Investigating the Function of Fimbriae-forming Lipoprotein in Porphyromonas gingivalis

牙龈卟啉单胞菌(Pg)是一种高度蛋白水解性的革兰氏阴性厌氧菌，与牙周病有关。 这是最常见的以生物膜为基础的慢性感染之一，会导致硬和软的破坏 牙周组织的损害，最终导致牙齿脱落。尽管PG在历史上被归类为 不能移动，最近发现菌毛菌株在被夹在中间时能够表面移位。 在软琼脂和玻璃或塑料表面之间。通过基因研究，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部分。我们的工作模式是 在表面转运的早期阶段，已知的T9SS货物蛋白被运输到 细胞表面和释放到环境中的外膜小泡(OMV)改变了周围环境 这促进了移民。然而，各种OMV货物蛋白的合作和功能在 向表面易位的转变尚不清楚。在转录组分析中，预测的菌毛PG1881- 形成脂蛋白，是表面形成初期最高表达的基因之一。 易位，但其功能尚不清楚。重要的是，PG1881已被证明富含鞘糖脂 (SL)装有OMV的油类。预测的PG1881的翻译后修饰包括棕榈酰化，提供了 链接到SLS，以及通过PPAD(卟啉单胞菌肽基精氨酸脱亚胺酶)进行的瓜氨酸化反应，它将L- 肽内精氨酸残基为L-瓜氨酸。以前的研究表明，PPAD的瓜氨酸化作用促进 OMV的生物发生以及表面易位。因此，本研究旨在探讨其在脑内的作用。 PG1881在细胞表面易位的早期阶段。我的初步数据从地面上检验OMV 缺乏PG1881的细胞的易位通过荧光和电子传输显示出明显的特性 显微镜。因此，中心假说是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，这影响了表面转运的初始阶段。这 假设将通过两个特定的目的来检验：1)表征PG1881和2)研究PG1881的功能 PG1881在表面易位的背景下。在目标1中，将通过使用以下方法确认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。瓜氨酸化 将使用重组PG1881作为底物确认PG1881，以进行比色分析，以测量 瓜氨酸化。在目标2中，PG1881在表面移位细胞上的空间定位将使用 我的多克隆抗体PG1881。我会从表面比较牙痛的蛋白质水平和酶活性 在亲本菌株和PG1881缺失突变体中进行细胞移位。上面概述的实验将给出 PG生理学的新视角和预测的菌毛形成相关脂蛋白的特征 已知的毒力决定因素包括OMV和T9SS。归根结底，这项研究建议是 博士论文，并将加强对口腔健康研究感兴趣的学生的培训。

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.