alpha-chymotrypsin and Periodontal-Pocket

Among periodontal anaerobic pathogens, the oral spirochetes, and especially Treponema denticola, have been associated with periodontal diseases such as early-onset periodontitis, necrotizing ulcerative gingivitis, and acute pericoronitis. Basic research as well as clinical evidence suggest that the prevalence of T denticola, together with other proteolytic gram-negative bacteria in high numbers in periodontal pockets, may play an important role in the progression of periodontal disease. The accumulation of these bacteria and their products in the pocket may render the surface lining periodontal cells highly susceptible to lysis and damage. T. denticola has been shown to adhere to fibroblasts and epithelial cells, as well as to extracellular matrix components present in periodontal tissues, and to produce several deleterious factors that may contribute to the virulence of the bacteria. These bacterial components include outer-sheath-associated peptidases, chymotrypsin-like and trypsin-like proteinases, hemolytic and hemagglutinating activities, adhesins that bind to matrix proteins and cells, and an outer-sheath protein with pore-forming properties. The effects of T. denticola whole cells and their products on a variety of host mucosal and immunological cells has been studied extensively (Fig. 1). The clinical data regarding the presence of T. denticola in periodontal health and disease, together with the basic research results involving the role of T. denticola factors and products in relation to periodontal diseases, are reviewed and discussed in this article.

Topics: Acute Disease; Adhesins, Bacterial; Aggressive Periodontitis; Animals; Bacterial Adhesion; Bacterial Outer Membrane Proteins; Chymotrypsin; Colony Count, Microbial; Epithelial Cells; Erythrocytes; Extracellular Matrix; Fibroblasts; Gingivitis, Necrotizing Ulcerative; Hemagglutinins; Hemolysin Proteins; Humans; Lymphocytes; Neutrophils; Peptide Hydrolases; Pericoronitis; Periodontal Diseases; Periodontal Pocket; Treponema; Treponemal Infections; Trypsin; Virulence

Treponema denticola, a periopathogen, evades complement-mediated killing by binding the negative complement regulatory protein factor H (FH) to its surface via the FhbB protein. Paradoxically, bound FH is cleaved by T. denticola's dentilisin protease, a process hypothesized to trigger localized dysregulation of complement activation in periodontal pockets. The ability of other oral treponemes to evade complement-mediated killing and bind and cleave FH has not been assessed. In this report, we demonstrate that representative isolates of Treponema socranskii, Treponema medium, Treponema pectinovorum and Treponema maltophilum are also serum resistant, whereas Treponema vincentii and Treponema amylovorum are serum sensitive. Although T. denticola's ability to evade complement-mediated killing is strictly dependent on FH binding, other serum-resistant treponemal species lack FhbB and do not bind FH, indicating an FH-independent mechanism of complement evasion. To assess the influence of FhbB sequence variation on FH binding and cleavage by T. denticola, fhbB sequences were determined for 30 isolates. Three distinct phyletic types were identified. All T. denticola strains bound FH and were serum resistant, but differences in binding kinetics, dentilisin activity and FH cleavage ability were observed. Based on these analyses, we hypothesize that the composition of the T. denticola population is a determining factor that influences the progression and severity of periodontal disease.

Topics: Antigens, Bacterial; Bacterial Proteins; Chymotrypsin; Complement Activation; Complement Factor H; Complement Inactivating Agents; Complement System Proteins; DNA, Bacterial; Genetic Variation; Humans; Immune Evasion; Mouth; Peptide Hydrolases; Periodontal Diseases; Periodontal Pocket; Phylogeny; Sequence Analysis, DNA; Sequence Homology, Nucleic Acid; Treponema; Treponema denticola

Treponema denticola is an anaerobic spirochete whose abundance in the subgingival crevice correlates with the development and severity of periodontal disease. The ability of T. denticola to survive and thrive in the hostile environment of the periodontal pocket is due, at least in part, to its ability to bind factor H (FH), a negative regulator of the alternative complement pathway. The FH binding protein of T. denticola has been identified as FhbB and its atomic structure has been determined. The interaction of FH with T. denticola is unique in that FH bound to the cell surface is cleaved by the T. denticola protease, dentilisin. It has been postulated that FH cleavage by T. denticola leads to immune dysregulation in periodontal pockets. In this study, we conduct a comparative assessment of the sequence, properties, structure and ligand binding kinetics of the FhbB proteins of strains 33521 and 35405. The biological outcome of the interaction of these strains with FH could differ significantly as 33521 lacks dentilisin activity. The data presented here offer insight into our understanding of the interactions of T. denticola with the host and its potential to influence disease progression.

