Binding mechanism of Streptococcus mutans glucan-binding domain of GbpC to saliva proteins

Objectives: Streptococcus mutans produces glucan-binding proteins (Gbps), which are thought to promote biofilm formation on tooth surfaces. Among them, GbpC encoded by the gbpC is responsible for dextran-dependent aggregation, thus contributing to biofilm formation. Previously, we used bioinformatics and identified the amino acid sequence DPTKTIF in gbpC as the glucan-binding domain. Salivary proteins interact with S. mutans during cell aggregation and contribute to mediate bacterial adherence to tooth surfaces. For the present study, we speculated that GbpC binds to salivary proteins for cell aggregation, and focused on the relationship between saliva proteins and the sequence DPTKTIF to clarify the mechanism of biofilm formation.
Methods: Recombinant GbpC (rGbpC) was prepared using the GST protein expression vector pET42a (+). Rabbit anti-DPTKTIF serum (anti-DPT) was generated using a commercially available peptide of DPTKTIF. First, we examined the interaction of rGbpC and lysozyme using GST pull-down assays. rGbpC was applied to a Glutathione Sepharose® 4B column followed by anti-DPT and lysozyme, then eluted by glutathione. The samples were separated by SDS-PAGE and subjected to Coomassie brilliant blue (CBB) staining and western blotting analysis using rabbit anti-lysozyme. Next, binding analysis was performed using ELISA plates to examine binding between rGbpC and lysozyme.
Results: In GST pull-down assays, CBB staining and western blotting analysis revealed the bands with the molecular size of lysozyme in samples with anti-DPT added to the column, while no such bands were seen without anti-DPT. Also, binding assay results showed that the absorbance of anti-DPT after reaction with lysozyme was significantly higher as compared to that of rGbpC after reaction with lysozyme.
Conclusions: Our results suggest that lysozyme binds to anti-DPT and may possess a molecular binding domain with high homology to the glucan-binding domain in gbpC.
This study was supported by a Grant-in-Aid for Young Scientists (B) 15K2059107 from Japan Society of Promotion of Science.