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

该应用的总体目标是研究唾液酸酶和唾液酸在关键蛋白中的作用 病原体牙龈卟啉单胞菌（Pg）及其对牙周炎发病机制的贡献。唾液酸 （SA）是一组结构相关的九碳糖酸，在宿主与病原体的相互作用中发挥着关键作用。 在宿主方面，哺乳动物的口腔、气道、肠道和阴道的粘膜表面和分泌物 尤其是富含唾液酸聚糖，具有多种生物学、生物物理、抗菌和免疫学活性 功能。此外，许多细胞表面受体（例如趋化因子、免疫球蛋白和 Toll 样受体） 受体）要么被唾液酸化，要么识别唾液酸化配体，其在免疫识别中发挥关键作用 和激活。在病原体方面，许多细菌病原体已经进化出不同的机制来靶向 宿主SA的粘附、侵袭、免疫调节和营养获取，从而促进其健康 及发病机制。具体来说，细菌病原体经常使用唾液酸酶来水解宿主唾液聚糖， 损害宿主免疫防御，并促进其在粘膜生态位中的生存。唾液和牙龈 裂隙液含有高浓度的 SA，与粘蛋白等多种蛋白质中的唾液酸聚糖结合。 临床研究表明，口腔唾液酸酶活性与口腔疾病的严重程度呈正相关。 牙周炎;因此，推荐作为牙周炎诊断的生物标志物。因此，唾液酸酶具有 在许多口腔细菌中都发现了这种细菌，包括主要病原体牙龈卟啉单胞菌 (Pg)。 PG 缺乏合成SA的基因。相反，它编码一种唾液酸酶（PG0352），该酶在所有唾液酸酶中高度保守。 基因组测序的 Pg 分离株。我们小组和其他人之前的研究表明 PG0352 发挥着 Pg 胶囊合成、生物膜、应激反应、先天免疫反应和毒力中发挥着至关重要的作用。 然而，这些表型背后的分子机制仍然难以捉摸。在这个应用程序中，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，这反过来又 用于修饰Pg细胞表面分子，如荚膜和脂多糖（LPS），从而拦截 宿主先天免疫防御，例如补体杀伤和中性粒细胞和巨噬细胞的吞噬作用。至 为了检验这一假设，专门设计并实施了以下研究： 目标 1：描绘 通过使用遗传学、生物化学和方法的方法研究 PG0352 的生化和结构特征 晶体学；目标 2：通过使用多学科方法研究 Pg 如何导入和利用 SA 遗传学、生物化学、糖生物学、免疫学和生物膜测定；目标 3：阐明 通过使用各种体外和体内方法研究PG0352对Pg的致病性。本项目完成情况 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制见解 Pg 或许还有其他口腔病原体，但也将为特定的未来发展铺平道路 针对口腔病原体的唾液酸酶抑制剂，可以提供减轻牙周疾病的替代方案。

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.