Porphyromonas gingivalis (P. gingivalis) is considered to be one of the main periodontal pathogens. The goal of this work was to confirm the ability of P. gingivalis to invade host cells. We detected P. gingivalis inside KB cells by confocal microscopy and analyzed the various aspects of the adherence and internalization process. Lysates of P. gingivalis-infected KB cells were also examined using anaerobic growth techniques. The results showed the viability and ability to replicate, inside the host cells, of the internalized pathogen. The production of vesicles was also tracked for the first time. Confocal microscopy revealed P. gingivalis in a perinuclear position.The literature is unanimous in assigning Porphyromonas gingivalis (P. gingivalis) a role as a major periodontal pathogen (17, 39). While it occasionally expresses its virulence on its own, P. gingivalis more commonly acts in cooperation with other microorganisms (21, 23). P. gingivalis is an opportunistic pathogen (22, 24) which can express an outstanding arsenal of virulence factors (6). As with many enteropathogens (5,7,26,32), invasion of host cells seems to be an important strategy used by P. gingivalis to protect itself against the host immune system and to advance through tissues (36). A possible connection has been made between the incidence of periodontal infections in pregnant women and an increased risk of their having preterm low-birth-weight babies (31). P. gingivalis has also been cited as a potential etiological factor in myocardial infarction and atherosclerosis (2, 27). The internalization was first quantified, and the ability of this pathogen to multiply within the eukaryotic cells was assessed. The survival conditions in the host cell were then determined.Epithelial cell growth. KB cells (ATCC CL17) derived from an epidermoid cancer of the oral cavity were used. The cells were inoculated into 24-well macroplates at a concentration of 10 5 cells/ml. The growth medium was replaced every day to maintain confluent cultures. For confocal microscopy, the cells were inoculated at the same concentration in glass culture dishes coated with 1% collagen I.Bacterial growth. P. gingivalis ATCC 33277 was maintained on blood agar plates in an anaerobic chamber at 37°C. ToddHewitt broth cultures were inoculated 48 h prior to each experiment.Bacterial contamination. The epithelial cells were washed twice with unsupplemented RPMI 1640 and covered with 500 l of a bacterial suspension for 1 h at 37°C. The bacterial concentration was adjusted by dilution in phosphate-buffered saline (PBS) containing 1 mM 2-mercaptoethanol to a ratio of 100 bacteria per epithelial cell as determined by optical density. RPMI alone and bacteria alone served as negative controls.Cell lysate culturing. The epithelial cells were washed and incubated with 500 l of metronidazole (100 g/ml) for 3 h (25). The cells were lysed in 1 ml of sterile distilled water for 15 min. The lysates were serially diluted, and 200 l of each dilution was spread on a blood agar plate. The plates we...
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