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（PG）是一种高度蛋白水解的厌氧菌，与牙周疾病有关， 这是最常见的基于慢性生物膜的感染，导致硬和软的破坏 尽管PG历史上被归类为 非运动型，最近表明，膜状菌株在夹心时能够表面易位 在软琼脂和玻璃或塑料表面之间。通过遗传研究，确定IX型 分泌系统（T9SS）和T9SS货物是PG迁移行为不可或缺的。我们的工作模型是 在表面易位的早期阶段，T9SS货物蛋白已知被转运到 细胞表面并释放到外膜蔬菜（OMV）上的环境中修改周围环境 这促进了迁移。但是，各种OMV货物蛋白的合作与功能 向表面易位的过渡仍然未知。在转录组分析中，PG1881，一个预测的fimbriae- 形成脂蛋白，是表面初始阶段中最高度更新的基因之一 易位，但其功能尚不清楚。重要的是，PG1881已显示在鞘脂中富集 （SL）包含OMV。 PG1881的预测翻译后修饰包括棕榈酰化，提供了 链接到SLS以及PPAD（卟啉单基因氨酸葡萄蛋白脱节酶）的柠檬酸链接，可转换L- 精氨酸在肽内保留至L-硫氨酸。以前证明，PPAD的柠檬化会促进 OMV生物发生以及表面易位。因此，本研究旨在研究 PG1881在表面易位的早期阶段。我的初步数据从表面检查OMV 缺乏PG1881的易位细胞揭示了通过荧光和透射电子的独特特性 显微镜。因此，中心假设是PG1881影响OMV的生物发生和特性， 特别是蛋白质货物携带在OMV上，这会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PG1881和2）研究的功能 PG1881在表面易位的背景下。在AIM 1中，PG1881的棕榈酰化将通过使用 我的PG1881多克隆抗体可进行免疫沉淀，然后进行质谱。柠檬化 PG1881将使用重组PG1881作为测量的比色测定法证实 柠檬化。在AIM 2中，PG1881在表面易位细胞上的空间定位将使用 我的多克隆PG1881抗体。我将比较表面的金aipan蛋白水平和酶活性 在父菌株中易位细胞和PG1881缺失突变体。上面概述的实验将提供 了解PG生理学的新方面，并表征预测的纤维化脂蛋白相关 有了已知病毒的决定，包括OMV和T9S。最终，该研究建议是 博士学位论文，并将增强对口腔健康研究感兴趣的学生的培训。