D-071. Role of Glucan-Binding Proteins in Biofilm Development by Streptococcus mutans

B. Olson, J. Banas, D. Drake;
Coll. of Dent., Univ. of Iowa, Iowa City, IA.

The cariogenic pathogen Streptococcus mutans (SM) produces four cell-surface proteins that are called glucan-binding proteins (Gbps). Evidence to date suggests that these proteins play major roles in the development of biofilms that form on teeth in the presence of sucrose. However, the precise contributions of each Gbp are far from being completely understood. The purpose of this study was to determine the effect of the loss of specific Gbps in the development of biofilms using a continuous culture/biofilm model system. Mutants in gbpA, gbpC, or gbpD were engineered using a strategy of allelic replacement with unique antibiotic resistance cassettes. Continuous cultures of SM wild-type and Gbp mutants were grown in BTRG media in a chemostat under equilibrium conditions at pH 7.0 and a MDT of 7.9 hr. Biofilms were formed in a modified Robbins device connected to the chemostat. The biofilms were developed for 24 hours, 48 hours, and 72 hours with and without sucrose pulsing (three times every 24 hours with 5% sucrose for 10 minutes). Biofilm discs were processed for enumeration using an Autoplate 4000 spiral-plating system and growth on appropriate selective media. WT, A-, and D- strains maintained equal numbers in planktonic phase while the C- mutant stabilized at 3 logs less than the others. Representation of the strains in non-sucrose biofilms mirrored their distribution in continuous culture. In sucrose biofilms the WT increased the most over non-sucrose levels, approximately one log more than the increase for the D- strain and about two logs more than for the A- strain. The C- strain also increased, more so than the A- strain but slightly less than the D- strain. The reduced levels of the C- strain may be attributable to a reduced growth rate. Loss of any of the three Gbps resulted in significant decreases in the ability of these organisms to form biofilms upon pulsing with sucrose, but the greatest magnitude occurred with the loss of GbpA perhaps indicating that GbpA possesses the strongest affinity for glucan. These data suggest that Gbps are important in biofilm development under conditions that simulate sucrose exposure in the oral cavity.