Characterization of the proteinase-dependent cytotoxicity of Porphyromonas gingivalis.
Cytotoxicity of proteins from Porphyromonas gingivalis towards gingival fibroblasts and epithelial cells was studied by methods measuring vital dye uptake, DNA synthesis, release of cytoplasmic components, and apoptosis. Microscopic examination of the cells was also performed for detection of morphological changes. Experiments were made with dialyzed culture supernatants of P. gingivalis as well as supernatant fractions obtained by isoelectric focusing. The main cell damage observed was rounding of the cells and detachment from each other and the underlying surface. The cell-damaging activity correlated with the cystein-dependent proteolytic activity of the various supernatant preparations as well as with the occurrence of gelatinolytic protein bands with molecular weights previously reported for the cysteine proteinases gingipains R and K of P. gingivalis. The activity was abolished by heat treatment or by the addition of cysteine proteinase inhibitors. The results indicate that the main cytotoxic effect towards fibroblasts and epithelial cells is degradation of the intercellular matrix. The gingipains released in P. gingivalis culture supernatants are the responsible factors for this degradation.
Johansson
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A
Department of Oral Biology, Faculty of Odontology, University of Umeå, Sweden. anders.johansson@oralbio.umu.se
Kalfas
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S
D018829:Adhesins, Bacterial / Q000302:isolation & purification; D017209:Apoptosis; D002448:Cell Adhesion; D048429:Cell Size; D002470:Cell Survival; D004396:Coloring Agents; D017077:Culture Media, Conditioned; D003546:Cysteine Endopeptidases / Q000187:drug effects / Q000302:isolation & purification; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D003593:Cytoplasm / Q000382:microbiology; D004247:DNA / Q000096:biosynthesis; D003956:Dialysis; D010450:Endopeptidases / Q000302:isolation & purification*; D004847:Epithelial Cells / Q000166:cytology / Q000382:microbiology; D005109:Extracellular Matrix / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000382:microbiology; D018093:Gelatinases / Q000302:isolation & purification; D000080867:Gingipain Cysteine Endopeptidases; D005881:Gingiva / Q000166:cytology / Q000382:microbiology; D006388:Hemagglutinins / Q000302:isolation & purification; D006358:Hot Temperature; D006801:Humans; D007525:Isoelectric Focusing; D008970:Molecular Weight; D016966:Porphyromonas gingivalis / Q000201:enzymology*
D018829:Adhesins, Bacterial; D004396:Coloring Agents; D017077:Culture Media, Conditioned; D015853:Cysteine Proteinase Inhibitors; D000080867:Gingipain Cysteine Endopeptidases; D006388:Hemagglutinins; D004247:DNA; D010450:Endopeptidases; D003546:Cysteine Endopeptidases; D018093:Gelatinases