Epithelial cell invasive and non-invasive subpopulations of Porphyromonas gingivalis

Porphyromonas gingivalis can invade and survive within epithelial cells. Using an antibiotic protection assay of invasion (APA), we have previously shown that cultures of P. gingivalis contain two populations, an invasive phenotype (I) and a poorly-invasive phenotype (NI). APAs actually measure survival after invasion rather than invasion alone and the characteristics of the two populations are unknown. Objectives: The aim of this study was to employ a fluorescent assay to assess cell invasion and to investigate levels of arg-gingipain activity and sensitivity to oxidative stress in recovered bacteria. Methods: Oral epithelial cells (H357; 107 cells/well) were incubated for 90 min with P.gingivalis NCTC 11834 and W50, either unlabeled or labelled with carboxyfluorescein succinimidyl ester. Bacteria recovered from the first round of infection, and bacteria in the supernatant that had not invaded, were each used to re-infect a second set of H357 cultures. The degree of invasion was assessed by flow cytometry (FACS) after quenching fluorescence of external bacteria with trypan blue. Sensitivity to H2O2 (0-3%) was determined as MBC by broth dilution and arg-gingipain (RGP) activity was determined by BAPNA assay. Results: The existence of an I and a NI population in cultures of both strains of P.gingivalis was confirmed by FACS (invasive NCTC 11834 42.4+2.7; W50 36.9+5.3. Non-invasive NCTC 11834 7.03+1.3, W50 5.73+0.3). The I phenotypes showed reduced RGP activity (NCTC 11834 0.35+0.03, W50 0.5+0.03) but higher resistance to H2O2 (MBC NCTC 11834 & W50 3%) compared with the NI phenotypes (RGP NCTC 11834 1.28+0.03, W50 0.89+0.02; H2O2 NCTC 11834 0.09%, W50 0.05%). Conclusions: Invasive and non-invasive phenotypes of epithelial cells are detectable in cultures of P.gingivalis and these differ in protease expression and sensitivity to oxidative stress. This may represent phase switching, which in vivo could provide the organism with a mechanism for persistence at a subgingival site.