Biotechnologie, Ghent University (GOA project nos. 12050700 and 12051501) and the commission of the European Communities, specific research, technological development and training activities program “Quality of Life and Management of Living Resources,” QLK1-2000-00146 “Probiotic strains with designed health properties.”

Author information

Authors and Affiliations


Corresponding author

Correspondence to Lothar Steidler.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Steidler, L., Neirynck, S., Huyghebaert, N. et al. Biological containment of genetically modified Lactococcus lactis for intestinal delivery of human interleukin 10. Nat Biotechnol 21, 785–789 (2003).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

This article is cited by


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing