Cleavage of protease-activated receptors on an immortalized oral epithelial cell line by Porphyromonas gingivalis gingipains.
Porphyromonas gingivalis activates protease-activated receptors (PARs) on oral keratinocytes, resulting in downstream signalling for an innate immune response. Activation depends on P. gingivalis gingipains, but could be confounded by lipopolysaccharide signalling through Toll-like receptors. We therefore hypothesized that P. gingivalis cleaves oral keratinocyte PARs in an Arg- (Rgp) or Lys- (Kgp) gingipain-specific manner to upregulate pro-inflammatory cytokines. Immortalized human oral keratinocytes (TERT-2) were incubated with wild-type P. gingivalis (ATCC 33277) or strains from a panel of isogenic gingipain deletion mutants: Kgp-deficient (KDP 129); Rgp-deficient (KDP 133); or Kgp- and Rgp-deficient (KDP 136). After incubation with P. gingivalis, keratinocytes were probed with specific antibodies against the N-terminus of PAR-1 and PAR-2. Using flow cytometry and immunofluorescence, receptor cleavage was marked by loss of specific antibody binding to the respective PARs. TERT-2 cells constitutively expressed high levels of PAR-1 and PAR-2, and lower levels of PAR-3. P. gingivalis ATCC 33277 cleaved PAR-1 and PAR-2 in a dose-dependent manner, while the receptors were unaffected by the protease-negative double mutant (KDP 136) at all m.o.i. tested. The single Kgp-negative mutant preferentially cleaved PAR-1, whereas the Rgp-negative mutant cleaved PAR-2. Wild-type or Kgp-negative mutant cleavage of PAR-1 upregulated expression of IL-1alpha, IL-1beta, IL-6 and TNF-alpha; the Rgp-negative mutant did not modulate these cytokines. Selective cleavage of PAR-1 on oral epithelial cells by P. gingivalis Rgp therefore upregulates expression of pro-inflammatory cytokines.
Giacaman
Rodrigo A
RA
The Mucosal and Vaccine Research Center, Minneapolis VA Medical Center, Minneapolis, MN 55417, USA.|Department of Diagnostic and Biological Sciences, School of Dentistry, University of Minnesota, Minneapolis, MN 55455, USA.
Asrani
Anil C
AC
The Mucosal and Vaccine Research Center, Minneapolis VA Medical Center, Minneapolis, MN 55417, USA.|Department of Diagnostic and Biological Sciences, School of Dentistry, University of Minnesota, Minneapolis, MN 55455, USA.
Ross
Karen F
KF
The Mucosal and Vaccine Research Center, Minneapolis VA Medical Center, Minneapolis, MN 55417, USA.|Department of Diagnostic and Biological Sciences, School of Dentistry, University of Minnesota, Minneapolis, MN 55455, USA.
Herzberg
Mark C
MC
The Mucosal and Vaccine Research Center, Minneapolis VA Medical Center, Minneapolis, MN 55417, USA.|Department of Diagnostic and Biological Sciences, School of Dentistry, University of Minnesota, Minneapolis, MN 55455, USA.
D018829:Adhesins, Bacterial / Q000235:genetics / Q000378:metabolism*; D000906:Antibodies / Q000494:pharmacology; D002460:Cell Line; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D004847:Epithelial Cells / Q000187:drug effects* / Q000276:immunology*; D005434:Flow Cytometry; D017353:Gene Deletion; D000080867:Gingipain Cysteine Endopeptidases; D006801:Humans; D007202:Indicators and Reagents / Q000494:pharmacology; D016966:Porphyromonas gingivalis / Q000201:enzymology* / Q000276:immunology*; D044463:Receptor, PAR-1 / Q000378:metabolism*; D044464:Receptor, PAR-2 / Q000378:metabolism*; D013194:Staining and Labeling / Q000379:methods
D018829:Adhesins, Bacterial; D000906:Antibodies; D000080867:Gingipain Cysteine Endopeptidases; D007202:Indicators and Reagents; D044463:Receptor, PAR-1; D044464:Receptor, PAR-2; D003546:Cysteine Endopeptidases