Response of Porphyromonas gingivalis to Environmental Stimuli Identifies Therapeutic Targets

Porphyromonas gingivalis is an oral pathogen linked to chronic periodontitis. Understanding of the changes to the proteome during different environmental stimuli could provide valuable insights into the molecular processes of survival and for the identification of new therapeutic targets. Objectives: To measure changes in protein abundance when P. gingivalis is grown under different conditions in order to identify novel potential therapeutic targets. Methods: P. gingivalis W50 was grown in continuous culture under i) heme-excess and limitation ii) biofilm and planktonic growth mode. Proteins were labeled with stable isotopes (Isotope Coded Affinity Tags or H₂¹⁸O), separated by gel electrophoresis, identified and quantified by mass spectrometry. A P. gingivalis mutant deficient in functional IrpI (PG1374) was tested for human epithelial cell invasion. Effects of a fumarate reductase inhibitor on P. gingivalis growth and biofilm formation were conducted in a 96 well assay. Results: During heme-limitation 142 proteins were identified of which 53 and 17 proteins significantly increased and decreased in abundance, respectively. During biofilm growth 116 P. gingivalis cell envelope proteins were identified of which 24 and 18 proteins significantly increased and decreased in abundance, respectively. Of particular interest were the coordinated change in abundance of the enzymes of the aspartate and glutamate catabolic pathways and the decrease in abundance of the fumarate reductase complex, FrdA-FrdB under heme-limitation and biofilm growth. The fumarate reductase inhibitor, oxantel inhibited P. gingivalis growth with a minimal inhibitory concentration of 112 µM and subminimal inhibitory concentrations inhibited early biofilm formation. There was an increase in abundance of three proteins potentially involved in invasion of epithelial cells and one of these, IrpI was demonstrated to be required for epithelial cell invasion activity. Conclusion: P. gingivalis proteins regulated by heme availability and biofilm growth show significant potential as targets to limit the growth and virulence of this bacterium.