Molecular Biotechnology

, Volume 28, Issue 2, pp 105–112 | Cite as


Expression of a functional single-chain Fv antibody on the surface of Streptococcus gordonii

  • Barbara Giomarelli
  • Tiziana Maggi
  • Justine Younson
  • Charles Kelly
  • Gianni Pozzi


Gram-positive bacterium Streptococcus gordonii, a human oral commensal, was engineered to display a single-chain Fv (scFv) antibody fragment at the cell surface. The previously developed host-vector system allowed expression of the Guy’s 13 scFv as a fusion with the streptococcal surface protein M6. Surface expression of the 515-amino acid M6/scFv fusion protein was confirmed by Western blot analysis on cellular fractions and flow cytometric analysis. Guy’s 13 scFv was derived from the Guy’s 13 monoclonal antibody, which was raised against streptococcal antigen I/II (SA I/II), the major adhesin of the caries-producing bacterium Streptococcus mutans. Surface plasmon resonance was used to test binding of scFv-expressing S. gordonii to SA I/II. Whole cells of recombinant S. gordonii were found to specifically bind to immobilised SA I/II and binding was inhibited by fluid-phase SA I/II in a dose-dependent manner. Production of a functional scFv in S. gordonii is the first step towards the development of genetically engineered commensal bacteria that, by colonizing mucosal surfaces, may provide the host with sustained delivery of recombinant antibodies.

Index Entries

Engineered Gram-positive bacteria recombinant proteins surface display single-chain Fv antibody fragments surface plasmon resonance binding activity 