Topics: Animals; Antigens, Bacterial; Bacterial Proteins; Base Sequence; Chymotrypsin; Complement Factor H; Computational Biology; Conserved Sequence; Disease Progression; Female; Host-Pathogen Interactions; Humans; Immune Sera; Immunologic Factors; Ligands; Mice; Mice, Inbred C3H; Mice, Inbred Strains; Mutagenesis, Site-Directed; Peptide Hydrolases; Periodontal Pocket; Promoter Regions, Genetic; Protein Binding; Rats; Rats, Sprague-Dawley; Sequence Analysis, DNA; Sequence Deletion; Sequence Homology, Nucleic Acid; Treponema denticola

Protease-activated receptor 2 (PAR2) is implicated in the pathogenesis of chronic inflammatory diseases, including periodontitis; it can be activated by gingipain and produced by Porphyromonas gingivalis and by neutrophil protease 3 (P3). PAR2 activation plays a relevant role in inflammatory processes by inducing the release of important inflammatory mediators associated with periodontal breakdown. The effects of periodontal treatment on PAR2 expression and its association with levels of proinflammatory mediators and activating proteases were investigated in chronic periodontitis patients. Positive staining for PAR2 was observed in gingival crevicular fluid cells and was reflective of tissue destruction. Overexpression of PAR2 was positively associated with inflammatory clinical parameters and with the levels of interleukin-6 (IL-6), IL-8, tumor necrosis factor alpha, matrix metalloprotease 2 (MMP-2), MMP-8, hepatocyte growth factor, and vascular endothelial growth factor. Elevated levels of gingipain and P3 and decreased levels of dentilisin and the protease inhibitors secretory leukocyte protease inhibitor and elafin were also associated with PAR2 overexpression. Healthy periodontal sites from individuals with chronic periodontitis showed diminished expression of PAR2 mRNA and the PAR2 protein (P < 0.05). Furthermore, periodontal treatment resulted in decreased PAR2 expression and correlated with decreased expression of inflammatory mediators and activating proteases. We concluded that periodontal treatment resulted in decreased levels of proteases and that proinflammatory mediators are associated with decreased PAR2 expression, suggesting that PAR2 expression is influenced by the presence of periodontal infection and is not a constitutive characteristic favoring periodontal inflammation.

Topics: Adhesins, Bacterial; Adult; Bacterial Proteins; Chymotrypsin; Cysteine Endopeptidases; Elafin; Female; Gingipain Cysteine Endopeptidases; Gingival Crevicular Fluid; Hepatocyte Growth Factor; Humans; Inflammation; Inflammation Mediators; Interleukin-6; Interleukin-8; Male; Matrix Metalloproteinase 2; Matrix Metalloproteinase 8; Middle Aged; Myeloblastin; Peptide Hydrolases; Periodontal Pocket; Periodontitis; Porphyromonas gingivalis; Receptor, PAR-2; RNA, Messenger; Secretory Leukocyte Peptidase Inhibitor; Tumor Necrosis Factor-alpha; Vascular Endothelial Growth Factor A; Young Adult

Treponema denticola has been shown to be associated with periodontitis in man and animals. The organism ferments amino acids and thrives on the proteins in the periodontal pocket. Accordingly, T. denticola possesses various proteolytic enzymes, including a chymotrypsin-like protease, capable of hydrolyzing a whole range of proteins, including immunoglobulins. Yet, it is not clear whether the intact cells of T. denticola can degrade immunoglobulins and albumin. The purpose of this study was to clarify this point. Three strains of T. denticola were cultured in liquid medium, and cells were harvested by centrifugation. Protein degradation in cell suspensions was assayed by capillary electrophoresis and immunonephelometry. None of the T. denticola strains appeared to be able to degrade IgA, IgG, or albumin, while a strain of P. gingivalis completely hydrolyzed these proteins. The findings suggest that, in the periodontal pocket, T. denticola depends on proteinases from other bacteria for utilization of the available serum proteins. This is in accordance with clinical data showing a close relationship between T. denticola and strongly proteolytic bacteria, such as Porphyromonas gingivalis and Bacteroides forsythus.

Topics: Bacteroides; Chymotrypsin; Electrophoresis; Humans; Immunoglobulin A; Immunoglobulin G; Nephelometry and Turbidimetry; Periodontal Pocket; Periodontitis; Porphyromonas gingivalis; Serum Albumin; Symbiosis; Treponema; Treponemal Infections

The 160, 190 and 270 kDa outer sheath proteases of Treponema denticola ATCC 35404 were found to be multiple forms of the major 91 kDa phenylalanine protease (PAP) by immunoblotting using anti-91 kDa specific antibodies. Multiple forms of the phenylalanine protease were also found in 2 other T. denticola strains studied, ATCC 33520 and the clinical isolate GM-1. Protein, proteolytic and Western blot analyses using antibodies against the PAP and the major outer sheath protein (MSP) indicated that the 190 and 270 kDa proteases were protein complexes formed by the MSP and the PAP. These complexes dissociated by storage in 0.3% or higher SDS concentrations. The purified PAP was found to completely degrade keratin, but was unable to degrade native actin either in its monomeric or polymerized form. The association of the MSP adhesin with a protease capable of degrading host native proteins may benefit the obtention of protein-based nutrients necessary to support the growth of these treponemes. These complexes may also play a role in the structural organization of T. denticola outer sheath.

Topics: Actins; Bacterial Proteins; Blotting, Western; Chymotrypsin; Electrophoresis, Polyacrylamide Gel; Humans; Keratins; Peptide Hydrolases; Periodontal Pocket; Phenylalanine; Treponema