Effects of Arginine Deiminase-Like Enzymes on Porphyromonas gingivalis Biofilm

A. Michael¹, K. Gregson², R.L. Gregory², ¹Indiana University School of Dentistry, Indianapolis, IN, ²Dept. of Oral Biology, Indiana University School of Dentistry, Indianapolis, IN.

Objective: According to the NIH, upwards of 50% of Americans have periodontal disease. The underlying etiology is biofilm formation. One of the more virulent species involved in periodontitis biofilms is Porphyromonas gingivalis. Previous studies have indicated that arginine deiminase (ADI), purified from the membrane of Streptococcus cristatus inhibits P. gingivalis biofilm formation via a cell-to-cell contact mechanism. Since ADI is not present in higher-level eukaryotes, it exhibits potential for drug targets in antimicrobial and antiparasitic therapy. The structure of ADI is similar to other arginine-modifying enzymes such as dimethyl arginine dimethylammohydrolase (DDAH) and human peptidylarginine deiminase 4 (PAD4). The objective of this study was to test these proteins for biofilm inhibition.

Methods: P. gingivalis was treated with PAD4 (375, 750, and 1500pg/ul), DDAH (333, 666, and 1332pg/ul), and ADI media (3.6uM) in media alone, in combination, or without protein or bacteria (negative and positive controls, respectively). The ADI protein was obtained by making aliquots of a filter sterilized S. cristatus supernatant. Growth of planktonic cells and biofilm formation was measured after a 48h incubation period with a crystal violet dye staining assay. Relative absorbance values were obtained using a microtiter plate spectrophotometer.

Results: P. gingivalis biofilm exposed to ADI media, PAD4 (1500pg/ul), and DDAH (666pg/ul) demonstrated significantly less growth than the negative control, while there was no significant difference in planktonic bacteria.

Conclusions: These results suggest that ADI media, PAD4, and DDAH slowed the growth of P. gingivalis biofilm formation at varying concentrations. None of the proteins had a significant effect on planktonic P. gingivalis. This study provides evidence for the potentially therapeutic aspects of P. gingivalis treated with arginine deiminase-like proteins.