Specific cell components of Bacteroides gingivalis mediate binding and degradation of human fibrinogen

Abstract

Bacteroides (Porphyromonas) gingivalis, which has been implicated as an etiologic agent in human periodontal diseases, has been shown to bind and degrade human fibrinogen. B. gingivalis strains bind fibrinogen reversibly and with high affinity and bind to a specific region of the fibrinogen molecule that appears to be located between the D and E domains (M. S. Lantz, R. D. Allen, P. Bounelis, L. M. Switalski, and M. Hook, J. Bacteriol. 172:716-726, 1990). We now report that human fibrinogen is bound and then degraded by specific B. gingivalis components that appear to be localized at the cell surface. Fibrinogen binding to bacterial cells occurred at 4, 22, and 37 degrees C. A functional fibrinogen-binding component (Mr, 150,000) was identified when sodium dodecyl sulfate-solubilized bacteria were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, and probed with 125I-fibrinogen. Fibrinogen degradation did not occur at 4 degrees C but did occur at 22 and 37 degrees C. When bacteria and iodinated fibrinogen were incubated at 37 degrees C, two major fibrinogen fragments (Mr, 97,000 and 50,000) accumulated in incubation mixture supernatant fractions. Two major fibrinogen-degrading components (Mr, 120,000 and 150,000) have been identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in substrate-containing gels. Fibrinogen degradation by the Mr-120,000 and -150,000 proteases was enhanced by reducing agents, completely inhibited by N-alpha-p-tosyl-L-lysyl chloromethyl ketone, and partially inhibited by n-ethyl maleimide, suggesting that these enzymes are thiol-dependent proteases with trypsinlike substrate specificity. The fibrinogen-binding component could be separated from the fibrinogen-degrading components by selective solubilization of bacteria in sodium deoxycholate.

Characterization of the binding activities of proteinase-adhesin complexes from Porphyromonas gingivalis

“…Evidence in this report and others (20,21) indicates that the active proteinase-adhesin complexes most likely degrade bound proteins, causing detachment of such complexes. This suggests a mechanism whereby the proteinase-adhesin complexes cause progressive attachment to, degradation of, and dissociation from host receptor molecules.…”

“…The results obtained here strongly support these conclusions. Fb-and Fn-binding proteins have previously been identified in studies of whole P. gingivalis cells (20,21) in which two proteins of 120 and 150 kDa which bound and degraded Fb and Fn were studied. Recently (4), an enzyme designated porphypain was isolated, also as 120-and 150-kDa forms, and found to be a fibrinogenase with Arg-Lyssplitting activity.…”

Analysis of the prtP gene encoding porphypain, a cysteine proteinase of Porphyromonas gingivalis

“…gingivalis HG66 was a gift from R. Arnold (School of Dentistry, University of North Carolina). All P. gingivalis strains were maintained as laboratory stocks and cultured as previously described (24). Culture purity was routinely confirmed by Gram staining.…”

“…To determine their effect on P. gingivalis W12 binding and hydrolysis of fibrinogen, cells were preincubated with preimmune IgGs, or various concentrations of immune IgGs, for 30 min at 22ЊC. Binding of 125 I-fibrinogen to the cells was determined as previously described (24,25). Briefly, cells pretreated with IgGs were incubated with 125 I-fibrinogen in the presence of phosphate-buffered saline and bovine serum albumin for 30 min at 22ЊC, and then the radioactivity associated with pelleted bacteria was determined in a gamma counter (LKB, Wallac, Turku, Finland).…”

Periodontopathic Bacteria and Their Potential Involvement in Atherosclerosis

“…Such sites may arise as a consequence of the flow-stress placed upon various areas of the vasculature (22). Since platelets and fibrinogen are present at inflamed sites and P. gingivalls has been demonstrated to attach to both (27,28), this could serve as a means of localizing at least one periodontopathogen in these areas.…”