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

项目摘要 牙周炎是一种高度流行的感染性、炎症性疾病，其支撑牙齿的组织可以 导致组织破坏、牙槽骨吸收、牙周袋形成和牙齿脱落。 病因学模型表明，牙周炎是由毒力细菌的协同群落驱动的，这些细菌引发 牙周线以下的宿主炎症反应导致疾病进展。在公认的病原体中， 革兰氏阴性、高度降解蛋白的厌氧菌牙龈卟啉单胞菌(Pg)一直强烈 与牙周炎有关。PG菌株主要产生内毒素(LPS)大分子 细胞最外层的成分，已多次被证明能刺激先天免疫 回应。耐人寻味的是，PG菌株可以对生物相关的刺激做出反应，使LPS的结构多样化。 暂时伪装自己，逃避免疫系统，保护自己免受压力，促进生存。 因此，在PG菌株中已鉴定出至少四种可能作为激动剂或拮抗剂的内毒素变体 来激活先天免疫反应。一种特定形式的LPS，称为A-LPS，用于锚定一系列 细胞表面的破坏性蛋白水解酶，称为牙龈痛。这些毒力因素共同发挥作用 通过参与各种活动，包括组织，在感染的病理结果中发挥重要作用 破坏、宿主防御系统紊乱、免疫系统逃避和骨吸收。然而，我们还没有 了解内毒素异质性的调节机制及其在发病机制中的生物学意义。 我们发现PG菌株已经进化为c-di-AMP信号通路，其中c-di-AMP合成酶 PGN_0523(Dacpg)和c-diamp磷酸二酯酶pgn_0521(Padeg)控制c-二AMP的基本周转。 Di-AMP，反过来，生物膜内的细胞活力，内毒素的异质性，牙龈痛向牙周的扩散 周围环境，以及毒力潜势。我们将调查c-di-AMP周转如何调节 LPS的异质性和免疫刺激特性，并影响牙龈痛相关毒力潜力。 为此，提出了两个具体目标，每个目标包括一套单独的实验和分析： 具体目的1：了解pG中c-diAMP控制的LPS的异质性。具体目标2： 了解依赖c-diAMP的内毒素变异对先天免疫反应和牙周疼痛的影响 相关的致命性。在完成拟议的研究后，我们将了解c-di-amp信号如何 控制内毒素的异质性，影响免疫刺激反应，以及PG的毒力潜力。因为c- Di-AMP信号在哺乳动物中不存在，我们的发现将为我们提供一个新的可药物靶点。

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.