title: Protease-active extracellular protein preparations from Porphyromonas gingivalis W83 induce N-cadherin proteolysis, loss of cell adhesion, and apoptosis in human epithelial cells.
abstract: BACKGROUND
  The protease-induced cytotoxicity of P. gingivalis may partly result from alteration of the extracellular matrix and/or surface receptors that mediate interaction between the host cells and their matrix. While P. gingivalis-induced degradation of E-cadherin has been documented, there is no information on the effects of P. gingivalis proteases on other members of this family of cell adhesion proteins.
  
  
  METHODS
  Human epithelial KB cells were exposed to protease-active extracellular protein preparations from isogenic mutants of P. gingivalis. Quantification of apoptosis was performed by visualization of nuclei stained with 4,6'-diamidino-2-phenylindole. Alteration of cell adhesion proteins was examined by immunoblotting of cell lysates using monoclonal antibodies to those proteins.
  
  
  RESULTS
  Treated cells exhibited loss of cell adhesion properties with apoptotic cell death subsequently observed. These effects correlated with the different levels of cysteine-dependent proteolytic activities of the isogenic mutants tested. Cleavage of N-cadherin was observed in immunoblots of lysates from detached cells. There was a direct correlation between the kinetics of N-cadherin cleavage and loss of cell adhesion properties. Loss of cell adhesion, as well as N-cadherin cleavage, could be inhibited by preincubation of P. gingivalis protease active extracellular protein preparations with the cysteine protease inhibitor TLCK. In control experiments, the cleavage of N-cadherin was detected after treatment of KB cells with trypsin but not after cell dissociation by a non-enzymatic method.
  
  
  CONCLUSIONS
  These results suggest that extracellular proteases from P. gingivalis can induce degradation of N-cadherin, which could have implications for the pathogenicity of this bacterium.
authors.affiliation: Department of Microbiology and Molecular Genetics, School of Medicine, Loma Linda University, CA 92350, USA.
authors.forename: Z
authors.initials: Z
authors.lastname: Chen
authors.forename: C A
authors.initials: CA
authors.lastname: Casiano
authors.forename: H M
authors.initials: HM
authors.lastname: Fletcher
mesh_terms: D000704:Analysis of Variance; D000911:Antibodies, Monoclonal; D017209:Apoptosis / Q000187:drug effects*; D015820:Cadherins / Q000378:metabolism*; D002448:Cell Adhesion / Q000187:drug effects; D002467:Cell Nucleus / Q000187:drug effects; D003546:Cysteine Endopeptidases / Q000494:pharmacology; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D010450:Endopeptidases / Q000494:pharmacology*; D004847:Epithelial Cells / Q000187:drug effects; D005456:Fluorescent Dyes; D006801:Humans; D015151:Immunoblotting; D007211:Indoles; D007624:KB Cells / Q000187:drug effects; D009154:Mutation / Q000235:genetics; D016966:Porphyromonas gingivalis / Q000201:enzymology* / Q000235:genetics / Q000472:pathogenicity; D014107:Tosyllysine Chloromethyl Ketone / Q000494:pharmacology; D014357:Trypsin / Q000494:pharmacology
chemical_list: D000911:Antibodies, Monoclonal; D015820:Cadherins; D015853:Cysteine Proteinase Inhibitors; D005456:Fluorescent Dyes; D007211:Indoles; D014107:Tosyllysine Chloromethyl Ketone; C007293:DAPI; D010450:Endopeptidases; D014357:Trypsin; D003546:Cysteine Endopeptidases