Effect of Gingipain and Fibronectin Receptors on Porphyromonas gingivalis invasion

P. gingivalis has been shown to invade epithelial cells rapidly, but it is not clear whether this involves a direct interaction between fimbrillin and the fibronectin receptor integrin, α5β1, or between the protease gingipain and fibronectin, which then binds to the integrin. Objectives: To evaluate the role of gingipain and an alternative fibronectin receptor αvβ6 in epithelial cell invasion by P.gingivalis. Methods: P.gingivalis strains W50, E8 (a rgpA:rgpB mutant), K1A (a kgp mutant), the type strain, NCTC 11834, and a clinical isolate, A02,were placed in contact with epithelial cells at a multiplicity of infection of 100 for 90 min and invasion was assessed using an antibiotic-protection assay (Metronidazole 300μg/ml). The epithelial cells employed were H357 and an αvβ6-transfected derivative (VB6). Protease was assessed in whole cells and sonic extracts using N-benzoyl-DL-arginine-2-naphthylamide (BANA) developed with Fast Blue salt. Results: Bacterial counts obtained from epithelial cell lysates before and after antibiotic exposure were not significantly different indicating high efficiency invasion by attached bacteria. All strains invaded both epithelial cell lines and expression of αvβ6 did not influence the degree of invasion. NCTC 11834 was the most invasive strain (30.8%), while the clinical isolate, A02, showed the lowest invasion (0.2%). W50 was also poorly invasive (1.3%), whereas the rgpA:rgpB mutant of this strain (E8) was significantly more invasive (20%) but the kgp mutant (K1A) was similar to the wildtype (1.34%). NCTC 11834 showed higher levels of BANA-hydrolysing activity (8.2U) than W50 (1.07U) and A02 (2.34 U). Conclusions: P.gingivalis invasion of epithelial cells is not mediated by the fibronectin receptor, αvβ6, and gingipain influences invasion but in a complex manner.