Investigating Environmental and Bacterial Factors that Influence PPAD Activity

Objectives: One of the etiological agents of periodontal disease is Porphyromonas gingivalis (Pg), an anaerobic bacterium that colonizes the subgingival biofilm. Pg secretes a peptidylarginine deiminase (PPAD), which is an enzyme that converts peptidylarginine to peptidylcitrulline. We have shown that deletion of the gene encoding PPAD results in greater biofilm formation, but regulation of PPAD activity in the wild type remains unclear. The aim of this study was to investigate how periodontitis-associated environmental and bacterial variables affect the enzymatic activity of PPAD and, ultimately, biofilm formation.
Methods: The effect of oxidative stress on planktonic growth and biofilm formation of Pg strain 381 was assessed by the addition of hydrogen peroxide at different concentrations. Planktonic growth was measured at an OD₆₀₀ over time. Cell-associated PPAD enzymatic activity was measured using a colorimetric assay. Biofilm formation was assessed using the static biofilm assay and safranin staining. The effect of hemin was assessed by passaging Pg to deplete hemin before diluting in media with or without hemin. The effect of gingipains was assessed by measuring PPAD activity in strain 381 compared to the ppad, rgp, and kgp deletion mutants.
Results: Our results showed that with increasing concentrations of hydrogen peroxide, PPAD enzymatic activity of planktonic cells decreased while biofilm formation increased. In contrast, hemin availability did not affect PPAD activity under the conditions tested. Similarly, gingipain activity did not affect PPAD enzymatic activity.
Conclusions: Our data show that oxidative stress is an environmental factor that regulates PPAD expression. Furthermore, this supports our model that when PPAD activity decreases, biofilm formation increases. Although hemin is necessary for the growth of Pg, changes in available hemin does not affect PPAD activity. In terms of gingipains, our data support the model that gingipains are important for the production of substrates for PPAD rather than directly impacting the activity of PPAD.