Invasive differences among Porphyromonas gingivalis strains from healthy and diseased periodontal sites.

BACKGROUND AND OBJECTIVE: The purpose of this study was to determine any difference between Porphyromonas gingivalis isolates from periodontally healthy sites as compared to those from diseased sites with respect to the ability to invade host cells. MATERIAL AND METHODS: Subgingival plaque samples were obtained from periodontally healthy and diseased sites using paper points. P. gingivalis colonies were isolated and tested, using an antibiotic protection assay, for their ability to invade KB cells. P. gingivalis 381 and Escherichia coli MC1061 were used as controls. RESULTS: Mean values of 16.79 +/- 0.86 x 10(3) colony-forming units/mL and 26.14 +/- 2.11 x 10(3) colony-forming units/mL were observed in invasion assays for isolates from periodontally healthy and diseased sites, respectively. P. gingivalis present in diseased sites had significantly greater invasive abilities than strains isolated from healthy sites. No statistical difference was noted between male or female subjects concerning the degree of invasion; isolates from diseased sites from both genders had significantly greater invasion abilities than those from healthy sites. A significant correlation was found between the increased invasive capabilities of P. gingivalis isolates vs. an increased probing depth. CONCLUSION: The increased invasion noted with P. gingivalis isolates from diseased sites vs. healthy sites, and the increased invasive capabilities with increasing probing depth, indicate that P. gingivalis isolates have a varying ability to invade host cells in the periodontal pocket.

Invasion of vascular cells in vitro by Porphyromonas endodontalis.

AIM: The objective of this study was to determine whether laboratory strains and clinical isolates of microorganisms associated with root canal infections can invade primary cultures of cardiovascular cells. METHODOLOGY: Quantitative levels of bacterial invasion of human coronary artery endothelial cells (HCAEC) and coronary artery smooth muscle cells (CASMC) were measured using a standard antibiotic protection assay. Transmission electron microscopy was used to confirm and visualize internalization within the vascular cells. RESULTS: Of the laboratory and clinical strains tested, only P. endodontalis ATCC 35406 was invasive in an antibiotic protection assay using HCAEC and CASMC. Invasion of P. endodontalis ATCC 35406 was confirmed by transmission electron microscopy. DISCUSSION: Certain microorganisms associated with endodontic infections are invasive. If bacterial invasion of the vasculature contributes to the pathogenesis of cardiovascular disease, then microorganisms in the pulp chamber represent potential pathogens.

Porphyromonas gingivalis traffics to autophagosomes in human coronary artery endothelial cells.

Porphyromonas gingivalis is a periodontal pathogen that also localizes to atherosclerotic plaques. Our previous studies demonstrated that P. gingivalis is capable of invading endothelial cells and that intracellular bacteria are contained in vacuoles that resemble autophagosomes. In this study, we have examined the trafficking of P. gingivalis 381 to the autophagic pathway. P. gingivalis 381 internalized by human coronary artery endothelial (HCAE) cells is located within vacuoles morphologically identical to autophagosomes. The progression of P. gingivalis 381 through intracellular vacuoles was analyzed by immunofluorescence microscopy. Vacuoles containing P. gingivalis colocalize with Rab5 and HsGsa7p early after internalization. At later times, P. gingivalis colocalizes with BiP and then progresses to a vacuole that contains BiP and lysosomal glycoprotein 120. Late endosomal markers and the lysosomal cathepsin L do not colocalize with P. gingivalis 381. The intracellular survival of P. gingivalis 381 decreases over 8 h in HCAE cells pretreated with the autophagy inhibitors 3-methyladenine and wortmannin. In addition, the vacuole containing P. gingivalis 381 lacks BiP but contains cathepsin L in the presence of wortmannin. These results suggest that P. gingivalis 381 evades the endocytic pathway to lysosomes and instead traffics to the autophagosome.

Invasion of endothelial and epithelial cells by strains of Porphyromonas gingivalis.

Porphyromonas gingivalis is a periodontal pathogen that may also be involved in the pathogenesis of coronary heart disease. This microorganism has the ability to invade several cell lines. In this study, 26 different strains of P. gingivalis were tested for invasion of human umbilical vein endothelial cells and KB cells, a human oral epidermoid cell line. Abilities to invade both cell lines by an individual strain were similar, and their invasion efficiencies could be assembled into four groups: high, moderate, low and non-invasive. Of the 26 strains, only P. gingivalis AJW4 was non-invasive. Since the fimbriae are implicated as having a key role in invasion by this species, the presence of fimbriae on strain AJW4 was investigated. Using polymerase chain reaction (PCR), strain AJW4 was found to contain the fimA gene. Sequence analysis revealed it to be type IV according to the typing scheme developed by Amano et al. Further, fimA is transcribed in this strain as demonstrated by reverse transcription PCR and is expressed on the cell surface as visualized by negative staining and electron microscopy. The adherence+invasion of strain AJW4 was 38.7% of the most invasive strain (strain 381). However, the CFU ml(-1) of strain AJW4 recovered from within cells was 2.9% of strain 381. Even though strains AJW4 and W50 have the same type IV fimbriae, strain AJW4 is 8.9-fold more adhesive yet is internalized 170-fold less. These data indicate that the invasion efficiency of P. gingivalis is variable among the different strains, and that the expression of FimA is not sufficient for invasion.

Invasion of human coronary artery cells by periodontal pathogens.

There is an emerging paradigm shift from coronary heart disease having a purely hereditary and nutritional causation to possibly having an infectious etiology. Recent epidemiological studies have shown a correlation between periodontal disease and coronary heart disease. However, to date, there is minimal information as to the possible disease mechanisms of this association. It is our hypothesis that invasion of the coronary artery cells by oral bacteria may start and/or exacerbate the inflammatory response in atherosclerosis. Since a few periodontal pathogens have been reported to invade oral epithelial tissues, we tested the ability of three putative periodontal pathogens-Eikenella corrodens, Porphyromonas gingivalis, and Prevotella intermedia-to invade human coronary artery endothelial cells and coronary artery smooth muscle cells. In this study we demonstrate by an antibiotic protection assay and electron microscopy that specific species and strains invade coronary artery cells at a significant level. Actin polymerization and eukaryotic protein synthesis in metabolically active cells were required since the corresponding inhibitors nearly abrogated invasion. Many intracellular P. gingivalis organisms were seen to be present in multimembranous vacuoles resembling autophagosomes by morphological analysis. This is the first report of oral microorganisms invading human primary cell cultures of the vasculature.

Invasion of human oral epithelial cells by Prevotella intermedia.

Invasion of oral epithelial cells by pathogenic oral bacteria may represent an important virulence factor in the progression of periodontal disease. Here we report that a clinical isolate of Prevotella intermedia, strain 17, was found to invade a human oral epithelial cell line (KB), whereas P. intermedia 27, another clinical isolate, and P. intermedia 25611, the type strain, were not found to invade the cell line. Invasion was quantified by the recovery of viable bacteria following a standard antibiotic protection assay and observed by electron microscopy. Cytochalasin D, cycloheximide, monodansylcadaverine, and low temperature (4 degreesC) inhibited the internalization of P. intermedia 17. Antibodies raised against P. intermedia type C fimbriae and against whole cells inhibited invasion, but the anti-type-C-fimbria antibody inhibited invasion to a greater extent than the anti-whole-cell antibody. This work provides evidence that at least one strain of P. intermedia can invade an oral epithelial cell line and that the type C fimbriae and a cytoskeletal rearrangement are required for this invasion.