rial strains and growth conditions P. gingivalis ATCC 33277 was used as a wild type strain in this study. This strain was grown at 37 C under anaer obic conditions on 5% horse blood agar plates and in 30 mg ml trypticase soy broth supple mented with 2. 5 mg ml yeast e tract, 5 ug ml hemin and 5 ug ml menadione. Bacterial growth was monitored by measuring the optical density at 660 nm. For invasion somehow assays, an inoculum with an infec tion ratio of 100 bacteria per cell was added to the cell culture medium. Cell culture The human gingival epithelial cell line Ca9 22 was ob tained from RIKEN Bioresource Center. Ca9 22 cells were cultured under standard conditions in Eagles minimal essential medium containing 10% fetal bovine serum, 1% penicillin and streptomycin at 37 C in a humidified atmosphere of 5% CO2.

The monocytic cell line THP 1 was obtained from Japanese Collection of Research Bioresources Cell Bank. THP 1 cells were cultured under standard condi tions in Roswell Park Memorial Institute 1640 Medium containing 10% FBS, 1% penicillin and streptomycin at 37 C in a humidified atmosphere of 5% CO2. Antibodies Antibodies were obtained from the following sources antiserum for P. gingivalis whole cells was kindly donated by Dr. Fuminobu Yoshimura, mouse monoclonal antibody specific for ICAM 1, goat polyclonal antibody specific for ICAM 1, mouse monoclonal antibody specific for TNFRI, mouse monoclonal antibody specific for TNFRII and mouse immunoglobulin G, mouse monoclonal antibody specific for Rab5, rabbit polyclonal antibody specific for ICAM 1, goat IgG, mouse monoclonal antibody specific for B actin, anti rabbit IgG Ale a 555 and anti rabbit IgG Ale a 633, mouse monoclonal antibody specific for GFP, anti mouse IgG HRP, anti rabbit IgG HRP and mouse monoclonal antibody specific for B actin.

Vector constructs GFP Rab5Q79L, GFP Rab5WT, and GFP Rab5S34N in pcDNA3 constructs were kindly provided by Dr. Yuji Yamamoto. The GST R5BD vector was kindly donated by Dr. Guangpu Li. P. gingivalis invasion assay Invasion of bacteria was quantitated by a standard anti biotic protection assay as described previously. Briefly, Ca9 22 cells were seeded in 12 well flat bottom culture plates and were incubated overnight before ad ministration of P. gingivalis. The cells then were washed twice with phosphate buffered saline and AV-951 incu bated for a further 1 h in OPTI MEM with out antibiotics.

The cells were treated with 10 ng ml of recombinant human TNF for 3 h. P. gingivalis suspended in OPTI MEM was added to the Ca9 22 cells at an MOI of 1 100 and further incubated at 37 C in 5% CO2 for 1 h. Unattached bacteria were removed by washing with PBS three times. OPTI MEM containing 200 ug ml of metronidazole and 300 ug etc ml of gentami cin was added to the plates and they were incubated for 1 h. The cells were washed twice with PBS, and then 1 ml of sterile distilled water per well was added and the cells were suspended persistently by pipetting to disrupt them. The lysates wer