title: Insertional inactivation of the prtP gene of Treponema denticola confirms dentilisin's disruption of epithelial junctions.
abstract: 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 cells were challenged with OM extracts.
authors.affiliation: Dental Research Institute and Medical Research Council Group in Periodontal Physiology, University of Toronto, Faculty of Dentistry, Ontario, Canada. richard.ellen@utoronto.ca
authors.forename: R P
authors.initials: RP
authors.lastname: Ellen
authors.forename: K S
authors.initials: KS
authors.lastname: Ko
authors.forename: C M
authors.initials: CM
authors.lastname: Lo
authors.forename: D A
authors.initials: DA
authors.lastname: Grove
authors.forename: K
authors.initials: K
authors.lastname: Ishihara
mesh_terms: D001422:Bacterial Adhesion / Q000235:genetics; D001426:Bacterial Proteins; D002455:Cell Division; D002462:Cell Membrane / Q000235:genetics / Q000648:ultrastructure; D002918:Chymotrypsin / Q000235:genetics* / Q000378:metabolism*; D004352:Drug Resistance, Microbial / Q000235:genetics; D017097:Electric Impedance; D004847:Epithelial Cells / Q000382:microbiology / Q000502:physiology; D005347:Fibroblasts / Q000382:microbiology / Q000502:physiology / Q000648:ultrastructure; D005881:Gingiva / Q000382:microbiology / Q000502:physiology / Q000648:ultrastructure; D006801:Humans; D007365:Intercellular Junctions / Q000382:microbiology* / Q000502:physiology*; D007700:Kinetics; D016254:Mutagenesis, Insertional; D010447:Peptide Hydrolases; D010510:Periodontal Diseases / Q000382:microbiology; D014208:Treponema / Q000166:cytology / Q000235:genetics / Q000502:physiology*; D014407:Tumor Cells, Cultured
chemical_list: D001426:Bacterial Proteins; D010447:Peptide Hydrolases; C102570:dentilisin; D002918:Chymotrypsin