Structural and Functional Studies on the Glucosyltransferases of the Dental Caries Pathogen Streptococcus mutans

SUMMARY Dental caries is a polymicrobial disease that affects much of the human population worldwide, where the equilibrium of a cooperative eco-organization among commensal microbes shifts towards a dysbiotic framework with an overrepresentation of pathogenic microorganisms, especially by Streptococcus mutans (SM). The progression of this disease begins with bacterial attachment and a complex cascade of events that include the production of soluble (a-1,6-linked) and insoluble (a-1,3-linked) glucans by glucosyltransferases (Gtfs) from the GH70 hydrolase family. This class of enzymes utilizes dietary sucrose to produce various isomeric glucan polymers through two important steps: (i) first is the sucrase (invertase) activity, where cleavage of sucrose results in glucose and fructose; (ii) subsequently, the polymerization of glucose molecules forms extended a- glucans. SM’s three different Gtfs, GtfB, GtfC, and GtfD, have been biologically well characterized, and their synthesis of glucans is crucial for biofilm formation, bacterial colonization, and virulence. However, despite more than 30 years of research and numerous studies, there remains a fundamental knowledge gap on their enzymatic mechanism; specifically, how do they produce the soluble and insoluble glucans via distinct functional domains? The goal of this application is to determine the specific molecular mechanism(s) that drive SM’s Gtfs to produce various glucans, particularly how they synthesize soluble and insoluble glucans. Our structural studies show that the sucrase activity pocket is very tight and cannot accommodate an a-retaining double displacement reaction like the GH13 enzymes. Therefore, polymerization must take place at another distinct site in the nearby vicinity. We hypothesize that ‘The GH70 glucosyltransferases of SM adopt a novel enzymatic mechanism to produce the soluble a-1,6-linked and insoluble a-1,3-linked glucans.’ We will address our hypothesis through three specific aims (SAs) by (a) determining the structures of Glucosyltransferases (GtfB & GtfD) of SM (SA1), (b) elucidating the catalytic mechanism of SM’s glucosyltransferases (SA2), and (c) characterizing the role of (i) the sucrase site, (ii) the polymerization site, (iii) the glucan binding domains and (iv) their inhibitors on biofilms, colonization, and virulence potential through in vitro and in vivo models of dental caries (SA3). The results from this study will (1) determine the novel structures adopted by these Gtf enzymes, (2) assign specific roles to domains/regions, (3) identify key residues involved in the dual-step enzymatic action, (4) specify how each GtfB and GtfD polymerize glucans, (5) reveal the importance of the sucrase site, the polymerization site, and the glucan binding domains on disease outcomes, and (6) develop inhibitors that selectively target each Gtf’s enzymatic activity. These investigations would provide a mechanistic foundation for the 800+ GH70 hydrolases, as multiple biotechnology interests exist to engineer these glucansucrases to dispense varied sizes of dextran for chromatography, food preservation, and pharmaceuticals. The long-term objective of this study is to explore innovative strategies for specifically addressing the cariogenic dysbiosis mediated by SM’s Gtfs.