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攻击的物理屏障，它们还通过激活先天免疫反应在免疫监视中发挥作用。通过模式识别受体（PRRs）的表达，这些细胞能够识别病原体相关的分子模式（PAMPs）。牙龈卟啉单胞菌（P. gingivalis）是破坏性牙周病的重要病原体，具有多种毒力因子，其中包括菌毛、膜锚定细胞附件。FimA基因编码牙龈假单胞菌的主要菌毛。这些菌毛使牙龈假单胞菌能够附着并侵入宿主细胞。牙龈假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。菌毛和菌壁成分被ppr识别并诱导细胞反应。这一过程与牙周病的发生和发展有关。b7 - h1是b7家族的共抑制分子，在细胞介导的免疫应答中具有重要的调控功能。结果表明，B7-H1介导的信号可诱导T细胞能量或凋亡。在先前的研究中，我们可以证明在口腔鳞癌细胞（SCC-25）和原代人牙龈角质形成细胞（PHGK）受到牙龈假单胞菌膜刺激后，B7-H1受体蛋白的表达呈浓度依赖性和时间依赖性上调。本项目旨在分析FimA在细胞和亚细胞水平上的致病潜力，阐明其潜在的分子机制，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；利用质粒克隆技术制备纯化的FimA；2 .牙龈假单胞菌膜组分的制备；3 .利用western blot和定量实时聚合酶链反应（PCR）分析受体的表达和活化，利用western blot和定量实时聚合酶链反应（PCR）分析信号通路。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).