Haemin-restriction influences haemin-binding haemagglutination and protease activity of cells and extracellular membrane vesicles of Porphyromonas gingivalis W50

Porphyromonas gingivalis strain W50 was grown in a chemostat either under haemin limitation or haemin excess at pH 7.3. Cells and the extracellular vesicle (ECV) and extracellular protein (EP) fractions were separated, quantified, and assayed for haemagglutination, protease activity and haemin binding. Under haemin-limitation, despite a reduction in cell yield, there was a 2.5-fold increase in the gravimetric yield of extracellular vesicles. Cells and vesicles from haemin-limited cultures, haemagglutinated sheep red blood cells to higher titres than their haemin-excess counterparts. Growth in haemin-excess conditions resulted in increased haemin-binding capacities of ECV, cells and EDTA-extracted outer membrane. Cells grown under haemin-excess showed a 2-fold elevation in specific activity towards the substrate N-α-benzoyl-l-arginine-p-nitroanilide (l-BAPNA) compared to haemin-limited cells. The specific activities against l-BAPNA for haemin-limited ECV were 3-fold greater than their haemin-excess counterparts. These vesicle activities represented 25% and 3% of the total culture protease activity under haemin limited and haemin excess conditions respectively.