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

基本信息

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
项目类别：
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

项目摘要

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革兰氏阴性厌氧菌，与牙周病有关，这是最常见的慢性生物膜感染之一，会导致软硬组织破坏尽管 Pg 历史上被归类为牙周组织，并最终导致牙齿脱落。不能运动，最近发现流苏状菌株在夹在中间时能够进行表面易位通过遗传学研究，确定了 IX 型。分泌系统（T9SS）和 T9SS 货物是 Pg 迁移行为不可或缺的一部分。在表面易位的早期阶段，已知 T9SS 货物蛋白被运输到细胞表面并通过外膜囊泡 (OMV) 释放到环境中，改变周围环境然而，这会促进迁移过程中各种 OMV 货物蛋白的合作和功能。在转录组分析中，PG1881（一种预测的菌毛）向表面易位的转变仍然未知。形成脂蛋白，是表面初始阶段最高度上调的基因之一易位，但其功能尚不清楚。重要的是，PG1881 已被证明在鞘脂上富集。 (SL) 包含 PG1881 的预测翻译后修饰，包括棕榈酰化，提供链接到 SL，以及通过 PPAD（卟啉单胞菌肽基精氨酸脱亚氨酶）进行瓜氨酸化，将 L- 先前表明，PPAD 促进瓜氨酸化。 OMV 生物发生以及表面易位因此，本研究旨在研究 OMV 的功能。 PG1881 在表面易位的早期阶段，我从表面检查 OMV 的初步数据。缺乏PG1881的易位细胞通过荧光和传输电子揭示了独特的特性因此，中心假设是 PG1881 影响 OMV 的生物发生和特性，特别是 OMV 上携带的蛋白质货物，会影响表面易位的初始阶段。假设将通过两个具体目标进行检验：1）表征 PG1881 和 2）研究 PG1881 的功能表面易位背景下的 PG1881 在目标 1 中，将通过使用确认 PG1881 的棕榈酰化。我的 PG1881 多克隆抗体进行免疫沉淀，然后进行质谱分析。将使用重组 PG1881 作为比色测定的底物来确认 PG1881，该比色测定可测量在目标 2 中，将使用以下方法确定 PG1881 在表面易位细胞上的空间定位。我的多克隆 PG1881 抗体将比较牙龈蛋白酶的蛋白质水平和表面的酶活性。上述实验将给出亲本菌株和 PG1881 缺失突变体中的细胞易位。深入了解 Pg 生理学的一个新方面，并表征预测的菌毛形成脂蛋白相关已知的毒力决定因素包括 OMV 和 T9SS 最终，该研究提案是该研究的基础。博士论文，将加强对口腔健康研究感兴趣的学生的培训。