Research Description:

Characterization of the Biofilm-Forming and Acid Tolerance Properties of the SM12 and SM13 strains of Streptococcus mutans

Streptococcus mutans, the principal etiological agent of human dental caries, thrives in multi-species biofilms on tooth surfaces, where it is subjected to a continuous assault by host defenses and to rapid and dramatic fluctuations in nutrient availability, carbohydrate source and pH. The organism has evolved multiple physiologic and genetic adaptations to optimize growth under these dynamic conditions, and it can grow and carry out glycolysis at low plaque pH values, gaining a selective advantage over less aciduric species when oral biofilms become acidified. Dissecting the pathways used to form stable biofilms and to tolerate environmental stresses is central to understanding the mechanisms of virulence of this organism.

The molecular chaperones GroEL and DnaK are central to the tolerance of environmental stresses by microorganisms. In S. mutans, groEL and dnaK are rapidly induced by acid shock, and elevated levels of DnaK expression are maintained under acidic conditions. Strains lacking DnaK or GroEL could not be isolated in S. mutans indicating that these proteins perform essential roles in cell metabolism. To distinguish the roles played by DnaK and GroEL in the physiology of S. mutans, we created a knocked-down strategy to lower the levels of GroEL and DnaK in this organism. For GroEL down-regulation, the native promoter was replaced by a polar kanamycin cassette followed by a non-native, weaker promoter (Km-PureI) generating strain SM13. For the dnaK knock-down strain (SM12), the Km-PureI cassette was introduced into the intergenic region of the dnaK operon immediately upstream of the dnaK gene. Western blot analysis confirmed that there were dramatic reductions in the levels of DnaK and GroEL in strains SM12 and SM13, respectively.

The goal of this proposal is to conduct a detailed characterization of the biofilm-forming and acid tolerance properties of the SM12 and SM13 strains with the use of physiologic and biochemical assays.