PROJECT SUMMARY Periodontitis is one of the most common human health conditions and is a chronic inflammatory disease affecting the tooth-supporting structures resulting from the actions of polymicrobial communities of organisms that induce destructive immune responses. Progression of periodontitis is associated with a microbial population shift and synergistic interactions between pathobionts such as Porphyromonas gingivalis and Treponema denticola. T. denticola emerges as one of the most abundant bacteria in diseased periodontal sites and is predictive of the severity and progression of periodontitis. The mechanisms that allow T. denticola to thrive in the diseased periodontium remain poorly defined. Two-component signal (TCS) transduction systems are ubiquitous sensory transduction systems in bacteria that often sense environmental stimuli to mediate cellular responses via genetic regulation. Our scientific premise is that TCSs allow T. denticola to sense and respond to changing periodontal environments, promoting persistence, interactions with other oral bacteria, and pathogenicity. We have initiated the characterization of the AtcS/AtcR and Hpk2/Rrp2 TCS of T. denticola, which represents half of the TCS encoded in the genome. We have characterized the AtcR binding motif and identified genes that contain the AtcR binding motif within their promoter. Interestingly, the promoter regulating the expression of the Hpk2/Rrp2 TCS is bound by AtcR. Yet, AtcR-mediated regulation of gene expression and the impact on T. denticola cellular processes are untested. We have observed that Hpk2 kinase activity is regulated by oxygen. However, the role of Rrp2 as a transcription factor and its regulon remains unstudied. Our ongoing studies indicate that both AtcR and Rrp2 may co-regulate the expression of genes with the alternative sigma factor, s54. However, no study has ever explored the role of s54 in T. denticola. The published literature supports our preliminary data suggesting that AtcR likely contributes to community interactions with P. gingivalis, and both AtcR and Rrp2 likely impact the fitness and virulence of T. denticola. Here, we propose three complementary Specific Aims that will fill these gaps in knowledge. Aim 1 will determine how phosphorylation of AtcR impacts DNA binding kinetic, affinity, and stoichiometry, while knockout of atcR in T. denticola will characterize the role of AtcR in gene regulation and physiology. Aim 2 will define the Rrp2 and s54 binding sites, demonstrate Rrp2 interacts with s54 to regulate gene expression and determine if Rrp2-mediated regulation responds to changes in environmental oxygen. Aim 3 will determine if AtcR and Rrp2 contribute to T. denticola colonization of the gingiva, induction of inflammatory markers, and alveolar bone loss in murine models of periodontitis. We will then determine if AtcR contributes to synergistic growth and pathogenicity with P. gingivalis. Completing this study will define half of t...