Cleavage of protease-activated receptors on an immortalized oral epithelial cell line by Porphyromonas gingivalis gingipains

Giacaman R.A.; Asrani A.C.; Ross K.F.; Herzberg M.C.

Keywords: sequence, expression, antibody, binding, cells, flow, protein, cell, gene, antibodies, mutant, alpha, specificity, bacterial, tumor, line, humans, human, receptor, strain, cysteine, deletion, level, immunofluorescence, interaction, necrosis, proteinase, affinity, indicators, mucosa, article, factor, cleavage, reagents, cytometry, keratinocyte, test, mouth, type, controlled, labeling, wild, par-2, study, 1, 3, interleukin, amino, priority, r, nonhuman, journal, 2, Receptor,, and, Epithelial, 6, staining, activated, terminal, 1beta, Porphyromonas, gingivalis, Endopeptidases, upregulation, 1alpha, Adhesins,, immortalization, gingipain, PAR-1

Abstract

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-1α, IL-1β, IL-6 and TNF-α; 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.

Más información

Título de la Revista: Microbiology
Volumen: 155
Asunto: 10
Editorial: Maik Nauka/Interperiodica Publishing
Fecha de publicación: 2009-01-01
Página de inicio: 3238
Página final: 3246
Idioma: eng
DOI/URL:

10.1099/mic.0.029132-0

Identificador: 10.1099/mic.0.029132-0
Notas: SCOPUS