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  1. 1.
    Pozzi, G. and Wells, J. M. (1997) Gram-positive Bacteria. Vaccine Vehicles for Mucosal Immunization. Landes Bioscience, Austin, TX, USA.Google Scholar
  2. 2.
    Oggioni, M. R., Medaglini, D., Maggi, T. and Pozzi, G. (1999) Engineering the Gram-positive cell surface for construction of bacterial vaccine vectors. Methods 19(1), 163–173.PubMedCrossRefGoogle Scholar
  3. 3.
    Oggioni, M. R., Ciabattini, A., Cuppone A. M. and Pozzi, G. (2003) Bacillus spores for vaccine delivery. Vaccine 21(S2), S96–101.PubMedCrossRefGoogle Scholar
  4. 4.
    Oggioni, M. R. and Pozzi, G. (1996) A host-vector system for heterologous gene expression in Streptococcus gordonii. Gene 169(1), 85–90.PubMedCrossRefGoogle Scholar
  5. 5.
    Medaglini, D., Ciabattini, A., Spinosa, M. R., et al. (2001) Immunization with recombinant Streptococcus gordonii expressing tetanus toxin fragment C confers protection from lethal challenge in mice. Vaccine 19(15–16), 1931–1939.PubMedCrossRefGoogle Scholar
  6. 6.
    Beninati, C., Oggioni, M. R., Boccanera, M., et al. (2000) Therapy of mucosal candidiasis by expression of an anti-idiotype in human commensal bacteria. Nat. Biotechnol. 18(10), 1060–1064.PubMedCrossRefGoogle Scholar
  7. 7.
    Giomarelli, B., Provvedi, R., Meacci, F., et al. (2002) The microbicide cyanovirin-N expressed on the surface of commensal bacterium Streptococcus gordonii captures HIV-1. AIDS 16(10), 1351–1356.PubMedCrossRefGoogle Scholar
  8. 8.
    Gunneriusson, E., Samuelson, P., Uhlen, M., Nygren, P. A. and Stahl, S. (1996) Surface display of a functional single-chain Fv antibody on staphylococci. J. Bacteriol. 178(5), 1341–1346.PubMedGoogle Scholar
  9. 9.
    Stahl, S. and Uhlen, M. (1997) Bacterial surface display: trends and progress. Trends Biotechnol. 15(5), 185–192.PubMedCrossRefGoogle Scholar
  10. 10.
    Oggioni, M. R., Beninati, C., Boccanera, M., et al. (2001) Recombinant Streptococcus gordonii for mucosal delivery of a scFv microbicidal antibody. Int. Rev. Immunol. 20(2), 267–269.Google Scholar
  11. 11.
    Kruger, C., Hu, Y., Pan, Q., et al. (2002) In situ delivery of passive immunity by lactobacilli producing single-chain antibodies. Nat. Biotechnol. 20(7), 702–706.PubMedCrossRefGoogle Scholar
  12. 12.
    Samuelson, P., Gunneriusson, E., Nygren, P. A. and Stahl, S. (2002) Display of proteins on bacteria. J. Biotechnol. 96(2), 129–154.PubMedCrossRefGoogle Scholar
  13. 13.
    Pozzi, G., Contorni, M., Oggioni, M. R., et al. (1992) Delivery and expression of a heterologous antigen on the surface of streptococci. Infect. Immun. 60(5), 1902–1907.PubMedGoogle Scholar
  14. 14.
    Pozzi, G., M. R. Oggioni, and Medaglini, D. (1997) Recombinant Streptococcus gordonii as live vehicle for vaccine antigens, in Gram-positive Bacteria. Vaccine Vehicles for Mucosal Immunization. Pozzi, G. and Wells, J. M., eds., Springer-Verlag, Berlin, Germany and Landes Bioscience, Georgetown, TX, pp. 35–60.Google Scholar
  15. 15.
    Medaglini, D., Pozzi, G., King, T. P. and Fischetti, V. A. (1995) Mucosal and systemic immune responses to a recombinant protein expressed on the surface of the oral commensal bacterium Streptococcus gordonii after oral colonization. Proc. Natl. Acad. Sci. USA 92(15), 6868–6872.PubMedCrossRefGoogle Scholar
  16. 16.
    Medaglini, D., Rush, C. M., Sestini, P. and Pozzi, G. (1997) Commensal bacteria as vehicles for mucosal vaccines against sexually transmitted diseases: vaginal colonization with recombinant streptococci includes local and systemic antibodies in mice. Vaccine 15(12–13), 1330–1337.PubMedCrossRefGoogle Scholar
  17. 17.
    Oggioni, M. R., Manganelli, R., Contorni, M., Tommasino, M. and Pozzi, G. (1995) Immunization of mice by oral colonization with live recombinant commensal streptococci. Vaccine 13(8), 775–779.PubMedCrossRefGoogle Scholar
  18. 18.
    Lehner, T., Caldwell, J. and Smith, R. (1985) Local passive immunization by monoclonal antibodies against streptococcal antigen I/II in the prevention of dental caries. Infect. Immun. 50(3), 796–799.PubMedGoogle Scholar
  19. 19.
    Ma, J. K., Hunjan, M., Smith, R. and Lehner, T. (1989) Specificity of monoclonal antibodies in local passive immunization against Streptococcus mutans. Clin. Exp. Immunol. 77(3), 331–337.PubMedGoogle Scholar
  20. 20.
    Ma, J. K., Hiatt, A., Hein, M., et al. (1995) Generation and assembly of secretory antibodies in plants. Science 268(5211), 716–719.PubMedCrossRefGoogle Scholar
