alpha-chymotrypsin and Dental-Plaque

PitA is the putative tip adhesin of the pilus islet 2 (PI-2)-encoded sortase-dependent pilus in the Gram-positive Streptococcus oralis, an opportunistic pathogen that often flourishes within the diseased human oral cavity. Early colonization by S. oralis and its interaction with Actinomyces oris seeds the development of oral biofilm or dental plaque. Here, the PI-2 pilus plays a vital role in mediating adherence to host surfaces and other bacteria. A recombinant form of the PitA adhesin has now been produced and crystallized. Owing to the large size (∼100 kDa), flexibility and complicated folding of PitA, obtaining diffraction-quality crystals has been a challenge. However, by the use of limited proteolysis with α-chymotrypsin, the diffraction quality of the PitA crystals was considerably enhanced to 2.16 Å resolution. These crystals belonged to space group P1, with unit-cell parameters a = 61.48, b = 70.87, c = 82.46 Å, α = 80.08, β = 87.02, γ = 87.70°. The anomalous signal from the terbium derivative of α-chymotrypsin-treated PitA crystals prepared with terbium crystallophore (Tb-Xo4) was sufficient to obtain an interpretable electron-density map via terbium SAD phasing.

Topics: Actinomyces; Adhesins, Bacterial; Biofilms; Chymotrypsin; Crystallization; Crystallography, X-Ray; Dental Plaque; Escherichia coli; Fimbriae, Bacterial; Gene Expression; Humans; Streptococcus oralis; X-Ray Diffraction

Topics: Adult; Animals; Bacteria; Benzoylarginine Nitroanilide; Case-Control Studies; Chymotrypsin; Culture Media; Dental Plaque; Dipeptides; Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; Endopeptidases; Female; Humans; Male; Middle Aged; Milk; Oligopeptides; Pancreatic Elastase; Periodontal Index; Periodontitis; Porphyromonas gingivalis; Prevotella intermedia; Substrate Specificity; Trypsin

Germ-free rats, fed a modified cariogenic diet 2000 with a reduced sucrose content of 25%, were inoculated with a strain of Streptococcus mutans, Streptococcus intermedius or Propionibacterium acnes, respectively. After 100 days on the diet, the rats were sacrificed. Microscopic examination of stained decalcified tooth sections revealed marked destruction of the outer zones of the dentin infected with S. mutans. The destruction was closely associated with the formation of glucan-mediated large aggregates of S. mutans. The aggregates, however, were rarely formed in the deep zones of the infected dentin. Inoculation with S. intermedius and P. acnes had little effect on the microscopic outline of the decalcified tooth sections. However, the staining behavior of the sections with Mallory's method, Alcian blue-PAS and silver was like that of the tooth sections infected with S. mutans, even in the central and inner zones of the dentin. The histochemical alterations of the deep zones of the dentin induced by the three infected bacteria appeared to be due to their proteolytic rather than to their acidogenic activities.

Topics: Animals; Chymotrypsin; Colony Count, Microbial; Dental Caries; Dental Enamel; Dental Plaque; Dentin; Diet, Cariogenic; Germ-Free Life; Male; Microbial Collagenase; Microradiography; Pepsin A; Propionibacterium acnes; Rats; Rats, Inbred Strains; Streptococcus; Streptococcus mutans; Tooth

Using an agar-skim milk gel method, over 300 oral bacterial isolates were tested for their ability to produce trypsin or chymotrypsin inhibitor. Eleven bacterial strains, including seven isolates identified as Bacteroides intermedius, were found to inhibit both proteolytic activities. The inhibitory factors of the 11 bacterial strains could be divided into two categories: i) heat-stable and dialysable, and ii) heat-labile and non-dialysable. The protease inhibitor activity of B. intermedius strains, which belongs to the latter category, was found in a membrane fraction as well as in a membrane-free extract. Furthermore, three strains of B. intermedius showed this inhibitory activity in the culture supernatant. In combination with plasma-derived protease inhibitors, the production of these additional protease inhibitors by oral bacteria could protect the host from tissue degradation.

Topics: Adult; Bacteria, Anaerobic; Bacterial Proteins; Bacteroides; Cell-Free System; Chymotrypsin; Dental Plaque; Eubacterium; Female; Fusobacterium; Gram-Negative Bacteria; Humans; Male; Periodontium; Protease Inhibitors; Trypsin Inhibitors