Assessment of Internalization and Viability of Porphyromonas gingivalis in KB Epithelial Cells by Confocal Microscopy

Abstract

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...

Identification of bistable populations of Porphyromonas gingivalis that differ in epithelial cell invasion

“…1b,. This is in agreement with other published data that used KB cells (Dorn et al, 2000;Eick et al, 2006;Houalet-Jeanne et al, 2001), and similar levels of invasion have been reported using normal oral keratinocytes (Lamont et al, 1995). When the invasion levels of the recovered bacteria were analysed after a second round of invasion with fresh cells we observed an increase in the percentage of bacteria able to invade the OSCC cells for both strains of over threefold (Fig.…”

Nuclear Targeting ofPorphyromonas gingivalisW50 Protease in Epithelial Cells

“…Nevertheless, HeLa cells, which were more resistant than KB cells to detachment at high concentrations of supernatant, still retained differences between their staining patterns with supernatant and HRgpA when these cells were exposed to comparable BApNA levels. Internalization of viable P. gingivalis organisms by epithelial cells in vitro has been described previously by a number of groups (1,13,23), and intracellular bacteria have been shown to release outer membrane vesicles both in monolayer cultures of KB cells (19) and in multilayered cultures of human pocket epithelium (33). Interestingly, the perinuclear localization of the viable whole organisms described in gingival epithelial cells and KB cells (1,19) resembled the nuclear targeting we observed with MAb 1A1-reactive material in W50 culture supernatant.…”

“…Internalization of viable P. gingivalis organisms by epithelial cells in vitro has been described previously by a number of groups (1,13,23), and intracellular bacteria have been shown to release outer membrane vesicles both in monolayer cultures of KB cells (19) and in multilayered cultures of human pocket epithelium (33). Interestingly, the perinuclear localization of the viable whole organisms described in gingival epithelial cells and KB cells (1,19) resembled the nuclear targeting we observed with MAb 1A1-reactive material in W50 culture supernatant. Belton et al (1) stated that the perinuclear region of keratinocytes contains many organelles, but the functional significance of the association remained to be determined.…”

Autophagy: A Highway for Porphyromonas gingivalis in Endothelial Cells

“…43,46,47 However, in KB cells the bacteria are found free in the cytosol or within single-membrane bound vacuoles. 19,[48][49][50] P. gingivalis has been shown to promote cell death in KB cells as well as in gingival fibroblasts. 51,52 Our data suggest that P. gingivalis does not promote cell death in HCAEC (unpublished observations).…”