IADR Abstract Archives
Characterisation of a novel Porphyromonas gingivalis fimbria
Objectives: Porphyromonas gingivalis is a non-motile, Gram-negative oral anaerobe that is implicated in chronic periodontitis, a bacterial biofilm-mediated disease. P. gingivalis major virulence factors include outer membrane vesicles (OMVs) that are adorned with proteases, haemagglutinins, lipopolysaccharide, and fimbriae. Fimbriae are crucial for bacterial adherence, such as that involved in biofilm formation and invasion of host cells.
Objective: To characterize the newly discovered P. gingivalis Ffp1 and its role in OMV biogenesis and biofilm formation.
Methods: Phyre2 was used for bioinformatic analysis of Ffp1. A Ffp1 knockout mutant (Ffp1-) was produced by allelic exchange mutagenesis. Purified Ffp1 and OMVs purified from wild-type (WT) and Ffp1- were analysed by transmission electron microscopy. Ffp1 polymerization was determined using NativePAGE and Western blotting. Ffp1 localization was studied using P. gingivalis whole cell dot blot assay and immune-flow cytometry. WT and Ffp1- biofilms were grown in a flow cell system, visualized by confocal laser scanning microscopy and analysed by COMSTAT. WT and Ffp1- dual species biofilms of mutant with Treponema denticola were analysed in a static biofilm assay.
Results: A P. gingivalis protein was identified to be a novel fimbria by bioinformatic analysis. This protein was present on the P. gingivalis cell surface and formed a polymer with a long filamentous structure, which we named filament-forming protein 1, Ffp1. Quantification of OMVs showed that Ffp1 had a 30% reduction in OMV production compared with WT. Despite lowered OMV production and absence of Ffp1, the Ffp1- mutant formed more extensive biofilms, with increased extracellular DNA compared to WT. This suggests the release of extracellular DNA by Ffp1- facilitated more robust biofilm formation. However, Ffp1 had a decreased ability to form dual species biofilm with T. denticola.
Conclusions: P. gingivalis possesses a previously unknown fimbria, Ffp1 which plays a role in OMV production and biofilm formation.
Objective: To characterize the newly discovered P. gingivalis Ffp1 and its role in OMV biogenesis and biofilm formation.
Methods: Phyre2 was used for bioinformatic analysis of Ffp1. A Ffp1 knockout mutant (Ffp1-) was produced by allelic exchange mutagenesis. Purified Ffp1 and OMVs purified from wild-type (WT) and Ffp1- were analysed by transmission electron microscopy. Ffp1 polymerization was determined using NativePAGE and Western blotting. Ffp1 localization was studied using P. gingivalis whole cell dot blot assay and immune-flow cytometry. WT and Ffp1- biofilms were grown in a flow cell system, visualized by confocal laser scanning microscopy and analysed by COMSTAT. WT and Ffp1- dual species biofilms of mutant with Treponema denticola were analysed in a static biofilm assay.
Results: A P. gingivalis protein was identified to be a novel fimbria by bioinformatic analysis. This protein was present on the P. gingivalis cell surface and formed a polymer with a long filamentous structure, which we named filament-forming protein 1, Ffp1. Quantification of OMVs showed that Ffp1 had a 30% reduction in OMV production compared with WT. Despite lowered OMV production and absence of Ffp1, the Ffp1- mutant formed more extensive biofilms, with increased extracellular DNA compared to WT. This suggests the release of extracellular DNA by Ffp1- facilitated more robust biofilm formation. However, Ffp1 had a decreased ability to form dual species biofilm with T. denticola.
Conclusions: P. gingivalis possesses a previously unknown fimbria, Ffp1 which plays a role in OMV production and biofilm formation.