Application of 16O/18O reverse proteolytic labeling to determine the effect of biofilm culture on the cell envelope proteome of Porphyromonas gingivalis W50

Porphyromonas gingivalis is an oral pathogen linked to chronic periodontitis. The bacterium exists as part of a polymicrobial biofilm accreted onto the tooth surface. An understanding of the changes to the proteome especially of the cell envelope of biofilm cells compared with planktonic cells could provide valuable insight into the molecular processes of biofilm formation. To establish which proteins changed in abundance between the planktonic and biofilm growth states, the cell envelope fractions of two biological replicates of P. gingivalis cultivated in a chemostat were analysed. Proteins were separated by 1-D S DS-PAGE, in-gel digested with trypsin in the presence of H-2 O-16 or H-2 O-18 and identified and quantified by LC-MALDI TOF/TOF-MS. Using a reverse labeling strategy we identified and quantified the changes in abundance of 81 P. gingivalis cell envelope proteins. No form of bias between the labels was observed. Twenty four proteins increased in abundance and 18 decreased in abundance in the biofilm state. A group of cell-surface located C-Terminal Domain family proteins including RgpA, HagA, CPG70 and PG99 increased in abundance in the biofilm cells. Other proteins that exhibited significant changes in abundance included transport related proteins (HmuY and IhtB), metabolic enzymes (FrdAB) and immunogenic proteins.