leupeptins and Periodontitis

To determine the levels of LL-37 in and its susceptibility to degradation by components of gingival crevicular fluid (GCF) in periodontal health and disease.. Levels of LL-37 in GCF from periodontitis patients and periodontally healthy subjects were determined by ELISA. In addition, degradation of synthetic/exogenous LL-37 by components of GCF in the presence and absence of inhibitors was determined by matrix-assisted laser desorption/ionization time of flight mass spectrometry.. The concentration of native LL-37 in GCF from Porphyromonas gingivalis positive (Pg+) and P. gingivalis negative (Pg-) sites in periodontitis patients was significantly higher than in GCF from healthy subjects. When synthetic LL-37 was added to healthy GCF, the peptide was not degraded. Conversely, GCF from Pg+ sites rapidly degraded synthetic LL-37 which was prevented in the presence of Arg- and Lys- gingipain inhibitors. Synthetic LL-37 was degraded more slowly by GCF from Pg- sites.. LL-37 is detectable in GCF in periodontal health and disease. The rapid degradation of synthetic LL-37 in periodontitis GCF, particularly in Pg+ sites, limits its role as a potential therapeutic in the gingival crevice. These results highlight the need to design stable peptide mimetics of LL-37 as future therapeutics in periodontitis.

Topics: Adhesins, Bacterial; Adult; Aged; Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Cathelicidins; Cysteine Endopeptidases; Cysteine Proteases; Cysteine Proteinase Inhibitors; Dental Plaque; Enzyme-Linked Immunosorbent Assay; Gingipain Cysteine Endopeptidases; Gingival Crevicular Fluid; Humans; Leupeptins; Middle Aged; Peptide Fragments; Periodontitis; Periodontium; Porphyromonas gingivalis; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Tosyllysine Chloromethyl Ketone

The association of periodontitis with atherosclerosis has been suggested from epidemiological studies. Recently, we have reported that macrophages stimulated by Porphyromonas gingivalis formed foam cells in the presence of low-density lipoproteins (LDL). In this study, we examined the direct interactions between LDL and P. gingivalis.. We investigated the aggregation of LDL with P. gingivalis and its outer membrane vesicles (OMVs), degradation of the apo B-100 protein of LDL by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analyses, as well as the effects of protease inhibitors or activators on the mobility of LDL by agarose gel shift assays. The binding of P. gingivalis or its OMVs with LDL was demonstrated by western blot analysis. We also examined whether or not the aggregated LDL induced foam cell formation from murine macrophages.. LDL was aggregated in a dose-dependent manner with P. gingivalis and its OMVs. Moreover, degradation of the apo B-100 protein of LDL was directly demonstrated in the presence of P. gingivalis or its OMVs. Furthermore, the gel shift assays indicated that the mobility of LDL was increased in the presence of P. gingivalis. This alteration was attenuated in the presence of the protease inhibitors TLCK and leupeptin and increased in the presence of reducing agents. Moreover, LDL was bound to specific proteins of P. gingivalis suggesting that these proteins may also play a role in aggregation. Finally, the aggregated LDL induced murine macrophages to form foam cells.. These results suggest that P. gingivalis may stimulate foam cell formation, in part, by aggregating LDL by proteolysis of apo B-100.

Topics: Adhesins, Bacterial; Animals; Apolipoprotein B-100; Apolipoproteins B; Arteriosclerosis; Cell Line; Cell Membrane; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Foam Cells; Gingipain Cysteine Endopeptidases; Hemagglutinins; Humans; Leupeptins; Lipoproteins, LDL; Macrophages; Mice; Periodontitis; Porphyromonas gingivalis; Protein Synthesis Inhibitors; Tosyllysine Chloromethyl Ketone; Vacuoles

