Porphyromonas gingivalis strain-dependent activation of human endothelial cells

Abstract

Porphyromonas gingivalis is an important bacterium involved in periodontal diseases. Colonization by periodontopathogens has been associated with severe local inflammatory reactions in the connective tissue. In this study we characterized P. gingivalis-mediated infection and activation of human umbilical vein endothelial cells by using two strains of different virulence capacities, strains ATCC 53977 and DSMZ 20709. Both strains were able to adhere to and infect endothelial cells with an infection rate of 0.48% for ATCC 53977 and 0.007% for DSMZ 20709. The triggering of two signal transduction pathways in P. gingivalis-infected endothelial cells was demonstrated for both strains, with a rapid increase of p38 mitogen-activated protein kinase phosphorylation and a more delayed degradation of IkappaBalpha, followed by nuclear translocation of NF-kappaB. In addition, both strains induced enhanced expression of endothelial adhesion molecules E-selectin and intracellular adhesion molecule 1 (ICAM-1). Target cell activation was independent of bacterial fimbriae expression since the fimA knockout strain A7436 DeltafimA induced the same level of ICAM-1 as the corresponding wild type (A7436-WT). Thus, two P. gingivalis strains, ATCC 53799 and DSMZ 20709, infect endothelial cells and trigger signaling cascades leading to endothelial activation, which in turn may result in or promote severe local and systemic inflammation.

FIG. 1.

Adherence and interactions of P. gingivalis DSMZ 20709 to HUVEC demonstrated by scanning electron microscopy. HUVEC were infected for 1.5 h at an MOI of 100 as described in Material and Methods. Note that P. gingivalis attaches to the surface of an endothelial cell (A). Subsequent invasion appeared to be mediated by a microvillus-like structure protruding from the endothelial cell, starting to engulf the bacterium (B). A magnification of ×100,000 was used for both pictures.

FIG. 2.

Invasion of P. gingivalis was analyzed by an antibiotic protection assay. ATCC 53977 or DSMZ 20709 at an MOI of 50 (5 × 10⁷ bacteria to 10⁶ endothelial cells) was added to HUVEC and further incubated as described in Materials and Methods. The total represents bacteria adhered or already invaded after 1.5 h of exposure to target cells; 1.4% of strain ATCC 53977 (7 × 10⁵ CFU/ml) bacteria and 0.12% of DSMZ 20709 (6.4 × 10⁴ CFU/ml) bacteria could be detected in the endothelial cell lysates. IC indicates bacteria found in endothelial cell lysates after 1.5 h of incubation followed by antibiotic treatment for 2.5 h. EC indicates bacteria that only adhered to HUVEC. Approximately 2.3 × 10⁵ CFU/ml and 3.5 × 10³ CFU/ml (34% and 5.4%, respectively) of adhered P. gingivalis ATCC 53977 and DSMZ 20709, respectively, invaded endothelial cells. Data presented are the means ± SEM of four separate experiments. #, P < 0.05 comparing ATCC 53977 versus DSMZ 20709.

FIG. 3.

Endothelial cell adhesion molecules quantified by cell surface ELISA were up-regulated in P. gingivalis-stimulated HUVEC in a dose- and time-dependent manner. E-selectin expression peaked at 5 h and declined to baseline after 24 h. Dose dependency was demonstrated 5 h postinfection (A). Expression of ICAM-1 peaked at 12 to 24 h. Dose dependency was demonstrated 24 h postinfection (C). An MOI of 10 (10⁶ bacteria/ml) was used for both time courses (B and D). Even the lowest concentrations were able to stimulate the expression of both adhesion molecules. Data presented are the means ± SEM of four separate experiments. *, P < 0.05 compared to control; #, P < 0.05 comparing ATCC 53977 versus DSMZ 20709; OD, optical density.

FIG. 4.

P. gingivalis-induced expression of ICAM-1 on HUVEC is independent of fimA. ICAM-1 expression was determined by cell surface ELISA after 24 h. An MOI of 10 (10⁶ bacteria/ml) was used for all strains. A7436-WT was equipotent to ATCC 53977. The fimA knockout strain A7436 ΔfimA induced the same level of ICAM-1 expression compared to the level in the corresponding wild-type strain. Data presented are the means ± SEM of three separate experiments. *, P < 0.05 compared to control. OD, optical density; n.s., not significant.

FIG. 5.

HUVEC were stimulated at an MOI of 10 for each strain. ELISA for phospho-p38 MAPK (for details, see Materials and Methods) revealed a time-dependent phosphorylation of p38 MAPK in ATCC 53977- and DSMZ 20709-treated endothelial cells (A). Strain ATCC 53977 induced a prolonged phosphorylation of p38 MAPK, while the signal in DSMZ 20709-stimulated cells decreased again after 60 min. Western blot analysis confirmed p38 MAPK phosphorylation 60 min postinfection in stimulated HUVEC (MOI of 10) (B). A 12.5% polyacrylamide gel was used; ERK2 served to confirm equal loading with proteins. TNF-α (10 ng/ml; 60 min) was used as a positive control. Data presented in panel A are the means ± SEM of three separate experiments. *, P < 0.05 compared to control; #, P < 0.05 comparing DSMZ 20709 versus ATCC 53977. In panel B one representative blot of three is shown.

FIG. 6.

Degradation of IκBα was demonstrated to confirm P. gingivalis-mediated NF-κB translocation. Cells were stimulated with strain ATCC 53977 (MOI of 10). Total cell protein was subjected to Western blotting, and IκBα and ERK2 were detected by specific antibodies as indicated in Materials and Methods. A 12.5% polyacrylamide gel was used. TNF-α (10 ng/ml; 60 min) was used as a positive control. One representative gel (of three) is shown.

FIG. 7.

NF-κB activation in P. gingivalis-stimulated HUVEC was analyzed by using immunofluorescence. HUVEC grown on Thermanox slides were stimulated with both strains for the times indicated. Cells were fixed and permeabilized as indicated in Materials and Methods, incubated with a polyclonal rabbit anti-human NF-κB p65 antibody, followed by the addition of an Alexa Fluor 488-conjugated goat anti-rabbit Ig antibody. The top row shows mock-treated control cells. Note a time-dependent increasing fluorescence intensity in the nuclei of P. gingivalis-stimulated HUVEC, demonstrating the nuclear translocation of NF-κB starting after 45 (ATCC 53977) to 90 min (DSMZ 20709) postinfection. Representative pictures (of three independent experiments; magnification, ×640) are shown.