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

本申请的首要目标是研究唾液酸酶和唾液酸在关键蛋白中的作用。 牙龈卟啉单胞菌（Porphyromonas gingivalis，Pg）在牙周炎发病中的作用唾液酸 (SA)是一组结构相关的九碳糖酸，在宿主-病原体相互作用中起着关键作用。 在宿主方面，哺乳动物的粘膜表面和口腔、气道、肠道和阴道的分泌物是 尤其是富含唾液酸聚糖的蛋白质，具有多种生物、生物物理、抗菌和免疫作用 功能协调发展的此外，许多细胞表面受体（例如，趋化因子、免疫球蛋白和Toll样 受体）是唾液酸化的或识别唾液酸化的配体，其在免疫识别中起关键作用 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， 宿主SA用于粘附、入侵、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，细菌病原体通常使用唾液酸酶来水解宿主唾液酸聚糖， 损害宿主的免疫防御，并促进它们在粘膜生态位中的存活。唾液和牙龈 龈沟液含有高浓度的SA，其与各种蛋白质如粘蛋白中的唾液酸聚糖结合。 临床研究表明，口腔中的唾液酸酶活性与口腔溃疡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 在许多口腔细菌中发现，包括关键病原体牙龈卟啉单胞菌（Porphyromonas gingivalis，Pg）。PG 缺乏合成SA的基因。相反，它编码唾液酸酶（PG 0352），这是高度保守的所有 基因组测序Pg分离物。我们小组和其他人以前的研究表明，PG 0352在体内起着重要的作用。 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，这些表型的分子机制仍然难以捉摸。在本申请中，我们 假设Pg使用唾液酸酶和SA特异性转运蛋白来转运宿主SA，这反过来 用于修饰Pg细胞表面分子，如囊膜和脂多糖（LPS），从而拦截 宿主先天性免疫防御，如补体杀伤和嗜中性粒细胞和巨噬细胞的吞噬作用。到 为了检验这一假设，专门设计并实施了以下研究：目标1： 利用遗传学、生物化学和分子生物学方法， 目的2：研究Pg如何通过使用多学科方法导入和利用SA， 遗传学、生物化学、糖生物学、免疫学和生物膜测定;以及目的3：阐明 通过多种体内、体外实验方法研究了PG 0352对Pg致病性的影响。完成本项目 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上的见解， Pg和其他口腔病原体，但也将为未来开发特定的 唾液酸酶抑制剂对口腔病原体，这可以提供替代，以减轻牙周疾病。

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.