Bactericidal effects of a fusion protein of llama heavy-chain antibodies coupled to glucose oxidase on oral bacteria.
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.
Department of Periodontology, Section Oral Microbiology, Academic Centre for Dentistry, van der Boechorststraat 7, 1081 BT Amsterdam, The Netherlands. a.szynol.omb.acta@med.vu.nl
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A
Szynol
J J
JJ
de Soet
E
E
Sieben-van Tuyl
J W
JW
Bos
L G
LG
Frenken
D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D000907:Antibodies, Bacterial / Q000494:pharmacology*; D001419:Bacteria / Q000187:drug effects*; D002161:Camelids, New World / Q000502:physiology*; D015723:Gene Library; D005949:Glucose Oxidase / Q000494:pharmacology*; D000922:Immunotoxins / Q000633:toxicity; D009055:Mouth / Q000382:microbiology*; D011485:Protein Binding; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000494:pharmacology; D013295:Streptococcus mutans / Q000201:enzymology / Q000235:genetics
D000900:Anti-Bacterial Agents; D000907:Antibodies, Bacterial; D000922:Immunotoxins; D016271:Proto-Oncogene Proteins c-myc; D011994:Recombinant Proteins; D005949:Glucose Oxidase