Porphyromonas gingivalis, the major etiologic agent of chronic periodontitis, produces a broad spectrum of virulence factors, including outer membrane vesicles. In this study, we investigated the capacity of P. gingivalis vesicles to promote the shedding or cleavage of the lipopolysaccharide (LPS) receptor CD14 from the surface of human U937 macrophage-like cells. SDS-PAGE/Western immunoblotting analysis of gingival crevicular fluid samples from patients affected by moderate or advanced periodontitis revealed the presence of soluble CD14 and CD14 fragments, thus supporting the hypothesis of an in vivo shedding and cleavage of CD14 receptors. Flow cytometry analysis of macrophage-like cells treated with a vesicle-containing culture supernatant of P. gingivalis showed a significant decrease in the binding of anti-human CD14 to the cell surface. However, no accumulation of soluble CD14 or immunoreactive CD14 fragments in the assay supernatant could be demonstrated by ELISA. Treatment of macrophage-like cells with various concentrations of P. gingivalis vesicles substantially suppressed TNF-alpha production triggered by Escherichia coli LPS. This suppressive effect was much less important using heat-treated vesicles or in the presence of leupeptin, a gingipain inhibitor, during the treatment. Recombinant human CD14 receptors were found to be susceptible to proteolytic degradation by P. gingivalis vesicles. A purified Arg-gingipain preparation produced much more degradation than a Lys-gingipain preparation. This study provides evidence that P. gingivalis outer membrane vesicles contribute to the loss of membrane-bound CD14 receptors and that gingipains degrade this LPS receptor. Such a phenomenon, which results in an hyporesponsiveness of macrophages to LPS stimulation, may contribute to an increased capacity of P. gingivalis, and other periodontopathogens, to evade the host immune system mechanisms.

Topics: Adhesins, Bacterial; Blotting, Western; Cell Line; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Escherichia coli; Gingipain Cysteine Endopeptidases; Gingival Crevicular Fluid; Hemagglutinins; Humans; Leupeptins; Lipopolysaccharide Receptors; Lipopolysaccharides; Macrophages; Periodontitis; Porphyromonas gingivalis; Tumor Necrosis Factor-alpha; Virulence Factors

Suffusion of gingipain RgpA (GRgpA) elicited a significant concentration-dependent increase in the clearance of macromolecules from in situ hamster cheek pouch which was attenuated by NPC 17647, a selective bradykinin B(2) receptor antagonist. Leupeptin and a mixture of proteinase inhibitors also attenuated GRgpA-induced responses. These data indicate that GRgpA elicits plasma exudation from in situ oral mucosa in a catalytic site-dependent fashion by elaborating bradykinin.

Topics: Adhesins, Bacterial; Animals; Bradykinin; Capillary Permeability; Catalytic Domain; Cricetinae; Cysteine Endopeptidases; Gingipain Cysteine Endopeptidases; Hemagglutinins; Indomethacin; Leupeptins; Male; Mesocricetus; Mouth Mucosa; Periodontitis; Receptor, Bradykinin B2; Receptors, Bradykinin

The degradation of human lactoferrin by putative periodontopathogenic bacteria was examined. Fragments of lactoferrin were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and measured by densitometry. The degradation of lactoferrin was more extensive by Porphyromonas gingivalis and Capnocytophaga sputigena, slow by Capnocytophaga ochracea, Actinobacillus actinomycetemcomitans and Prevotella intermedia, and very slow or absent by Prevotella nigrescens, Campylobacter rectus, Campylobacter sputorum, Fusobacterium nucleatum ssp. nucleatum, Capnocytophaga gingivalis, Bacteroides forsythus and Peptostreptococcus micros. All strains of P. gingivalis tested degraded lactoferrin. The degradation was sensitive to protease inhibitors, cystatin C and albumin. The degradation by C. sputigena was not affected by the protease inhibitors and the detected lactoferrin fragments exhibited electrophoretic mobilities similar to those ascribed to deglycosylated forms of lactoferrin. Furthermore a weak or absent reactivity of these fragments with sialic acid-specific lectin suggested that they are desialylated. The present data indicate that certain bacteria colonizing the periodontal pocket can degrade lactoferrin. The presence of other human proteins as specific inhibitors and/or as substrate competitors may counteract this degradation process.

Topics: Aggregatibacter actinomycetemcomitans; Bacteria; Bacteroides; Campylobacter; Capnocytophaga; Enzyme Inhibitors; Fusobacterium nucleatum; Humans; Iodoacetamide; Lactoferrin; Leupeptins; Peptostreptococcus; Periodontitis; Phenylmethylsulfonyl Fluoride; Porphyromonas gingivalis; Prevotella intermedia; Serine Proteinase Inhibitors; Tosyllysine Chloromethyl Ketone; Tosylphenylalanyl Chloromethyl Ketone