Effects of Arginine, Proline or Glucose on Protease and Peptidase Activity from in vitro Oral Biofilm Models

Objectives: Oral bacteria grown in a mixed aerobic-anaerobic environment demonstrated proteolytic degradation of salivary proteins when sterilised saliva is used as a growth-medium. Metabolomic analysis revealed these biofilms produced significant amounts of proline. We were therefore interested to know if proline could alter these biofilms in a similar way to the previously published effects of arginine and glucose. The aim of this study was to analyse salivary protein degradation using SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry of in vitro oral bacterial biofilms when grown in sterilised saliva and in addition to arginine, proline and glucose.
Methods: Biofilms were grown using a previously described method on hydroxyapatite (HA) discs (Cleaver, Moazzez & Carpenter, 2019). Sterilised saliva growth-medium was supplemented with 50mM of either arginine, proline or glucose. This was refreshed every 72-hours. Spent-saliva growth-medium was retained for SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and filtered using a 10kDa filter for mass spectrometry.
Results: The SDS-PAGE average (n=3) total lane volume intensity (Int) for all treatments and controls was calculated using ImageLab. When compared to the positive control (un-supplemented biofilm), which demonstrated proteolytic activity regardless of exposure, proline had an increased lane intensity of 0.9%, arginine an increase of 4%, and glucose a decrease of 3%.
The total number of peptides per sample was calculated for all treatments and controls. The positive control sample contained 1,600 peptides, proline 1,600, arginine 3,500, and glucose 2,200.

Conclusions: The SDS-PAGE results suggest that protease activity in un-supplemented biofilms was induced by glucose and inhibited by arginine, but unaffected by proline. The mass spectrometry results suggest glucose induces protease and peptidase activity, that arginine may inhibit proteases as shown by SDS-PAGE, but it may activate peptidases. Proline has no effect on proteolytic activity within the biofilm. Peptidase activity within the biofilms will be further analysed.