Binding Capacity and Antimicrobial Activity of a Fusion Protein of Llama Heavy-chain Antibodies against S. mutans Coupled to Glucose Oxydase

Objectives: Dental caries is a multifactorial disease, in which cariogenic bacteria such as Streptococcus mutans play an essential role. In order to prevent dental caries by controlling the amount of S. mutans, an antimicrobial agent was designed. This agent is a fusion protein GOx-V_HH that consists of a variable domain of the llama heavy chain antibody (V_HH) and a glucose oxidase (GOx). The immunoglobulin moiety of GOx-V_HH is in charge of the recognition antigens on S. mutans and GOx is responsible for the production of hydrogen peroxide in the presence of glucose and oxygen. The fusion protein has been produced in Saccharomyces cerevisiae. The purpose of this study was to examine the binding specificity and the killing activity of the fusion protein. Methods: Fifteen different oral bacteria were tested for the binding specificity of the fusion protein and GOx by modified ELISA. Antimicrobial activity was tested by coating the bacteria with GOx-V_HH, GOx or V_HH, followed by an incubation with a killing mixture, containing peroxidase, glucose and potassium iodide, and then the determination of the minimum inhibitory concentration (MIC) by measuring CFU/ml. Results: Among tested bacteria, only S. mutans and Campylobacter concisus were able to bind GOx-V_HH. The interaction of C. concisus with the fusion protein was proved to be caused by cross reaction of the GOx with these bacteria. The MIC for GOx-V_HH, expressed as activity units of GOx (Sigma, G9010 was the standard), was 0.6 U/ml for S. gordonii and 0.02 U/ml for S. mutans. Moreover GOx-V_HH did not affect the survival of the third tested bacterium, S. sanguinis, indicating that the MIC was higher than 2.4 U/ml. Conclusions: The results confirm the biological activity of the GOx-V_HH. Moreover, we suggest that the fusion protein may be used to specifically recognise and subsequently kill the target bacteria.