P. gingivalis and S. gordonii interactions with epithelial cell multilayers

Objectives: The objective of this study was to compare the physical interactions of periodontal pathogen P. gingivalis and the commensal S. gordonii, with multilayers of human gingival epithelial cells. Methods: Human immortalized gingival keratinocyte (HIGK) cells were grown on collagen coated 12 mm polytetrafluoroethylene membranes in an air-liquid interface for three weeks, yielding three cell layers. P. gingivalis strain 33277 was labeled with CellTracker Green BODIFY. S. gordonii strain DL1 was labeled with FITC. Both strains were reacted with HIGK multilayers at an MOI 200 for 2 h. Cell actin was labeled with Texas Red-phalloidin. Images were collected using a Leica DM IRB confocal microscope along with VoxCell Scan v4.11.0.2 software. Analysis of images was conducted with Imaris v6.0.0. Four depths were chosen for analysis, spanning the multilayer at regular intervals. Assessment of bacteria location was performed by utilizing the three dimensional imaging properties of the Imaris software. Results: The majority (72%) of P. gingivalis resided within the HIGK cells. In contrast S. gordonii was unable to internalize was located exclusively outside of the HIGK cells. P. gingivalis penetrated the top two layers of HIGK cells intracellularly, whereas S. gordonii demonstrated a greater (p<0.05) maximum extent of penetration through the intercellular route. Conclusions: P. gingivalis invades multilayer HIGK cells intracellularly and can penetrate two cells layers after two hour exposure. S. gordonii resides between the HIGK cells in a multilayer. However, S. gordonii can penetrate deeper than P. gingivalis into the multilayer by movement between the cells. These results have relevance for local and systemic spread of P. gingivalis and S. gordonii. Supported by NIDCR DE18307, DE11111 and T32DE007200