Identification of bistable populations of Porphyromonas gingivalis that differ in epithelial cell invasion.

Bistable populations of bacteria give rise to two or more subtypes that exhibit different phenotypes. We have explored whether the periodontal pathogen, Porphyromonas gingivalis, exhibits bistable invasive phenotypes. Using a modified cell invasion assay, we show for the first time that there are two distinct sub-types within a population of P.ginigivalis strains NCTC 11834 and W50 that display differences in their ability to invade oral epithelial cells. The highly-invasive sub-types invade cells at 10-30 fold higher levels than the poorly-invasive subtype and remain highly invasive for approximately 12-16 generations. Analysis of the gingipain activity of these sub-types revealed that the highly invasive type had reduced cell-associated arginine specific protease activity. The role of arg-gingipain activity in invasion was verified by enhancement of invasion by rgpAB mutations and by inclusion of an arg-gingipain inhibitor in invasion assays using wild-type bacteria. In addition a population of DeltargpAB bacteria did not contain a hyperinvasive sub-type. Screening of the protease activity of wild-type populations of both strains identified high and low protease sub-types which also showed the corresponding reduction or enhancement of invasive capabilities. Microarray analysis of these bistable populations revealed a putative signature set of genes that include oxidative stress resistance and iron transport genes that might be key to invasion of or survival within epithelial cells. Porphyromonas gingivalis comparison of Invasive V Non-invasive sub-types of NCTC11834 and W50 (samples 1-12 AND High V low protease subtypes (samples 13-24) Samples 1-3 were compared top 4-6 and 7-9 V 10-12 for invasive experiments and similarly for protease strain experiments