Daspher Stuart, University of Melbourne, Australia

The effect of biofilm culture on the cell envelope proteome of Porphyromonas gingivalis

Stuart G. Dashper, Ching Seng Ang, Paul Veith and Eric C. Reynolds
Principal Research Fellow, Co-operative Research Centre for Oral Health Science, The School of Dental Science, The University of Melbourne, 720 Swanston st., Parkville 3010, Vic. Australia, e-mail stuartgd@unimelb.edu.au

Porphyromonas gingivalis is an oral pathogen linked to chronic periodontitis. This bacterium exists as part of a biofilm accreted onto the tooth surface in periodontal pockets. The aim of this study was to determine changes in the cell envelope proteome that are associated with biofilm growth state of this bacterium. These changes could provide valuable insight into the molecular processes of biofilm formation and maintenance and the metabolism of cells within the biofilm. P. gingivalis was grown in planktonic and biofilm states in a single continuous culture system. A differential proteomic analysis of the P. gingivalis cell envelope fraction of planktonic and biofilm cells was conducted using a 16O/18O labelling method. Proteins were separated by 1-D SDS-PAGE, in-gel digested with trypsin in the presence of H216O or H218O 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. Fourty two proteins were significantly and consistently regulated in abundance in the biofilm growth state of two biological replicates. 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 indicating that there is considerable change in the cell surface. Other proteins that exhibited significant changes in abundance included iron complex transport related proteins (HmuY and IhtB), metabolic enzymes (FrdAB) and immunogenic proteins. These data indicate that the majority of cells in a mature biofilm are haem-limited and this has a significant effect on their ability to catabolise amino acids. They also demonstrate the applicability of heavy water labelling to determine environmentally controlled changes in bacterial proteomes.