Extracellular DNA Influences Matrix Protein Release in Streptococcus mutans Biofilms

Objectives: Biofilm matrix is composed of various amounts of eDNA, proteins, polysaccharides and membrane vesicles. This study was designed to further analyze the roles of membrane vesicles (MVs) and the effects of eDNA-protein interactions on in S. mutans biofilm formation.
Methods: MVs were prepared from overnight broth cultures of S. mutans, and their effect on biofilm formation was assessed using 96-well plate model with or without inclusion of human whole saliva. For eDNA-protein interactions, S. mutans biofilms were resuspended in buffer of pH 5.0 and 7.5 with or without DNase I (100 ug/mL) or Benzonase (25 U/mL). Following centrifugation, the proteins in the supernatant were separated by SDS-PAGE and stained using silver staining kit.
Results: When included in growth medium, MVs significantly increased S. mutans biofilm formation, and the most significant increases were measured when grown sucrose-containing medium, and such effects were shown to be independent of human whole saliva. Substantial amount of proteins was detected when the biofilms were resuspended in buffer of pH 7.5, but only few proteins were seen at pH 5. When cells first resuspended at pH 5 were resuspended at pH 7.5, there was a great increase in proteins, but no such effects were seen with cells initially resuspended at pH 7.5. However, following DNase I treatment, substantial amount of proteins was detected at both pH 5.0 and 7.5. Similar results were obtained with biofilms treated with DNase I during growth, although major differences existed between the treated and untreated samples in protein content as shown in SDS-PAGE.
Conclusions: These results suggest that MVs significantly influence S. mutans biofilm formation independent of human saliva and that eDNA and proteins in biofilms interact, although the nature of the proteins interactive with eDNA and how such interactions influence biofilm formation await further investigation.