Porphyromonas gingivalis strain specific interactions with human coronary artery endothelial cells: a comparative study

Abstract

Both epidemiologic and experimental findings suggest that infection with Porphyromonas gingivalis exacerbates progression of atherosclerosis. As P. gingivalis exhibits significant strain variation, it is reasonable that different strains possess different capabilities and/or mechanisms by which they promote atherosclerosis. Using P. gingivalis strains that have been previously evaluated in the ApoE null atherosclerosis model, we assessed the ability of W83, A7436, 381, and 33277 to adhere, invade, and persist in human coronary artery endothelial (HCAE) cells. W83 and 381 displayed an equivalent ability to adhere to HCAE cells, which was significantly greater than both A7436 and 33277 (P<0.01). W83, 381, and 33277 were more invasive than A7436 (P<0.0001). However, only W83 and A7436 were able to remain viable up to 48 hours in HCAE cell cultures, whereas 381 was cleared by 48 hours and 33277 was cleared by 24 hours. These differences in persistence were in part due to strain specific differences in intracellular trafficking. Both W83 and 381 trafficked through the autophagic pathway, but not A7436 or 33277. Internalized 381 was the only strain that was dependent upon the autophagic pathway for its survival. Finally, we assessed the efficacy of these strains to activate HCAE cells as defined by production of IL-6, IL-8, IL-12p40, MCP-1, RANTES, TNF-α, and soluble adhesion molecules (sICAM-1, sVCAM-1, and sE-selectin). Only moderate inflammation was observed in cells infected with either W83 or A7436, whereas cells infected with 381 exhibited the most profound inflammation, followed by cells infected with 33277. These results demonstrate that virulence mechanisms among different P. gingivalis strains are varied and that pathogenic mechanisms identified for one strain are not necessarily applicable to other strains.

Figure 1. Adherence of P. gingivalis to HCAE cells detected by qPCR (A) and ELISA (B).

Values represent the mean ± SD of six biological replicates from two independent experiments. Inoculated HCAE cells were incubated at 4°C for 30 min without agitation. (A) P. gingivalis 16S DNA copy number was normalized to HCAE cell 18S copy number. (B) Absorbance values at 450 nm for each replicate were normalized to their corresponding inoculums. **Values were significantly less than W83 and 381 (P<0.0001).

Figure 2. Invasion and persistence of P. gingivalis in HCAE cells.

Values represent mean log CFU ± SD of six biological replicates from two independent experiments. **Values were significantly different from other groups (P<0.0001). * Values were significantly different (P<0.05).

Figure 3. Ultrastructural evaluation of internalized P. gingivalis at 6 hours post-inoculation.

Transmission electron microscopic images (6000× magnification) of uninfected (control) and infected cells are representative of 3 independent experiments. Block arrows indicate internalized bacteria in CMPase negative vacuoles. Thin arrow depicts bacteria undergoing degradation within CMPase positive vacuoles. Scale bar represents 2 µm.

Figure 4. Representative microscopic images of P. gingivalis strain W83 within LC3 positive or LAMP-1 positive vacuoles at 6 hours post-inoculation.

Arrows indicate magnified inserts. Scale bar is equivalent to 10 µm. Representative images of other P. gingivalis strains can be viewed in supporting file Figure S4.

Figure 5. Number of internalized P. gingivalis in HCAE cells and their distribution in LC3 or LAMP-1 positive vacuoles.

A) Mean number ± SD of internalized bacteria. **Significantly less than other strains (P<0.0001). B) Mean percent ± SD bacteria in LC3 positive vacuoles. *Significantly different from W83 and 381 (P<0.01). C) Mean percent ± SD bacteria in LAMP-1 positive vacuoles. *Significantly different from other strains (P<0.02). All values were obtained from samples collected at 6 h post inoculation and represent three biological replicates obtained from three independent experiments.

Figure 6. Impact of 3-MA treatment on the intracellular trafficking and survival of P. gingivalis.

A) Mean percent ± SD of internalized bacteria found in LAMP-1 positive vacuoles obtained from 3 biological replicates from 3 independent experiments. *Values were significantly different (P<0.05). B) Mean log CFU ± SD (n = 5) of internalized P. gingivalis recovered from HCAE cell lysates at 6 h post-inoculation. ** Values were significantly different (P<0.02).

Figure 7. Endothelial response profile to P. gingivalis infection at 24 hours post-inoculation.

Values represent the mean ± SD of 3 biological replicates from 3 independent experiments. **Values were significantly different from all other groups (P<0.05). *Values were significantly different from control (P<0.05).