Cultivation of P. gingivalis Strains

P. gingivalis strains (wt-Pg ATCC 33277 and its isogenic mutant Δppad and C351A expressing catalytically inactive PPAD (PPAD^C351A) were grown on blood agar plates (BHI, brain heart infusion medium supplemented with 5% sheep blood, 5 μg mL⁻¹ hemin and 0.5 μg mL⁻¹ vitamin K in an anaerobic chamber (90% N₂, 10% CO₂, and 5% H₂). The media were additionally supplemented with erythromycin (5 μg mL⁻¹) or tetracycline (1 μg mL⁻¹) for the growth of ΔPPAD or C351A mutants, respectively. After cultivation at 37°C for 7 days, bacterial cells were inoculated into enriched BHI broth (Becton Dickinson) for overnight culture. Prior to inoculation, bacteria were washed with phosphate-buffered saline (PBS) and resuspended in a fresh culture medium. Bacterial cell counts were determined using a spectrophotometer; an optical density of 1.0 at 600 nm corresponded to a concentration of 1 × 10⁹ colony-forming units (CFU) per mL. Bacterial suspensions were used for further experiments.