Purification and Biochemical Characterization of Outer-Membrane-Vesicles of Porphyromonas gingivalis Strains

Objectives: Porphyromonas gingivalis is a periodontal pathogen, which secretes outer membrane vesicles (OMV) containing virulence factors such proteolytic enzymes and toxins that could be helpful to infection establishment. OMV can even be more harmful than the bacteria itself. OMV are produced in all strains of P. gingivalis and it has been reported to have gingipains proteases as the main component. The aim was to purify OMV and characterize their protease activity in two P.gingivalis laboratory strains
Methods: Cultures of P. gingivalis ATCC 33277 and P. gingivalis W83 were standardized with an identical initial bacteria concentration and rocked in anaerobe atmosphere for 48h, then culture supernatants were used to obtain OMV by ultracentrifugation. Precipitated OMV were analyzed by transmission electronic microscopy (TEM), protein content, SDS-PAGE protein profile and integrity and arginine gingipain (Rgp) activity using the fluorogenic substrate (CAAM).
Results: P. gingivalis W83 showed a growth rate more than thrice the P.gingivalis 33277. Despite the slower growth of P.gingivalis 33277, the 48 h culture had 15-fold more Rgp activity than W83 strain (25.3 U/µg v. 1.54 U/µg). The standardized OMV purification protocol yield samples with no proteolytic degradation and, despite the W83 strain vesicles showing more and better bands in electrophoresis, were the P.gingivalis 33277 OMV vesicles which had a higher Rgp activity regarding W83 strain (0,6 U/ µg v. 0.46 U/ µg). TEM images showed the classic aspect of OMV like irregular spheres with diameters between 50-250 nm.
Conclusions: P.gingivalis W83 had a faster growth rate than P.gingivalis. 33277, however the Rgp activity is higher in the OMV of P. gingivalis 33277. These results contribute to understand the infection models in vivo and in vitro with different strains of P. gingivalis because OMV enzyme content vary depending on the nature of the evaluated strain.