Dissecting the role of sialic acid and sialidase in the pathophysiology of Porphyromonas gingivalis

这项申请的首要目标是研究唾液酸酶和唾液酸在Keystone中的作用 牙龈卟啉单胞菌及其在牙周炎发病机制中的作用唾液酸 (SA)是一类结构上相关的九碳糖酸，在宿主与病原菌的相互作用中起着关键作用。 在宿主一侧，哺乳动物的口腔、呼吸道、肠道和阴道的粘膜表面和分泌物是 尤其是富含唾液酸的多糖，具有多种生物、生物物理、抗菌和免疫学作用 功能。此外，许多细胞表面受体(如趋化因子、免疫球蛋白和Toll样受体) 受体)是唾液酸化的或识别唾液酸化的配体，它们在免疫识别中起着关键作用 和激活。在病原体方面，许多细菌病原体进化出不同的机制来靶向 宿主SA用于黏附、侵袭、免疫调节和营养获取，从而促进其适合性 和发病机制。具体地说，细菌病原体通常使用唾液酸酶来降解宿主唾液酸聚糖， 损害宿主的免疫防御，并促进它们在粘膜利基中的生存。唾液和牙龈 阴沟液中含有高浓度的SA，与粘蛋白等各种蛋白质中的唾液聚糖结合。 临床研究表明口腔唾液酸酶活性与牙周炎的严重程度呈正相关 牙周炎；因此，建议将其作为牙周炎诊断的生物标志物。因此，唾液酸酶有 在许多口腔细菌中发现，包括基石病原体牙龈卟啉单胞菌(PG)。皮格 缺乏合成SA的基因。相反，它编码唾液酸酶(PG0352)，该酶在所有 对PG分离株进行基因组测序。我们小组和其他人之前的研究表明，PG0352在 在PG胶囊合成、生物膜、应激反应、先天免疫反应和毒力中起关键作用。 然而，这些表型背后的分子机制仍然难以捉摸。在此应用程序中，我们 假设PG使用唾液酸酶和SA特异性转运蛋白来清除宿主SA，而宿主SA又 用于修饰PG细胞表面分子，如胶囊和内毒素，从而拦截 宿主的先天免疫防御，如杀死补体和吞噬中性粒细胞和巨噬细胞。至 检验这一假设，以下研究是专门设计并将实施的：目标1：描绘 用遗传学、生物化学和生物化学方法研究PG0352的生化和结构特征 结晶学；目标2：通过使用多学科方法研究PG如何引入和利用SA 遗传学、生物化学、糖生物学、免疫学和生物膜分析；以及目标3：阐明 用多种体外和体内方法研究PG0352对PG的致病性。本项目竣工 不仅为理解SA和唾液酸酶在病理生理学中的作用提供了机械性的见解 PG，也许还有其他口腔病原体，但也将为未来的特定发展铺平道路 唾液酸酶抑制剂对口腔病原体的抑制作用，可为缓解牙周病提供替代方案。

The overarching goal of this application is to investigate the role of sialidase and sialic acid in the keystone pathogen Porphyromonas gingivalis (Pg) and their contributions to the pathogenesis of periodontitis. Sialic acid (SA), a group of structurally related nine-carbon sugar acids, plays critical roles in host-pathogen interactions. On the host side, mammalian mucosal surfaces and secretions of the mouth, airway, gut, and vagina are especially sialoglycan-rich, which have a variety of biological, biophysical, antimicrobial, and immunological functions. In addition, a number of cell surface receptors (e.g., chemokine-, immunoglobulin-, and toll-like- receptors) are either sialylated or recognize sialylated ligands, which play critical roles in immune recognition and activation. On the pathogen side, many bacterial pathogens have evolved different mechanisms to target host SA for adherence, invasion, immune modulation and nutrient acquisition, thereby promoting their fitness and pathogenesis. Specifically, bacterial pathogens often use sialidases to hydrolyze host sialoglycans, compromise host immune defenses, and promote their survival in the mucosal niche. Salivary and gingival crevicular fluids contain a high concentration of SA bound to sialoglycans in various proteins such as mucins. Clinical studies indicate that sialidase activity in the oral cavity is positively associated with the severity of periodontitis; thus, it is recommended as a biomarker for periodontitis diagnosis. Accordingly, sialidases have been found in numerous oral bacteria including the keystone pathogen Porphyromonas gingivalis (Pg). Pg lacks genes to synthesize SA. Instead, it encodes a sialidase (PG0352), which is highly conserved among all genome sequenced Pg isolates. Previous studies from our group and others have shown that PG0352 plays a crucial role in Pg capsule synthesis, biofilms, stress response, innate immune responses, and virulence. However, the molecular mechanism underlying these phenotypes remains elusive. In this application, we hypothesize that Pg employs a sialidase and a SA specific transporter to scavenge host SA, which is in turn used to modify Pg cell surface molecules such as capsule and lipopolysaccharide (LPS), thereby intercepting host innate immune defenses, such as complement killing and phagocytosis of neutrophils and macrophage. To test this hypothesis, the following studies are specifically designed and will be implemented: Aim 1: To delineate the biochemical and structural features of PG0352 by using an approach of genetics, biochemistry, and crystallography; Aim 2: To investigate how Pg imports and utilizes SA by using a multidisciplinary approach of genetics, biochemistry, glycobiology, immunology, and biofilm assays; and Aim 3: To elucidate the role of PG0352 in the pathogenicity of Pg by using various in vitro and in vivo approaches. Completion of this project will not only provide mechanistic insights into understanding the role of SA and sialidase in the pathophysiology of Pg and perhaps other oral pathogens as well, but also will pave a way for future development of specific sialidase inhibitors against oral pathogens, which can provide alternatives to mitigate periodontal diseases.