Role of Porphyromonas gingivalis Fimbriae in infection of oral epithelial cells

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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).