The Control of LPS Heterogeneity and Virulence by C-di-AMP Signaling in P. gingivalis

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Project Summary Periodontitis is a highly prevalent infectious, inflammatory disease of the tissues supporting the teeth that can lead to tissue destruction, alveolar bone resorption, formation of the deep periodontal pocket, and tooth loss. Etiological models suggest that periodontitis is driven by a synergistic community of virulent bacteria that trigger host inflammatory responses below the gum line resulting in disease progression. Among recognized pathogens, the Gram-negative, highly proteolytic anaerobe Porphyromonas gingivalis (Pg) has been strongly implicated in periodontitis. Pg strains produce lipopolysaccharide (LPS) macromolecules as the main components of the outermost layer of the cells that have been repeatedly shown to stimulate innate immune responses. Intriguingly, Pg strains can diversify the structure of LPSs in response to biologically relevant stimuli to temporarily disguise themselves, evade the immune system, protect from stresses, and promote survival. Accordingly, at least four LPS variants have been identified in Pg strains that may act as agonists or antagonists to activate innate immune responses. A specific form of LPSs, called A-LPS, serves to anchor a series of destructive proteolytic enzymes known as gingipains on the cell surface. These virulence factors collectively play prominent roles in the pathological outcome of infection by being involved in various activities, including tissue destruction, host-defense perturbation, immune system evasion, and bone resorption. However, we do not yet know the mechanisms of the regulation of LPS heterogeneity and its biological importance during pathogenesis. We discovered that Pg strains have evolved with c-di-AMP signaling pathway in which the c-di-AMP synthase PGN_0523 (dacpg) and the c-di-AMP phosphodiesterase PGN_0521 (pdepg) control the essential turnover of c- di-AMP and, in turn, cell viability within biofilms, LPS heterogeneity, the diffusion of gingipains into the surrounding milieu, and virulence potential. We will investigate how c-di-AMP turnover regulates the heterogeneity and immunostimulatory properties of LPSs and affects gingipain-associated virulence potential. To this end, two specific aims are proposed, and each aim includes a set of separate experiments and assays: Specific Aim 1: To understand c-di-AMP-controlled heterogeneity of LPSs in Pg. Specific Aim 2: To understand the impact of c-di-AMP-dependent variation of LPS on the innate immune response and gingipains- associated virulence. Upon completion of the proposed studies, we will learn how c-di-AMP signaling controls LPS heterogeneity, impacts immunostimulatory responses, and the virulence potential of Pg. Since c- di-AMP signaling does not exist in mammals, our findings will inform about a novel druggable target.