Characterization of sodium dodecyl sulfate-stable Bacteroides gingivalis proteases by polyacrylamide gel electrophoresis

Abstract

Profiles of the proteolytic activities found in Bacteroides gingivalis culture supernatants, outer membranes, vesicles, and cell extracts were analyzed in sodium dodecyl sulfate-polyacrylamide gels containing covalently bound bovine serum albumin. A total of eight distinct bands of proteolytic activity could be detected. Four of these were found in the culture supernatant (P1, P2, P3, and P4). The outer membranes, vesicles, and the cell extract each contained seven major proteolytic bands (P1, P3, P4, P5, P6, P7, and P8). No activity was found in the membrane-free extract, suggesting that the proteases were associated with the cell envelope. With the exception of P7 and P8, all the proteolytic bands were dependent on reducing agents for activity. The eight proteolytic bands were distributed in an identical manner in all four strains of B. gingivalis studied. The effects of protease inhibitors, pH, and heat were determined. SulThydryl group reagents and N-a-p-tosyl-L-lysine chloromethyl ketone reduced proteolytic activity. The optimum pH was found to be between 7 and 8. A 30-min preincubation at 50°C inactivated the P6, P7, and P8 proteolytic bands. All proteolytic activity was lost after the samples were heated at 75°C for 30 min.

Phagocytosis and virulence of different strains of Porphyromonas gingivalis

“…A large increase in the number of C3 molecules bound to W83 was, however, noted in assays carried out in the presence of the protease inhibitor TLCK (30). We found that the presence of this inhibitor, and other known inhibitors of the proteolytic activity oi P. gingivalis (13,19), during opsonization did not increase the phagocytosis of the strain W83. This suggests that opsonically active C3 was not attached to the true external surface of the bacterial cells as has been shown for encapsulated bacteria (31).…”

“…A number of studies have shown that there is a considerable variation in virulence between strains within the species and it has been suggested that this may be due to differences in collagenolytic and proteolytic activities (16,17). However, recent results indicate that the protease and collagenolytic activities do not account for the diflferences between the invasive and non-invasive strains (18,19). It has also been suggested that differences in virulence might be due to the degree of encapsulation and resistance to phagocytosis (20)(21)(22).…”

Helicobacter pylori and proteolytic activity

“…Enzyme activity was demonstrated by a modification of the method of Kelleher & Juliano [8] and Grenier et al [12].…”

Pathogenicity and virulence of black-pigmented Gram-negative anaerobes

“…P. gingivalis synthesizes a number of different proteolytic enzymes. However, no major differences in the protease profile have so far been found which may differentiate between the virulent and avirulent strains within the species [72]. Proteinases from black-pigmented Gram-negative anaerobes can degrade human plasma proteins.…”