alpha-chymotrypsin and Periodontal-Diseases

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

Topics: Adult; Chymotrypsin; Clinical Trials as Topic; Edema; Humans; Male; Oral Hemorrhage; Periodontal Diseases; Tablets; Trypsin; Wound Healing

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, a spirochete associated with periodontitis, is abundant at the leading edge of subgingival plaque, where it interacts with gingival epithelia. T. denticola produces a number of virulence factors, including dentilisin, a protease which is cytopathic to host cells, and FhbB, a unique T. denticola lipoprotein that binds complement regulatory proteins. Earlier analyses suggested that FhbB specifically bound to factor H (FH)-like protein 1 (FHL-1). However, by using dentilisin-deficient mutants of T. denticola, we found that T. denticola preferentially binds FH and not FHL-1, and that FH is then cleaved by dentilisin to yield an FH subfragment of approximately 50 kDa. FH bound to dentilisin-deficient mutants but was not cleaved and retained its ability to serve as a cofactor for factor I in the cleavage of C3b. To assess the molecular basis of the interaction of FhbB with FH, mutational analyses were conducted. Replacement of specific residues in widely separated domains of FhbB and disruption of a central alpha helix with coiled-coil formation probability attenuated or eliminated FH binding. The data presented here are the first to demonstrate the retention at the cell surface of a proteolytic cleavage product of FH. The precise role of this FH fragment in the host-pathogen interaction remains to be determined.

Topics: Amino Acid Sequence; Animals; Bacterial Proteins; Chymotrypsin; Complement Factor H; Host-Pathogen Interactions; Humans; Lipoproteins; Molecular Sequence Data; Mutagenesis, Site-Directed; Mutation; Peptide Hydrolases; Periodontal Diseases; Sequence Analysis, DNA; Treponema denticola; Treponemal Infections; Virulence Factors

The purified chymotrypsin-like protease of Treponema denticola, designated dentilisin or PrtP (DDBJ accession no. D83264), can disrupt cell-cell junctions and impair the barrier function of epithelial monolayers in vitro. Serine protease inhibitors block these effects. Yet, the protease is apparently less significant in perturbing intracellular signaling pathways and cytoskeletal rearrangement in fibroblasts. The purpose of this study was to use a PrtP-deficient mutant of T. denticola to confirm that the cytopathic effects of whole bacteria and its outer membrane on epithelial cell junctions were primarily accounted for by the activity of this protease. The prtP gene of ATCC 35405 was inactivated by insertion of an erythromycin-resistance cassette, yielding mutant K1. In contrast to wildtype ATCC 35405, mutant K1 grew in tight cell aggregates; the cells had a disrupted outer sheath, as determined by electron microscopy. When compared by silver stained SDS-PAGE of sonicated extracts of whole cells, the extract of mutant K1 was missing a band at approximately 90 kDa that was present in the wildtype ATCC 35405 strain. Whole cells and Triton X-100 outer membrane (OM) extracts of K1 and the wildtype strains were compared 1) for SAAPNA degrading activity by a colorimetric assay, 2) for stress fiber disruption in human gingival fibroblasts (HGF) by fluorescence microscopy of TRITC-phalloidin stained cells, and 3) the OM extracts only for perturbation of HEp-2 epithelial monolayers by electrical cell-substrate impedance sensing (ECIS). Mutant K-1 cells and OM had no SAPPNA degrading activity that is characteristic of dentilisin. K1 cells had HGF stress fiber disrupting activity (86 +/- 4.5% of HGFs affected) equivalent to both 35405 wildtype strains (84 +/- 3.9% and 71 +/- 14.1% of HGF, respectively). Yet, mutant K1 OM had diminished stress fiber disrupting activity (12.9 +/- 4.6% of HGF) compared with its parent 35405's OM (94.6 +/- 2.9%). The major cytopathogenic difference between the K1 mutant and wildtype strains was in their OM's effect on epithelial cell junctions. ATCC 35405 OM completely disrupted epithelial resistance in a concentration - dependent manner; mutant K1 OM had negligible effects. These data confirm that inactivation of the prtP gene completely reverses T. denticola's disruption of epithelial junctions, but there are pleiotropic effects of the mutation that may account for its apparently diminished effects on the cytoskeleton of HGF when the

Topics: Bacterial Adhesion; Bacterial Proteins; Cell Division; Cell Membrane; Chymotrypsin; Drug Resistance, Microbial; Electric Impedance; Epithelial Cells; Fibroblasts; Gingiva; Humans; Intercellular Junctions; Kinetics; Mutagenesis, Insertional; Peptide Hydrolases; Periodontal Diseases; Treponema; Tumor Cells, Cultured

Prevotella intermedia has been reported to be associated with periodontal disease whilst P. nigrescens has predominantly been isolated from more specific conditions and healthy sites. The aim of the present study was to compare the enzyme activity of these species.. Nine strains of P. intermedia and 12 strains of P. nigrescens were studied. Lipolytic, saccharolytic, nucleolytic and proteolytic activity was determined by traditional microbiological and chromogenic substrate methods.. All strains hydrolysed gelatine, casein, DNA and RNA. Lipase activity was produced by all strains except P. nigrescens ATCC 33563T. Lipolytic activity of P. nigrescens strains decreased as the environmental glucose concentration was increased. Only two strains, both P. intermedia, hydrolysed benzyl-arg-rho-nitroanilide. All strains hydrolysed alkaline rho-nitrophenolphosphate (except P. intermedia DAL100), produced glycylprolyl dipeptidase activity and demonstrated elastase-like activity. All but three strains (2 P. intermedia and I P. nigrescens) hydrolysed suc-ala-ala-pro-phe-rho-nitroanilide. Overall, no qualitatively analysed enzyme activity was exclusive to all strains of either species. Quantitatively analysed activity exhibited a high degree of variability both within and between species.. P. intermedia and P. nigrescens degrade natural and synthetic substrates, but intra- and interspecies activity is variable.

Topics: Alkaline Phosphatase; Chymotrypsin; Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; Glycosaminoglycans; Hot Temperature; Hydrolases; Lipase; Periodontal Diseases; Porphyromonas gingivalis; Prevotella; Prevotella intermedia; Species Specificity; Virulence

Topics: Adolescent; Adult; Aged; Anti-Inflammatory Agents; Child; Chymotrypsin; Drug Combinations; Evaluation Studies as Topic; Female; Humans; Male; Middle Aged; Periodontal Diseases; Trypsin

Topics: Chymotrypsin; Electrosurgery; Occlusive Dressings; Periodontal Diseases; Trypsin

Topics: Anti-Inflammatory Agents; Chymotrypsin; Cortisone; Drug Combinations; Humans; Injections, Intra-Articular; Neomycin; Ointments; Periodontal Diseases; Phenylbutazone; Prednisolone; Tablets; Temporomandibular Joint Dysfunction Syndrome; Triamcinolone Acetonide; Trypsin

Topics: Alveolar Process; Angiography; Anti-Inflammatory Agents; Arteriosclerosis; Arteriovenous Anastomosis; Capillary Permeability; Chymotrypsin; Endarteritis; Gingiva; Gingivitis; Humans; Maxillary Artery; Periodontal Diseases; Periodontium; Plethysmography; Protein Deficiency; Sodium Isotopes; Trypsin; Vascular Diseases

Topics: Administration, Topical; Anti-Inflammatory Agents; Chymotrypsin; Gingivitis; Microscopy, Fluorescence; Periodontal Diseases; Prednisolone; Rolitetracycline; Trypsin