Investigating the Function of Fimbriae-forming Lipoprotein in Porphyromonas gingivalis

牙龈卟啉单胞菌菌毛形成脂蛋白的功能研究

• 批准号: 10749647
• 负责人: Christina Rothenberger
• 金额: $ 4.27万
• 依托单位: ADA FORSYTH INSTITUTE, INC.
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-12-01 至 2025-11-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10749647
• 关键词: Adult; Agar; Anaerobic Bacteria; Antibodies; Arginine; Bacteria; Bacteroidetes; Behavior; Biogenesis; Biological Assay; Cardiovascular Diseases; Cell surface; Cells; Chronic; Citrulline; Classification; Communities; Complex; Cysteine; Data; Development; Diabetes Mellitus; Disease; Disease Progression; Elements; Ensure; Environment; Eukaryota; Fluorescence; Fluorescence Microscopy; Genes; Genetic study; Glass; Goals; Human; Immunoprecipitation; Infection; Inflammatory; Life Style; Link; Lipids; Lipoproteins; Mass Spectrum Analysis; Measures; Membrane; Microbial Biofilms; Modeling; Oral Microbiology; Oral health; Parents; Pathogenicity; Peptides; Periodontal Diseases; Periodontium; Physiology; Population; Porphyromonas; Porphyromonas gingivalis; Post-Translational Protein Processing; Prevention; Proliferating; Property; Protein-arginine deiminase; Proteins; Recombinants; Research; Research Personnel; Research Proposals; Role; Scientist; Sphingolipids; Stroke; Students; Surface; System; Systemic disease; Testing; Tooth Loss; Training; Transmission Electron Microscopy; United States; Vesicle; Virulence; Work; cell motility; chronic inflammatory disease; cost; dysbiosis; experimental study; gingipain; innovation; insight; interest; member; migration; mutant; palmitoylation; pathogen; pathogenic bacteria; polyclonal antibody; protein function; protein structure; soft tissue; subgingival biofilm; transcriptome

Porphyromonas gingivalis (Pg) is a highly proteolytic Gram-negative anaerobe implicated in periodontal disease, which is one of the most common chronic biofilm-based infections that result in destruction of hard and soft tissues of the periodontium and, ultimately, in loss of teeth. Although Pg has been historically classified as nonmotile, it was recently shown that fimbriated strains are capable of surface translocation when sandwiched between soft agar and a glass or plastic surface. Through genetic studies, it was determined that the type IX secretion system (T9SS) and T9SS cargo are integral to Pg’s migration behavior. Our working model is that during the early stages of surface translocation, T9SS cargo proteins which are known to be transported to the cell surface and released into the environment on outer membrane vesicles (OMVs) modify the surroundings and this promotes migration. However, the cooperation and function of the various OMV cargo proteins during the transition to surface translocation is still unknown. In a transcriptome analysis, PG1881, a predicted fimbriae- forming lipoprotein, was one of the most highly upregulated genes during the initial stages of surface translocation, yet its function is not clear. Importantly, PG1881 has been shown to be enriched on sphingolipid (SL) containing OMVs. Predicted post-translational modifications of PG1881 include palmitoylation, providing a link to SLs, as well as citrullination by PPAD (Porphyromonas peptidylarginine deiminase) which converts L- arginine residues to L-citrulline within peptides. It was previously shown that citrullination by PPAD promotes OMV biogenesis as well as surface translocation. Therefore, this study aims to investigate the function of PG1881 during the early stages of surface translocation. My preliminary data examining OMVs from surface translocating cells lacking PG1881 revealed distinct properties via fluorescence and transmission electron microscopy. Therefore, the central hypothesis is that PG1881 influences the biogenesis and properties of OMVs, in particular the protein cargo carried on OMVs, which impacts the initial stages of surface translocation. This hypothesis will be tested through two specific aims: 1) Characterize PG1881 and 2) Investigate the function of PG1881 in the context of surface translocation. In Aim 1, palmitoylation of PG1881 will be confirmed by using my PG1881 polyclonal antibody to perform immunoprecipitation followed by mass spectrometry. Citrullination of PG1881 will be confirmed using recombinant PG1881 as a substrate for a colorimetric assay that measures citrullination. In Aim 2, the spatial localization of PG1881 on surface translocating cells will be determined using my polyclonal PG1881 antibody. I will compare gingipains protein levels and enzymatic activity from surface translocating cells in the parent strain and PG1881 deletion mutant. The experiments outlined above will give insight on a new aspect of Pg physiology and characterize a predicted fimbriae-forming lipoprotein associated with known virulence determinants including OMVs and T9SS. Ultimately, this research proposal is the basis of a doctoral dissertation and will enhance the training of a student with an interest in oral health research.

牙龈卟啉单胞菌（Porphyromonas gingivalis，Pg）是一种高度蛋白水解的革兰氏阴性厌氧菌，与牙周病有关， 这是一种最常见的慢性生物膜感染，导致硬和软组织的破坏， 牙周组织，并最终导致牙齿脱落。虽然PG在历史上被归类为 不运动，最近表明，当夹在中间时，有菌毛的菌株能够进行表面移位。 在软琼脂和玻璃或塑料表面之间。通过遗传学研究，确定IX型 分泌系统（T9 SS）和T9 SS货物是不可或缺的Pg的迁移行为。我们的工作模式是， 在表面易位的早期阶段，已知被转运到细胞表面的T9 SS货物蛋白， 细胞表面并释放到外膜囊泡（OMV）上的环境中， 这就促进了移民。然而，各种OMV货物蛋白的合作和功能， 向表面易位的转变仍然是未知的。在转录组分析中，PG 1881，一个预测的菌毛- 形成脂蛋白，是最高度上调的基因之一，在初始阶段的表面 易位，但其功能尚不清楚。重要的是，PG 1881已被证明富含鞘脂 (SL)包含OMV。预测的PG 1881的翻译后修饰包括棕榈酰化，提供了一种新的修饰。 连接到SL，以及瓜氨酸通过PPAD（卟啉单胞菌肽基精氨酸脱亚氨酶），其转化L- 精氨酸残基转化为L-瓜氨酸。以前的研究表明，瓜氨酸通过PPAD促进 OMV的生物发生以及表面易位。因此，本研究旨在探讨 PG 1881在表面易位的早期阶段。我的初步数据从表面检查OMV 缺乏PG 1881的易位细胞通过荧光和透射电子显微镜显示出不同的特性 显微镜因此，中心假设是PG 1881影响OMV的生物起源和性质， 特别是OMV上携带的蛋白质货物，其影响表面易位的初始阶段。这 假设将通过两个具体目标进行检验：1）表征PG 1881和2）研究 PG 1881在表面易位的背景下。在目标1中，PG 1881的棕榈酰化将通过使用 我的PG 1881多克隆抗体进行免疫沉淀，然后质谱。的瓜氨酸化 将使用重组PG 1881作为比色测定的底物来确认PG 1881， 瓜氨酸。在目的2中，将使用以下方法确定PG 1881在表面易位细胞上的空间定位： my polyclonal PG 1881 antibody.我将比较牙龈卟啉酶的蛋白水平和酶活性从表面 在亲本菌株和PG 1881缺失突变体中易位细胞。上述实验将给出 对Pg生理学新方面的认识和预测的菌毛形成脂蛋白相关的特征 已知的毒力决定因子包括OMV和T9 SS。最终，这项研究提案是 博士论文，并将加强对口腔健康研究感兴趣的学生的培训。