Antimicrobial Assay of Proteins Against P. gingivalis

Porphyromonas gingivalis ATCC 33277 (ATCC, Manassas, VA, USA) were grown anaerobically for at least 24 h (80% N₂, 10% CO₂, 10% H₂) at 37 °C in Todd-Hewitt broth (THB) (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 0.001% hemin (Thermo Fisher Scientific, Waltham, MA, USA) and 0.0001% vitamin K (Thermo Fisher Scientific, Waltham, MA, USA). 20 µM of each of the purified proteins (SCPPPQ1 and ODAM) were exposed in a test tube to 800 µl of a suspension of bacteria at a OD₆₆₀ of 1 (CFU = 10⁹ cells/ml^{13 (link)}) for 2 h at 37 °C. This concentration of P. gingivalis was previously used to experimentally induce periodontitis in mice^{38 (link)}. The protein concentration of 20 μM (160 μg/ml) was selected based on our own prior work showing resistance of SCPPPQ1 to degradation by P. gingivalis^{13 (link)}. Also, it represented a concentration within the range used in other studies looking at the effects of antimicrobial effects of peptides^{17 (link),18 (link),34 (link),42 (link),43 (link)}. The mixture was then sampled for fluorescence microscopy, SEM and TEM characterization. As a negative control, buffer without the proteins was incubated with P. gingivalis under the same conditions.