  21. 21.
    Pozzi, G., Musmanno, R. A., Renzoni, E. A., Oggioni, M. R. and Cusi, M. G. (1988) Host-vector system for integration of recombinant DNA into chromosomes of transformable and nontransformable streptococci. J. Bacteriol. 170(4), 1969–1972.PubMedGoogle Scholar
  22. 22.
    Ricci, S., Medaglini, D., Rush, C. M., et al. (2000) Immunogenicity of the B monomer of the Escherichia coli heat-labile toxin expressed on the surface of Streptococcus gordonii. Infect. Immun. 68(2), 760–766.PubMedCrossRefGoogle Scholar
  23. 23.
    Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227(259), 680–685.PubMedCrossRefGoogle Scholar
  24. 24.
    Towbin, H., Staehelin, T. and Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76(9), 4350–4354.PubMedCrossRefGoogle Scholar
  25. 25.
    Ma, J. K. C., Kelly, C., Munro, G. H., Whiley, R. A. and Lehner, T. (1991) Conservation of the gene encoding streptococcal antigen I/II in oral streptococci. Infect. Immun. 59(8), 2686–2694.PubMedGoogle Scholar
  26. 26.
    Kelly, C., Evans, P., Bergmeier, L. A., et al. (1989) Sequence analysis of the cloned streptococcal cell surface antigen I/II. FEBS Lett. 258(1), 127–132.PubMedCrossRefGoogle Scholar
  27. 27.
    Kelly, C. G., Todryk, S., Kendal, H. L., Munro, G. H. and Lehner, T. (1995) T-cell, adhesion, and B-cell epitopes of the cell surface Streptococcus mutans protein antigen I/II. Infect. Immun. 63(9), 3649–3658.PubMedGoogle Scholar
  28. 28.
    Kelly, C. G., Younson, J. S., Hikmat, B. Y., et al. (1999) A synthetic peptide adhesion epitope as a novel antimicrobial agent. Nat. Biotechnol. 17(1), 42–47.PubMedCrossRefGoogle Scholar
  29. 29.
    Ma, J. K. C., Hikmat, B. Y., Wycoff, K., et al. (1998) Characterization of a recombinant plant monoclonal secretory antibody and preventive immunotherapy in humans. Nat. Medicine. 4(5), 601–606.CrossRefGoogle Scholar
  30. 30.
    Holmes, S. D., May, K., Johansson, V., Markey, F. and Critchley, I. A. (1997) Studies on the interaction of Staphylococcus aureus and Staphylococcus epidermis with fibronectin using surface plasmon resonance (BIAcore). J. Immunol. Methods. 28, 77–84.Google Scholar
  31. 31.
    Hirmo, S., Artursson, E., Puu, G., Wadstrom, T. and Nilsson, B. (1998) Characterization of Helicobacter pylori interactions with sialylglycoconjugates using a resonant mirror biosensor. Analyt. Biochem. 257, 63–66.PubMedCrossRefGoogle Scholar
  32. 32.
    Hirmo. S., Artursson, E., Puu, G., Wadstrom, T. and Nilsson, B. (1999) Helicobacter pylori interactions with human gastric mucin studied with a resonant mirror biosensor. J. Microbiol. Methods. 37, 177–182.PubMedCrossRefGoogle Scholar
  33. 33.
    Quinn, J. G., O’Neill, S., Doyle, A., et al. (2000) Development and application of surface plasmon resonance-based biosensors for the detection of cell-ligand interactions. Analyt. Biochem. 281, 135–143.PubMedCrossRefGoogle Scholar
  34. 34.
    Wang, Y., Kelly, C. G, Karttunen, J. T., et al. (2001) CD40 is a cellular receptor mediating mycobacterial heat shock protein 70 stimulation of CC-chemokines. Immunity. 15(6), 971–983.PubMedCrossRefGoogle Scholar
  35. 35.
    Medaglini, D., Ricci, S., Maggi, T., et al. (1997) Recombinant Gram-positive bacteria as vehicles of vaccine antigens. Biotechnol. Ann. Rev. (3), 297–312.CrossRefGoogle Scholar
  36. 36.
    Pozzi, G., M. R. Oggioni, R. Manganelli, and Plevani, P. (1990) Genetic exchange and heterologous gene expression in streptococci: the bacterial chromosome as vector of recombinant DNA molecules, in Recombinant DNA Technologies in Industry and Agriculture: State-of-Art in Italy. Amaldi, F., De Felice, M., Di Fonzo, N., Merlin, P., Polsinelli, A. and Viotti A. eds., Amity, Milano, pp. 45–48.Google Scholar
  37. 37.
    Navarre, W.W. and Schneewind, O. (1999) Surface proteins of Gram-positive bacteria and mechanisms of their targeting to the cell wall envelope. Microbiol. Mol. Biol. Rev. 63(1), 174–229.PubMedGoogle Scholar

Copyright information

© Humana Press Inc 2004

Authors and Affiliations

  • Barbara Giomarelli
    • 1
  • Tiziana Maggi
    • 1
  • Justine Younson
    • 2
  • Charles Kelly
    • 2
  • Gianni Pozzi
    • 1
  1. 1.LAMMB Department of Molecular BiologyUniversity of SienaSienaItaly
  2. 2.Department of Oral ImmunologyGKT Dental Institute, King’s CollegeLondonUK

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