Role of Porphyromonas gingivalis Fimbriae in infection of oral epithelial cells

上皮细胞不仅提供抵抗毒性影响或细菌挑战的物理屏障，它们还通过激活先天免疫应答在免疫监视中发挥作用。通过模式识别受体（PRR）的表达，这些细胞能够识别病原体相关分子模式（PAMP）。牙龈卟啉单胞菌（Porphyromonas gingivalis，P. gingivalis）是破坏性牙周病的重要致病菌，具有多种毒力因子，其中包括菌毛、膜锚定细胞附件等。FimA基因编码牙龈卟啉单胞菌的主要菌毛。这些菌毛使牙龈卟啉单胞菌能够粘附并侵入宿主细胞。牙龈卟啉单胞菌感染可诱导促炎细胞因子如白细胞介素（IL）-6，IL-8，肿瘤坏死因子（TNF）-α和粒细胞-巨噬细胞-集落刺激因子（GM-CSF）的表达。菌毛和细菌壁的组分被PPR识别并诱导细胞应答。这一过程与牙周病的发生和发展有关。B7-H1是B7家族的一种共抑制分子，在细胞免疫应答中具有重要的调节作用。研究表明，B7-H1介导的信号可以诱导T细胞的无能或凋亡。在以前的研究中，我们可以证明，在口腔鳞状细胞癌细胞（SCC-25）和原代人牙龈角质形成细胞（PHGK）刺激牙龈卟啉单胞菌膜后，表达或B7-H1受体蛋白的上调浓度和时间依赖性。本研究的目的是从细胞和亚细胞水平分析FimA的致病潜力，阐明其致病的分子机制，并探讨FimA在口腔上皮细胞感染中的作用。将执行以下步骤：1.使用质粒克隆生产纯化的FimA，2.牙龈卟啉单胞菌膜级分的制备，3.使用蛋白质印迹和定量真实的时间聚合酶链反应（PCR）分析受体表达和活化，使用蛋白质印迹和定量真实的时间PCR分析信号传导途径，以及4.使用下一代测序（NSG）的转录组学。

Epithelial cells do not only provide a physical barrier against toxic influences or bacterial challenge, they also play a role in immune surveillance by activating innate immune response. By expression of pattern recognition receptors (PRRs), these cells are able to recognize pathogen associated molecular patterns (PAMPs). Porphyromonas gingivalis (P. gingivalis), a keystone pathogen in destructive periodontal disease, possesses a variety of virulence factors, amongst them fimbriae, membrane anchored cell appendices. The FimA gene is coding the major fimbriae of P. gingivalis. These fimbriae enable P. gingivalis to adhere to and invade host cells. Infection with P. gingivalis may induce the expression of pro-inflammatory cytokines such as interleukin (IL)-6, IL-8, tumor necrosis factor (TNF)-alpha and granulocyte-macrophage-colony-stimulating factor (GM-CSF). Fimbriae and components of the bacterial wall are recognized by PPRs and induce a cell response. This process is linked to the initiation and progression of periodontal disease. B7-H1is a co-inhibitory molecule belonging to the B7-family, that has important regulatory functions in cell-mediated immune response. It was shown, that B7-H1 mediated signals may induce anergy or apoptosis in T cells. In previous studies we could demonstrate that in oral squamous carcinoma cells (SCC-25) and primary human gingival keratinocytes (PHGK) after stimulation with P. gingivalis membrane, the expression or B7-H1 receptor protein is up-regulated concentration- and time-dependently. The aims of this project are to analyse the pathogenic potential of FimA on the cellular and subcellular level, to elucidate the underlying molecular mechanisms and to investigate the role of FimA in infection of oral epithelia cells. The following steps will be performed, 1. production of purified FimA using plasmid cloning, 2. preparation of P. gingivalis membrane fractions, 3. analysis of the receptor expression and activation using western blot and quantitative real time polymerase chain reaction (PCR), analysis of the signalling pathway using western blot and quantitative real time PCR and 4. transcriptomics using next generation sequencing (NSG).