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Bactericidal Effects of a Fusion Protein of Llama Heavy-Chain Antibodies Coupled to Glucose Oxidase on Oral Bacteria

Department of Periodontology, Section Oral Microbiology, Academic Centre for Dentistry, Amsterdam,¹ Biotechnology Group, Unilever Research Vlaardingen, Vlaardingen, The Netherlands²

Received 31 October 2003/ Returned for modification 22 January 2004/ Accepted 19 May 2004

Enzymes such as lactoperoxidase and glucose oxidase (GOx) are used as antimicrobial agents in oral care products. Their low specificities and substantiveness can be reduced by covalent coupling of antimicrobial molecules to antibodies. Variable domains (V_HH) derived from llama heavy-chain antibodies are particularly suited for such an approach. The antibodies are composed solely of heavy-chain dimers; therefore, production of active fusion proteins by using molecular biology-based techniques is less complicated than production by use of conventional antibodies. In this study, a fusion protein consisting of V_HH and GOx was constructed and expressed by Saccharomyces cerevisiae. A llama was immunized with Streptococcus mutans strain HG982. Subsequently, B lymphocytes were isolated and cDNA fragments encoding the V_HH fragments were obtained by reverse transcription-PCR. After construction of a V_HH library in Escherichia coli and screening of the library against mutans group streptococci and Streptococcus sanguinis strains, we found two V_HH fragments with high specificities for S. mutans strains. A GOx gene was linked to the two V_HH genes and cloned into S. cerevisiae yeasts. The yeasts expressed and secreted the recombinant proteins into the growth medium. The test of binding of fusion proteins to oral bacteria through their V_HH fragments showed that S. mutans had been specifically targeted by GOx-S120, one of the fusion protein constructs. A low concentration of the fusion protein was also able to selectively kill S. mutans within 20 min in the presence of lactoperoxidase and potassium iodide. These findings demonstrate that the fusion protein GOx-V_HH is potentially valuable in the selective killing of target bacteria such as S. mutans.

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