Heme and Susceptibility of Porphyromonas gingivalis to Reactive Oxygen Species

Neutrophils constitute the predominant class of professional phagocytes protecting the gingival tissues. Neutrophils kill bacteria principally by the generation of reactive oxygen species, in particular the superoxide radical (O₂^.-) and hydrogen peroxide (H₂O₂). As a redox-active molecule, heme may protect heme-accumulating bacteria such as Porphyromonas gingivalis from the effects of reactive oxygen species. AIM: To determine the influence of bacterial heme status on the survival of P. gingivalis following exposure to either H₂O₂ or O₂^.-. METHODS: Heme-replete (H+) cells of P. gingivalis strains ATCC 33277 and W50 were prepared by growth in tryptic soy broth containing menadione (TSB-M) and hemin (5µg/ml) whereas heme-starved (H-) cells were grown in TSB-M. Washed bacteria were resuspended to a concentration of approximately 10⁸/ml in Tris-buffered saline (TBS) containing peptone (0.1%). Either pyrogallol (which generates O₂^.-), or H₂O₂ was added and the tubes vortexed aerobically for 60 seconds (pyrogallol) or 10 seconds (H₂O₂). Tubes were incubated at 37C for 1 hour, before plating onto brain-heart infusion agar. After five days incubation colonies were enumerated to determine bacterial survival. RESULTS: There was no difference in survival between H+ and H- bacteria when each was exposed to H₂O₂ (0 – 100 µM); less than 1% of cells of either preparation survived exposure to 100 µM. Similarly, there was no difference in survival following exposure to various concentrations of pyrogallol (0 - 100 µM); both H+ and H- preparations experienced less than 5% survival at 100 µM. The addition of either superoxide dismutase (1 mg/ml) or catalase (1 mg/ml) to assays involving pyrogallol, raised bacterial survival to more than 60% confirming the involvement of O₂^.-. Both strains of P. gingivalis responded to reactive oxygen species in the same way. CONCLUSIONS: There was no evidence that bacterial heme status influences resistance of P. gingivalis to either O₂^.- or H₂O₂.