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.
