P. gingivalis is an oral bacterium that causes pathology in a number of dental infections. A common sequelae of infection is death of fibroblasts. Previous investigators had reported that gingipains produced by P. gingivalis induced apoptosis. Objective: To test the hypothesis that protease activity was responsible for P. gingivalis induced fibroblast apoptosis. Method: Human gingival fibroblasts (HGF) were incubated in vitro with wild type P. gingivalis (W83) or P. gingivalis lacking genes encoding both the cysteine proteases R-gingipain and K-gingipain. The incubation period was six hours with bacterial concentration of 2x10^9/ml. Apoptosis was measured by the amount of cytoplasmic histone-associated DNA as determined by ELISA. The impact of P. gingivalis produced protease activity was established by cell rounding. A protease inhibitor leupeptin was also used to suppress the effect of proteases in both groups. Results: Wild type and mutant P. gingivalis significantly increased fibroblast apoptosis by 60% compared to cells not incubated with P. gingivalis (P<0.05). However, there was no difference in the level of apoptosis in the wild type and mutant P. gingivalis (p>0.05). In contrast to effects on cell death, the mutant P. gingivalis had a delayed effect in cell rounding consistent with deletion of two gingipain genes. Application of the protease inhibitor leupeptin prevented cell rounding. However, it had no effect on P. gingivalis stimulated apoptosis (P>0.05). Conclusion: The principal mechanism by which P. gingivalis induces human gingival fibroblast apoptosis is not dependent of gingipain activity. NIH Grant Number: DE13191

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