Regulation of LPS structure and function in Porphyromonas gingivalis

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Project Summary/Abstract Periodontitis is a highly prevalent infectious, inflammatory disease of the tissues supporting the teeth that can lead to tissue destruction, formation of a deep periodontal pocket, alveolar bone resorption, and tooth loss. Etiological models suggest that periodontitis is driven by a synergistic community of virulent bacteria that trigger host inflammatory responses in the gingival tissues resulting in disease progression. Among recognized pathogens, the Gram-negative anaerobe Porphyromonas gingivalis (Pg) has been strongly implicated in periodontitis. Lipopolysaccharide (LPS) macromolecules produced by Pg strains have been repeatedly shown to stimulate pro-inflammatory innate immune responses. Intriguingly, Pg strains can diversify the structure of LPS 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 in the interaction with the innate immune system. Agonistic LPS plays prominent roles in the pathological outcome of infection by being involved in the activation of TLR signaling pathways leading to production of proinflammatory cytokines, tissue destruction, 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 possess a c-di-AMP signaling mechanism 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 consequently LPS heterogeneity and virulence potential. This study will investigate how c-di-AMP turnover regulates the heterogeneity and immunomodulatory properties of Pg LPS using in vitro and in vivo models. To this end, two independent but related specific aims are proposed: Specific Aim 1: To understand how c-di-AMP-controls heterogeneity of LPS in Pg. Specific Aim 2: To understand the impact of c- di-AMP-dependent variation of LPS on the innate immune response. Upon completion of the proposed studies, we will learn how c-di-AMP signaling controls Pg LPS heterogeneity and determines the innate immune responses. Since c-di-AMP signaling does not exist in humans, it is a potential novel druggable target. Our findings will inform the discovery of potent antagonistic LPS isoforms as a foundation for development of anti- inflammatory therapeutics.