Project Summary Periodontitis is a polymicrobial-driven and prevalent inflammatory disease of the tissues supporting the teeth in adults. Studies revealed highly active biosynthesis of the lipopolysaccharide (LPS) components of the Gram-negative bacteria, and also components of cyclic di-3', 5'-adenylic acid (c-di-AMP) second messenger signaling in the periodontal pockets of the periodontitis subjects. Findings from our laboratory demonstrated, for the first time, that the LPS structure and function is regulated by c-di-AMP signaling in Porphyromonas gingivalis (Pg) strongly implicated in the onset and development of periodontitis. Our studies indicate that c- di-AMP regulation of LPS structure and therefore, function, is an essential aspect of the pathogenesis of the periodontal pathogens. We have identified genes encoding some of the key components constituting the c-di- AMP signaling pathway in Pg, including the essential c-di-AMP synthase gene (dacpg; PGN_0523), the c-di-AMP phosphodiesterase gene (pdepg; PGN_0521), and the predicted regulatory gene cdaR (PGN_1486), all of which regulate Pg LPS structure and function. However, we do not yet know the mechanisms of the regulation of LPS structure and function by c-di-AMP signaling and its contribution to the pathogenesis of the periodontal pathogens and periodontitis progression. We will investigate these mechanisms in Pg in molecular detail through these aims: Specific Aim 1: We will conduct a comprehensive analysis of structural differences in LPS synthesized by WT (strain 381), the strain lacking the c-di-AMP phosphodiesterase gene (∆pdepg) and a regulator of c-di-AMP levels (∆cdaR) mutants. This will involve large-scale LPS purification, followed by chromatographic fractionation, and thorough characterization of LPS constituents using FLATn, MALDI-ToF MS, and NMR techniques. Specific Aim 2: We will determine the protein interaction network of the c-di-AMP-regulated LPS structure and function and identify c-di-AMP receptors and effectors through various genetic and biochemical approaches. The significance of the interactome will be demonstrated by creating mutations in the corresponding genes in Pg and determining their effect on LPS structure and function. Specific Aim 3: We will use highly pure individual lipid A/LPS fractions isolated from WT, ∆pdepg and ∆cdaR mutants, purified through chromatographic fractionation, to test their immunostimulatory activities in relation to the TLR4 signaling pathway, the complement system, and cytokine production. These assessments will involve cell culture assays, human whole blood studies, and an animal model of inflammation. Upon completion of the proposed studies, we will learn how c-di-AMP signaling controls LPS structure-function, impacts immunostimulatory potential of LPS variants, and subsequently host defense responses. Since c-di-AMP signaling does not exist in mammals, our findings will inform about a novel druggable target.

PROJECT NARRATIVE Periodontitis afflicts ~ 50% of the adult population in the United States with substantial socio- economic impacts. Porphyromonas gingivalis has been strongly implicated in periodontitis. The proposed studies will decipher the regulatory role of a novel signaling pathway that controls the lipopolysaccharide driven pathoadaptation and pathogenesis of Pg, contributing to periodontitis progression.