PROJECT SUMMARY Dental caries is the most prevalent chronic disease in both children and adults, affecting 80–90% of the world population3.4. The bacterium, Streptococcus mutans, is the primary causative agent of this disease and is frequently found together with Streptococcus sobrinus in situations of more severe dental caries12,18,19. While many of the virulence factors contributing to the cariogenicty of S. mutans have been well studied, the mechanisms that regulate virulence gene expression has received less attention. Understanding the mechanisms by which S. mutans regulates its virulence are critical for developing therapeutic approaches to mitigate dental caries. Many bacteria, including streptococci, are known to regulate gene expression through a cell-to-cell communication mechanism called quorum sensing (QS) 21. Previous work by our lab has demonstrated that Rgg-type QS pathways regulate biofilm formation and virulence expression in Streptococcus pyogenes and genetic competence in S. mutans 26, 35. We have identified additional Rgg pathways in S. mutans and our preliminary data has identified putative virulence genes controlled by each Rgg thereby providing insight on the biological function of Rgg QS circuits. Moreover, the virulence of S. mutans may also be influenced through interactions with S. sobrinus. The current proposal will explore the mechanisms by which S. mutans regulates gene expression alone and in the presence of S. sobrinus. The central hypothesis of this proposal is that virulence gene expression by S. mutans is modulated through Rgg-mediated QS pathways and through its interaction with S. sobrinus. The specific aims of this research are (1) Describe regulatory function and chemical signaling for Rgg paralogs in S. mutans. (2) Characterize the synergistic effects of S. mutans and S. sobrinus interaction. To elucidate the functional roles of each Rgg regulon, we will perform a series of phenotypic experiments, each of which exploits a virulence trait suggested by our preliminary data, on rgg deletion mutants. We will then analyze the genome-wide expression profile of each mutant using RNA-seq to determine gene networks underpinning the phenotypes observed and to identify additional transcriptional targets of each Rgg. The influence of S. sobrinus on S. mutans’ gene expression will also be assessed. We will preform RNA-seq on dual-species biofilms of S. mutans and S. sobrinus to determine the influence of this interaction on gene expression. Genetic pathways showing differential expression will be top candidates that contribute to interspecies interaction, and mutants of these pathways will be generated and tested for their contribution to virulence. It is anticipated that this research will delineate critical aspects of S. mutans pathogenesis that will help improve strategies of prevention and treatment of dental caries. This research is also an important step in achieving the l...