Trypsin-like Enzyme Activity of the Extracellular Membrane Vesicles of Bacteroides gingivalis W50

Abstract

Trypsin-like enzyme activity in spent culture media from 3-d-old batch cultures of Bacteroides gingivalis W50 was measured by using the hydrolysis of Na-benzoyl-L-arginine-p-nitroanilide. The cell-free culture medium was fractionated by differential centrifugation at 10000 g and 75000 g, yielding two particulate fractions and a soluble supernatant fraction. About 80% of the total recoverable activity was associated with the particulate fractions, the remainder being in the supernatant. Electron microscopy of ruthenium-red/osmium stained ultrathin sections of the pellet fractions showed them to be composed of vesicular particles (extracellular vesicles), between 50 and 250 nm in diameter. Enzyme activity in all three fractions was enhanced by dithiothreitol. Gel-permeation chromatography of the soluble fraction yielded one peak of activity which contained 64 kDa and 58 kDa polypeptides. Enzyme activity from the vesicular fractions could be solubilized by sonication, giving a similar chromatographic profile to the supernatant fraction. The main peak of activity was composed of 64 kDa and 58 kDa polypeptides. In addition, there was a higher molecular mass enzyme activity peak composed of the 64 kDa and 58 kDa components along with 11 1 kDa, 93 kDa and 70 kDa polypeptides. We conclude that the trypsin-like enzyme of B. gingivalis is released as a soluble protein and is also associated with extracellular vesicles, in which it may exist as a soluble component and also as a protein complex.

Characterization of the trypsin-like enzymes of Porphyromonas gingivalis W83 using a radiolabeled active-site-directed inhibitor

“…Both enzymes are cell-associated and released into the supernatant of batch cultures of the organism. We cannot yet state whether they are both components of the extracellular vesicle trypsin-like activity (Smalley & Birss, 1987). These data are complementary to those recently presented by Hinode et al (1991) who described the purification of three proteases from the culture supernatant of P. gingivalis 381.…”

Pathogenesis of periodontitis: a major arginine-specific cysteine proteinase from Porphyromonas gingivalis induces vascular permeability enhancement through activation of the kallikrein/kinin pathway.

“…Most of the trypsin-like activity of P. gingivalis (54,55), including RGP-1, is localized in the extracellular membrane vesicles and cell membranes of invasive strains of the bacteria (Potempa, J., unpublished observation). Hence, RGP-1 should be in contact with PK and HMWK present in periodontal interstitial fluid or plasma leaked into periodontitis sites, thereby producing kallikrein and, ultimately, the liberation of BK from HMWK.…”

Possibility of Bacteroides gingivalis Hemagglutinin Possessing Protease Activity Revealed by Inhibition Studies

“…Therefore, it is suggested that the HA of B. gingivalis has trypsin-like protease activity and that the site of the molecule participating in the hemagglutination is also involved in the catalysis. Since it has been reported by several researchers including us that B. gingivalis produces protease(s) hydrolyzing synthetic substrates of trypsin-like proteases such as benzoyl-L-arginine p-nitroanilide (BAPA) (2,4,7,10,12,14), it is of great interest from both biochemical and clinical points of view to examine the relationship between the HA and this BAPA-hydrolyzing protease of B. gingivalis. In this report, as a first step, we examined effects of various compounds on the HA and the BAPA-hydrolyzing protease activities in the membrane fraction of B.…”